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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 β-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.

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.

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.

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.

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.