Why cytoplasmic signalling proteins should be recruited to cell membranes.

It has been suggested that localization of signal-transduction proteins close to the cell membrane causes an increase in their rate of encounter after activation. We maintain that such an increase in the first-encounter rate is too small to be responsible for truly enhanced signal transduction. Instead, the function of membrane localization is to increase the number (or average lifetime) of complexes between cognate signal transduction proteins and hence increase the extent of activation of downstream processes. This is achieved by concentrating the proteins in the small volume of the area just below the plasma membrane. The signal-transduction chain is viewed simply as operating at low default intensity because one of its components is present at a low concentration. The steady signalling level of the chain is enhanced 1000-fold by increasing the concentration of that component. This occurs upon 'piggyback' binding to a membrane protein, such as the activated receptor, initiating the signal-transduction chain. For the effect to occur, the protein translocated to the membrane cannot be free but has to remain organized by being piggyback bound to a receptor, membrane lipid(s) or scaffold. We discuss an important structural constraint imposed by this mechanism on signal transduction proteins that might also account for the presence of adaptor proteins.

Comparative studies on nicotinamide nucleotide transhydrogenase from different sources.

(1) Nicotinamide nucleotide transhydrogenases in submitochondrial particles from beef heart mitochondria, chromatophores from Rhodospirillum rubrum and membrane preparations from Escherichia coli and Pseudomonas aeruginosa have been compared with respect to the following properties: stereospecificity for the 4-hydrogen of NADH, reactivity with 3'-NADP, inhibition by palmityl-CoA, sensitivity tot rypsin, and effects of Ca2+ and 2'-AMP on the reaction rates. (2) Transhydrogenases from submitochondrial particles, R. rubrum chromatophores and E. coli membrane preparations have A-side stereospecificity for NADH, do not react with 3'-NADP, are inhibited by palmityl-CoA and are extremely sensitive to trypsin treatment. No effects of Ca2+ or 2'-AMP on the reaction rates were observed. In R. rubrum chromatophores trypsin-sensitive sites are present both in the soluble transhydrogenase factor and in the membrane preparation devoid of transhydrogenase factor. (3) In contrast, P. aeruginosa transhydrogenase is allosterically regulated by Ca2+ and 2'-AMP, is reactive with 3'-NADP, has B-side stereospecificity for NADH and is insensitive to palmityl-CoA or trypsin treatment. (4) It is concluded that the properties characterizing the transhydrogenase in E. coli, R. rubrum chromatophores and submitochondrial particles are closely connected with the interaction of the enzyme with an energy-conserving membrane system.

Unilateral nephrectomy selectively stimulates phospholipase D in the remaining kidney.

The activation of phospholipase D in the kidney could be detected in vivo in rats treated with ethanol by the accumulation of phosphatidylethanol. Unilateral nephrectomy stimulated the activity of phospholipase D in the remaining kidney as indicated by an increase in the level of phosphatidylethanol. A significant increase in phosphatidylethanol level was observed as early as 5 min after contralateral nephrectomy and peak accumulation (200% of control) was observed after 15 min. The phosphatidylethanol level decreased again to the basal level after 2 h. The accumulation of phosphatidylethanol was specific for kidney and the product was localized primarily in the cortex. Phospholipase D activity in kidney cortical slices from untreated rats was stimulated in vitro by plasma obtained from unilaterally nephrectomized rats, indicating that circulating factors in the plasma are responsible for the activation of phospholipase D. The phospholipase D activation by plasma from uninephrectomized animals was selectively inhibited by the tyrosine kinase inhibitor genistein, but not by the protein kinase C inhibitor H7. It is concluded that phospholipase D activity is stimulated as an early signal transduction event in compensatory kidney growth.

Effect of chronic ethanol ingestion on pancreatic protein synthesis.

