Cigarette toxin 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induces experimental pancreatitis through α7 nicotinic acetylcholine receptors (nAChRs) in mice.

Clinical studies have shown that cigarette smoking is a dose-dependent and independent risk factor for acute pancreatitis. Cigarette smoke contains nicotine which can be converted to the potent receptor ligand and toxin, NNK [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone]. Previously, we have shown that NNK induces premature activation of pancreatic zymogens in rats, an initiating event in pancreatitis, and this activation is prevented by pharmacologic inhibition of nicotinic acetylcholine receptors (nAChR). In this study, we determined whether NNK mediates pancreatitis through the α7 isoform of nAChR using α7nAChR knockout mice. PCR analysis confirmed expression of non-neuronal α7nAChR in C57BL/6 (WT) mouse and human acinar cells. NNK treatment stimulated trypsinogen activation in acini from WT but not α7nAChR-/- mice. NNK also stimulated trypsinogen activation in human acini. To further confirm these findings, WT and α7nAChR-/- mice were treated with NNK in vivo and markers of pancreatitis were measured. As observed in acini NNK treatment induced trypsinogen activation in WT but not α7nAChR-/- mice. NNK also induced other markers of pancreatitis including pancreatic edema, vacuolization and pyknotic nuclei in WT but not α7nAChR-/- animals. NNK treatment led to increased neutrophil infiltration, a marker of inflammation, in WT mice and to a significantly lesser extent in α7nAChR-/- mice. We also examined downstream targets of α7nAChR activation and found that calcium and PKC activation are involved down stream of NNK stimulation of α7nAChR. In this study we used genetic deletion of the α7nAChR to confirm our previous inhibitor studies that demonstrated NNK stimulates pancreatitis by activating this receptor. Lastly, we demonstrate that NNK can also stimulate zymogen activation in human acinar cells and thus may play a role in human disease.

Tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone initiates and enhances pancreatitis responses.

Clinical studies indicate that cigarette smoking increases the risk for developing acute pancreatitis. The nicotine metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a major cigarette smoke toxin. We hypothesized that NNK could sensitize to pancreatitis and examined its effects in isolated rat pancreatic acini and in vivo. In acini, 100 nM NNK caused three- and fivefold activation of trypsinogen and chymotrypsinogen, respectively, above control. Furthermore, NNK pretreatment in acini enhanced zymogen activation in a cerulein pancreatitis model. The long-term effects of NNK were examined in vivo after intraperitoneal injection of NNK (100 mg/kg body wt) three times weekly for 2 wk. NNK alone caused zymogen activation (6-fold for trypsinogen and 2-fold for chymotrypsinogen vs. control), vacuolization, pyknotic nuclei, and edema. This NNK pretreatment followed by treatment with cerulein (40 µg/kg) for 1 h to induce early pancreatitis responses enhanced trypsinogen and chymotrypsinogen activation, as well as other parameters of pancreatitis, compared with cerulein alone. Potential targets of NNK include nicotinic acetylcholine receptors and ß-adrenergic receptors; mRNA for both receptor types was detected in acinar cell preparations. Studies with pharmacological inhibitors of these receptors indicate that NNK can mediate acinar cell responses through an nonneuronal α(7)-nicotinic acetylcholine receptor (α(7)-nAChR). These studies suggest that prolonged exposure to this tobacco toxin can cause pancreatitis and sensitize to disease. Therapies targeting NNK-mediated pathways may prove useful in treatment of smoking-related pancreatitis.

Effects of increased intracellular cAMP on carbachol-stimulated zymogen activation, secretion, and injury in the pancreatic acinar cell.

A characteristic of acute pancreatitis is the premature activation and retention of enzymes within the pancreatic acinar cell. Because ligands linked to cAMP production may prevent some forms of pancreatitis, we evaluated the effects of increased intracellular cAMP in the rat pancreatic acinar cell. Specifically, this study examined the effects of the cholinergic agonist carbachol and agents that increase cAMP [secretin and 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP)] on zymogen activation (trypsin and chymotrypsin), enzyme secretion, and cellular injury in isolated pancreatic acini. Although cAMP agonists affected the responses to physiological concentrations of carbachol (1 microM), their most prominent effects were observed with supraphysiological concentrations (1 mM). When secretin was added to 1 mM carbachol, there was a slight increase in zymogen activation, but no change in the secretion of amylase or chymotrypsin. Furthermore, coaddition of secretin increased parameters of cell injury (trypan blue exclusion, lactic dehydrogenase release, and morphological markers) compared with carbachol (1 mM) alone. Although directly increasing cellular cAMP by 8-Br-cAMP caused much greater zymogen activation than carbachol (1 mM) alone or with secretin, 8-Br-cAMP cotreatment reduced all parameters of injury to the level of unstimulated acini. Furthermore, 8-Br-cAMP dramatically enhanced the secretion of amylase and chymotrypsin from the acinar cell. This study demonstrates that increasing acinar cell cAMP can overcome the inhibition of enzyme secretion caused by high concentrations of carbachol and eliminate acinar cell injury.

Effect of ligands that increase cAMP on caerulein-induced zymogen activation in pancreatic acini.

The pathological activation of proteases within the pancreatic acinar cell is critical to initiating pancreatitis. Stimulation of acinar cells with supraphysiological concentrations of the CCK analog caerulein (CER) leads to protease activation and pancreatitis. Agents that sensitize the acinar cell to the effects of CCK might contribute to disease. The effects of physiological ligands that increase acinar cell cAMP [secretin, VIP, and pituitary adenylate cyclase activating peptide (PACAP)] on CER-induced responses were examined in isolated rat pancreatic acini. Each ligand sensitized the acinar cell to zymogen activation by physiological concentrations of CER (0.1 nM). VIP and PACAP but not secretin also enhanced activation by supraphysiological concentrations of CER (0.1 muM). A cell-permeable cAMP analog also sensitized the acinar cell to CER-induced activation. The cAMP antagonist Rp-8-Br-cAMP inhibited these sensitizing effects. These findings suggest that ligands that increase acinar cell cAMP levels can sensitize the acinar cell to the effects of CCK-induced zymogen activation.

Prostaglandin f2alpha treatment in vivo, but not in vitro, stimulates protein kinase C-activated superoxide production by nonsteroidogenic cells of the rat corpus luteum.

Luteal regression is associated with the generation of reactive oxygen species (ROS). To determine the nature of the ROS generator, cells isolated from luteinized rat ovaries were examined for ROS production using luminol-amplified chemiluminescence (LCL). Cells cultured for 2-48 h exhibited minimal LCL, but there was a significant (30- to 50-fold), rapid (maximum at 3-5 min), and dose-dependent increase in LCL in response to phorbol ester (phorbol 12-myristate 13-acetate; TPA; ED50 = 0.03 microM) and diacylglycerol (1,2-dioctanoyl-glycerol; ED50 = 30 microM). The TPA-induced response was cell number dependent and was virtually abolished by superoxide dismutase, freezing, or heating (95 degrees C for 5 min). Zymosan, known to induce a phagocytic response in leukocytes, stimulated a superoxide (O2-.) response with a slow onset (maximum at 40 to 60 min) and a maximum about one third of that observed for TPA. The response to TPA and zymosan was inhibited by the NADPH/NADH-oxidase inhibitor, diphenylene iodonium (ID50 = 5 microM for TPA), but not by the mitochondrial inhibitors, potassium cyanide, rotenone, or sodium azide. Fractionation of cells by centrifugal elutriation showed that TPA-stimulated O2-. production coeluted with the nonsteroidogenic cells and that little, if any, O2-. generation coeluted with the steroidogenic cells. Cells isolated 1, 2, and 4 h after in vivo treatment with a luteolytic dose of prostaglandin F2alpha (PGF2alpha) showed a significant increase in TPA-stimulated O2-. production at 2 h, whereas luteal cells or corpora lutea incubated directly with 1 microM PGF2alpha did not show any increase in response. Corpora lutea isolated from naturally regressed ovaries (18 days after ovulation) showed a significantly elevated level of TPA-stimulated O2-. production. In conclusion, there is a superoxide generator in luteinized ovaries that is activated through a protein kinase C pathway, localized in nonsteroidogenic cells, transiently increased during PGF2alpha-induced luteolysis in vivo, and elevated during natural luteal regression.