The effect of chronic ethanol feeding on pancreatic protein synthesis was assessed by studying the rate of incorporation of [3H]leucine into proteins in isolated rat pancreatic acini in vitro. Chronic ethanol feeding increased the rate of protein synthesis (2-3-fold) compared to controls fed an isocaloric diet. The onset of the increase in protein synthesis was detectable 2 days after the beginning of ethanol feeding, reached a maximum after 7 days and remained constant for up to 4 months. The increased incorporation of [3H]leucine was not due to an increased turnover of proteins as measured in pulse-chase experiments. After separation of individual digestive enzymes by SDS-polyacrylamide gel electrophoresis and determination of the distribution of radioactivity in different proteins, a general increase in the rate of incorporation of the label into all of the proteins was observed. In contrast to the observations made with isolated acini, there was no significant difference between the control and ethanol-fed groups when the rate of pancreatic protein synthesis was measured in vivo. However, overnight withdrawal of ethanol led to an increase of approx. 70% in protein synthesis in the ethanol-fed group. These results suggest that chronic ethanol ingestion modifies the control of pancreatic protein synthesis; the enhanced protein synthesis is expressed in isolated acini, i.e., in the absence of physiological factors present during chronic ethanol ingestion and in vivo after ethanol withdrawal.

Calcium ion-dependent signalling and mitochondrial dysfunction: mitochondrial calcium uptake during hormonal stimulation in intact liver cells and its implication for the mitochondrial permeability transition.

Hormones that elevate cytosolic Ca2+ concentrations ([Ca2+]cyt) often use Ca2+ as a messenger to activate intramitochondrial metabolic processes. However, the mitochondrial Ca2+ level also regulates the activation of the mitochondrial permeability transition (MPT), a process that involves the assembly of a high conductance proteinaceous pore across the inner and outer membrane. Studies on intact liver cells indicate that the MPT is a critical step in the cell killing induced by anoxia or respiratory inhibitors. In this study, we used freshly isolated hepatocytes to investigate to what extent the elevation of [Ca2+]cyt by vasopressin or other agonists causes Ca2+ accumulation in the mitochondria and how this treatment affects the mitochondrial susceptibility to undergo the MPT. Hepatocytes were incubated with vasopressin, glucagon, or with thapsigargin (an inhibitor of the endoplasmic reticulum Ca2+ pump) prior to permeabilization with digitonin. Mitochondrial Ca2+ accumulation was determined by following the ionomycin-induced Ca2+ release in permeabilized cells and mitochondrial swelling was studied by following cyclosporin A-sensitive light scattering changes induced by phenyl-arsenoxide and rotenone. The results indicate that agents that elevate [Ca2+]cyt cause a significant Ca2+ accumulation in the mitochondria. Excessive Ca2+ accumulation (> 10-fold increase over basal levels) was obtained with the combination of vasopressin and glucagon or with incubations containing thapsigargin. These conditions were also associated with a marked increase in rotenone-induced mitochondrial swelling. However, the more modest increase in mitochondrial Ca2+ content after treating cells with vasopressin alone did not enhance the swelling response; instead, vasopressin suppressed mitochondrial swelling compared to control incubations. Vasopressin also partly suppressed the swelling associated with thapsigargin treatment, although it did not significantly affect the Ca2+ accumulation under these conditions. This effect of vasopressin was mimicked by phorbol ester, suggesting a role for protein kinase C. The data indicate that mitochondrial Ca2+ accumulation following elevation of elevation of [Ca2+]cyt enhances the susceptibility for activation of the MPT, a response that may increase cell injury during anoxia or in response to other challenges. However, hormones also activate protective responses in the cell that suppress the MPT.

Phosphatidylethanol as a 13C-NMR probe for reporting packing constraints in phospholipid membranes.

13CH2-ethyl labeled phosphatidylethanol (PEth), a rare naturally occurring anionic phospholipid, was used to probe the interleaflet packing density difference in small and large unilamellar phospholipid vesicles (SUVs and LUVs, respectively). The intrinsically tighter lipid packing in the inner leaflet of the SUVs resulted in the splitting of the CH2-ethyl 13C-resonance into two distinct components originating from PEth molecules residing in the inner and outer leaflets. The splitting of the 13C-NMR signal from the PEth headgroup appears to be unique among naturally occurring phospholipids. We present data suggesting that the splitting of the PEth signal reports on transleaflet packing density difference modulated by unequal electrostatic interactions and structured water on the inner and outer surfaces of the SUV. The PEth resonance splitting was insensitive to pH changes over the range 5.3-8.6 and cannot be accounted for by differences in the pKa of PEth in the inner and outer monolayers of the SUV. In 13C-NMR spectra of LUVs, where packing constraints in both monolayers are approximately similar, only a single, narrow symmetrical CH2-ethyl signal was observed, which was shifted downfield at higher PEth concentrations. The carbonyl and C3-glycerol backbone PEth resonances were shifted upfield compared to those of phosphatidylcholine or phosphatidylglycerol, suggesting a more tightly packed/hydrophobic environment for these segments of the PEth molecule in the membrane. We conclude that the unique splitting of the PEth 13C-resonance reported here can be used to characterize the lipid packing conditions in various membranes and to monitor the transbilayer distribution/movement of PEth.