Ascorbic acid-dependent cytoprotection of ovarian cells by leukocyte and nonleukocyte peroxidases.

Luteal cells contain high levels of ascorbic acid that is secreted by stimulation with agents like luteinizing hormone (LH) and prostaglandin F2 alpha (PGF2 alpha). One role for interstitial ascorbic acid, we propose, may be the detoxification of H2O2 by regeneration of catalytically active peroxidase. By serving as a preferred secondary substrate, ascorbic acid regenerates the catalytically active peroxidase that is inhibited irreversibly by H2O2 alone. To test this hypothesis, luteal cells were incubated in the absence and presence of peroxidases and H2O2, and the maximal cyclic AMP and steroidogenic response to LH was examined. In luteal cells, H2O2 is known to severely inhibit LH-sensitive cyclic AMP accumulation and steroidogenesis, and the addition of lactoperoxidase, myeloperoxidase, eosinophil peroxidase, or ascorbic acid (1 mM) alone had no effect on these responses to H2O2. However, co-incubation of ascorbic acid and the peroxidases completely reversed the inhibition of cyclic AMP accumulation and steroidogenesis produced by H2O2. These findings and the results that show direct oxidation of ascorbic acid in the presence of peroxidase and H2O2, but not with H2O2 alone, support the conclusion that ascorbic acid released from cells may detoxify H2O2 by regenerating the catalytically active state of peroxidases.

Modulation of cholesteryl ester hydrolase messenger ribonucleic acid levels, protein levels, and activity in the rat corpus luteum.

Previous studies have shown that the induction of functional luteolysis (loss of progesterone production) with either prostaglandin F2 alpha (PGF2 alpha) treatment or hypophysectomy (APX) diminished neutral cholesteryl ester hydrolase (CEH) activity in the corpus luteum (CL) and that prolactin (PRL) replacement of APX animals prevented luteolysis and maintained CEH activity at control levels. More recent studies have shown that CEH is the same protein as hormone-sensitive lipase (HSL) and that CEH/HSL activity may be regulated by phosphorylation. However, the possibility that CEH/HSL activity may be under transcriptional and/or translation control has not been excluded. Therefore, in the present study we examined whether PGF2 alpha treatment, APX, or inhibition of PRL secretion by bromocryptine (BrC) treatment modulated CEH/HSL mRNA and/or protein levels in a coordinate fashion with CEH activity. Furthermore, we examined whether CEH/HSL mRNA and/or protein levels changed after luteinization of the ovary and after natural functional regression. PGF2 alpha treatment and APX significantly reduced CEH activity; and PGF2 alpha treatment, APX, and BrC treatment significantly reduced CEH/HSL protein and mRNA levels. PRL replacement after APX substantially blocked the reductions in CEH activity, CEH/HSL protein, and CEH/HSL mRNA levels. PRL replacement during BrC treatment significantly inhibited the reductions in CEH/HSL protein and mRNA levels. CEH/HSL mRNA levels increased twofold after luteinization. Whereas CEH/HSL mRNA levels remained elevated after natural luteal regression, CEH/HSL protein significantly decreased. In summary, the luteolytic actions of PGF2 alpha, APX, and BrC resulted in coordinate reductions in luteal CEH activity, protein levels, and mRNA levels; PRL replacement significantly reversed the luteolytic effects of APX and BrC; natural luteal regression resulted in a reduction in CEH/HSL protein without a concomitant reduction in CEH/HSL mRNA. These results suggest that ovarian CEH activity is controlled at the level of both transcription and translation, and that PRL is important for continued CEH/HSL mRNA transcription in the CL.

A cell adhesion receptor antiserum abolishes, whereas laminin and fibronectin glycoprotein components of extracellular matrix promote, luteinization of cultured rat granulosa cells.

We recently showed that structural regression is marked by an endocrine-induced increase in matrix metalloproteinase activity specific for basement membrane, which suggests that extracellular matrix (ECM) may play an important role in sustaining luteal cell function. Such a role for ECM has been demonstrated for cultured mammary epithelial cells, hepatocytes, and keratinocytes. To test this hypothesis, granulosa cells from preovulatory follicles that were induced to luteinize by gonadotropin stimulation in vivo were examined. Initial studies established that cells cultured on plastic in medium supplemented with 1% fetal bovine serum, LH (100 pg/ml), PRL (1 microgram/ml), and insulin-like growth factor-I (5 ng/ml) showed a time-dependent increase in the secretion of progesterone (P4) and total progestin (P4 plus 20 alpha-dihydroprogesterone) for at least 10 days and that replacement of fetal bovine serum with 0.1% BSA stimulated P4 secretion and reduced the 20 alpha-dihydroprogesterone to P4 ratio from 10:1 to as low as 3:1. The inclusion of an anticell adhesion receptor subunit sera (Lenny IV, against the integrin beta 1-subunit) in the culture medium for the first 2 days resulted in an irreversible loss of progestin secretion by the cultured granulosa cells, but the inclusion of a bacterial collagenase (form III) had no effect. Granulosa cells from preovulatory follicles cultured on ECM (Matrigel matrix) formed cell aggregates and projected cellular sprouts, but secreted less P4 than those cultured on plastic. The inclusion of laminin in the culture medium or laminin coating the culture wells stimulated P4 secretion by granulosa cells and promoted the enlargement of steroidogenic cells (3 beta-hydroxysteroid dehydrogenase). Fibronectin-coated, but not collagen-I-coated, wells similarly promoted P4 secretion. These results suggest that a cell adhesion receptor (an integrin), and laminin and fibronectin, major glycoprotein components of ECM, play important roles in the differentiation of granulosa cells to luteal cells.

Ovarian vitamin E accumulation: evidence for a role of lipoproteins.

Reactive oxygen species, such as superoxide, hydrogen peroxide, and lipid peroxides, impair luteal function. Vitamin E, a lipophilic antioxidant vitamin, provides a major avenue of protection by scavenging free radicals and terminating lipid peroxidation. We previously showed that ovarian vitamin E levels increase after functional regression (loss of progesterone production) of the corpus luteum in the pseudopregnant rat and the objective of the present studies was to determine the mechanism(s) that resulted in such increased levels of vitamin E. Luteal vitamin E levels were significantly elevated after function regression and remained elevated. Luteal cholesterol ester levels, in contrast, decreased in parallel with the decrease in plasma progesterone levels, whereas plasma vitamin E, cholesterol, and cholesterol ester levels did not change. Because vitamin E is transported in blood by chylomicrons and lipoproteins, ovarian vitamin E levels were determined after treatments known to modify ovarian lipoprotein receptor content and serum lipoproteins. Acute treatment with aminoglutethimide during the mid-luteal phase decreased serum progesterone levels and increased luteal vitamin E and cholesterol ester levels. Daily treatment with 4-amino-pyrazolo-(3,4-d)pyrimidine reduced serum vitamin E and cholesterol ester levels, diminished the accumulation of vitamin E associated with luteal regression, significantly reduced luteal cholesterol esters levels, and increased luteal high density lipoprotein-binding sites. Analysis of the distribution of vitamin E between a membrane/particulate pellet and a lipid droplet/granule cytosol before and after luteal regression revealed no changes. Vitamin E levels were divided 60:40 between a crude particulate/membrane fraction and a cytosol/lipid droplet fraction, although functional regression produced a 2.5-fold increase in total luteal vitamin E levels. In conclusion, the uptake of vitamin E by the corpus luteum appears to be mediated by lipoprotein receptors and the increase in vitamin E that follows functional regression, we suggest, may be due to a diminished consumption of vitamin E by oxidative radicals, most likely generated during steroidogenesis.