Evidence for a lipid dependence of mitochondrial nicotinamide nucleotide transhydrogenase.

1. The lipid dependence of mitochondrial nicotinamide nucleotide transhydrogenase from beef heart was investigated. With submitochondrial particles digestion of phospholipids by phospholipases A and C led to a partial inhibition that could not be readily reversed by phospholipids. 2. Extraction of neutral lipids including ubiquinone from lyophilized submitochondrial particles with pentane did inhibit the transhydrogenase, whereas further extraction with water/acetone led to a complete and apparently irreversible inhibition. 3. A partially purified preparation of transhydrogenase, depleted of lipids (and inactivated) by treatment with cholate and ammonium sulphate, was reactivated by various purified phospholipids but not by detergents or triacylglycerols. 4. It is concluded that mitochondrial transhydrogenase, catalyzing the nonenergy-linked transhydrogenase reaction, requires phospholipids specifically for its catalytic activity and not as dispersing agents. A mixture of phospholipids appears to fulfill this requirement better than the individual phospholipids.

Ethanol withdrawal stimulates protein synthesis in rat pancreatic lobules.

Recent studies from our laboratory (Ponnappa et al. (1988) Biochim. Biophys. Acta 966, 390-402), indicate that in the pancreas of rats fed ethanol chronically, an overnight withdrawal of ethanol stimulated the rate of protein synthesis in vivo, whereas, during continuous ethanol ingestion, the rate of protein synthesis was the same as in the control group which did not receive ethanol. However, a stimulation of protein synthesis was also observed when isolated acini were prepared from the pancreas of continuously ethanol-fed rats. In the present studies, preparations of pancreatic lobules were used to further characterize the stimulatory effect observed in the ethanol-fed group. The rate of protein synthesis was studied in vitro by determining the rate of incorporation of [3H]leucine into proteins. Similar to in vivo observations, chronic ethanol feeding did not alter the rate of protein synthesis, but an overnight withdrawal of ethanol stimulated the rate of protein synthesis by 84%. The stimulation of protein synthesis reflected a general enhancement in the rate of synthesis of most of the digestive enzymes ranging from 60 to 110%. The maximal stimulation of protein synthesis occurred within 24 h of ethanol withdrawal and the rates rapidly decreased to control levels within 3 days. During the overnight ethanol withdrawal there was also a 30-40% decrease in the activities of most of the pancreatic digestive enzymes. This observation indicates that ethanol withdrawal also initiated the secretion and/or degradation of pancreatic digestive enzymes in vivo. The observation that the enhanced rate of protein synthesis can be observed in isolated acini but not in vivo or in lobular preparations from the continuously ethanol-fed rats indicates that the pancreas contains factors which supress this stimulatory effect of ethanol intake. The stimulation of protein synthesis, brought about either by ethanol withdrawal or by collagenase digestion of the tissue, may reflect the removal of such factors.

Stimulation of protein synthesis in isolated pancreatic acini from chronically ethanol-fed rats is due to alterations in post-transcriptional regulation.

In an earlier study, we reported that isolation of acini from the pancreas of rats fed ethanol chronically, led to a 2- to 3-fold increase in the rate of protein synthesis compared to acini from rats fed the control diet. In the present study, we wanted to investigate whether the enhanced rate of protein synthesis was due to an increased rate of degranulation, reflecting a stimulation of cellular signal transduction processes, and/or to changes at the level of transcription/translation. The rate of degranulation was monitored by initially prelabelling the secretory proteins in vivo with [3H]leucine followed by determination of their fate in the intact tissue as well as in the subsequently isolated acini. The recovery of the label in isolated acini as a fraction of that incorporated into the tissue was similar for control and ethanol-fed groups, suggesting that ethanol feeding had no effect on the rate of degranulation during the isolation of acini. The rate of incorporation of [3H]uridine into total RNA was about 70% higher in acini from the ethanol-fed group as compared to the control group, suggesting a higher rate of transcription. However, the steady-state level of mRNA for trypsinogen, a representative digestive enzyme mRNA, showed only a moderate increase of 20% in acini from the ethanol-fed group compared to those from the intact tissue. These results suggest that the increased rate of protein synthesis in isolated acini from ethanol-fed rat pancreas is primarily due to post-transcriptional modifications.

Leupeptin inhibits phospholipases D and C activation in rat hepatocytes.

The relationship between phospholipase D and C activation was studied in intact rat hepatocytes and rat liver plasma membranes. In intact hepatocytes, in the presence of ethanol, vasopressin, phorbol ester, and calcium independently stimulated phosphatidylethanol (PETH) formation, a specific marker of phospholipase D activity. Leupeptin (10-1500 microM) inhibited PETH formation induced by vasopressin, but was ineffective in response to phorbol ester or calcium. Leupeptin also inhibited the formation of inositol phosphates in intact cells in response to vasopressin. In liver plasma membranes, GTP[S] induced the production of phosphatidic acid and, in the presence of ethanol, PETH. Plasma membrane-associated phospholipase D did not require calcium and was insensitive to protein kinase C inhibitors. Leupeptin inhibited PETH formation in response to GTP[S]. The inhibition by leupeptin could be overcome by increasing the concentration of GTP[S]. In plasma membranes, the inhibitory effects of leupeptin on phospholipase D occurred at doses that far exceed those required to maximally inhibit proteolysis. These data highlight a central role for phospholipase C in the activation of phospholipase D, and a minor role for a direct G-protein activation. The findings also demonstrate a novel use of leupeptin as an inhibitor of phospholipases D and C, perhaps at the level of a G protein.

On the use of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine in the study of lipid polymorphism.

The change in the fluorescence properties of dioleoyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanola mine (N-NBD-PE) as an indicator of the (liquid-crystalline) bilayer-to-non-bilayer hexagonalII (HII) phase transition has been investigated. Lipid bilayer systems which are known to undergo the bilayer-to-HII phase transition on addition of Ca2+ were compared with systems which can undergo aggregation and fusion but not HII phase formation. The former included Ca2+-triggered non-bilayer transitions in cardiolipin and in phosphatidylethanolamine mixed with phosphatidylserine. The latter type of system investigated included the addition of polylysine to cardiolipin and Ca2+ to phosphatidylserine. Freeze-fracture electron microscopy was used to confirm that under the experimental conditions used, the formation of HII phase was occurring in the first type of system, but not in the second, which was stable in the bilayer state. It was found that the fluorescence intensity of N-NBD-PE (at 1 mol% of the phospholipids) increased in both types of system, irrespective of the formation of the HII phase. A dehydration at the phospholipid head group is a common feature of the formation of the HII phase, the interaction of divalent cations with phosphatidylserine and the interaction of polylysine with lipid bilayers, suggesting that this may be the feature which affects the fluorescence properties of the NBD. The finding of a fluorescence intensity increase in systems lacking HII phase involvement clearly indicates that the effect is not unique to the formation of the HII phase. Thus, while offering high sensitivity and the opportunity to follow kinetics of lipid structural changes, changes in the N-NBD-PE fluorescence properties should be interpreted with caution in the study of the bilayer-to-HII phase transition.

Amygdalar neuronal plasticity and the interactions of alcohol, sex, and stress.

Alcohol abuse and alcoholism are major medical problems affecting both men and women. Previous animal studies reported a difference in c-Fos neuronal activation after chronic alcohol exposure; however, females remain an understudied population. To model chronic alcohol exposure match-pair fed adult male and female rats were administered 14 days of a liquid ethanol containing diet. Analysis focused on the central nucleus of the amygdala (CeA), a region integral to stress sensitivity and substance abuse. Immunocytochemical approaches identified cells containing ΔFosB, a marker of sustained neuronal activation, and activity patterns within the CeA were mapped by subdivision and rostral-caudal extent. Significant interactions were present between all groups, with gender differences noted among control groups, and ethanol exposed animals having the greatest number of ΔFosB immunoreactive cells indicating baseline dysregulation. Compared with c-Fos, a marker of recent neuronal activation, male ethanol treated animals had similar activity to controls, indicating a neuronal habituation not seen in females. Next, a cohort of animals were exposed to the forced swim test (FST), and c-Fos was examined in addition to FST behavior. Neuronal activity was increased in ethanol exposed animals compared to controls, and control females compared to males, indicating a potentiated stress response. Further, a population of activated neurons were shown to contain either corticotropin releasing factor or enkephalin. The present data suggest that dysregulation in the CeA neuronal activity may underlie some of the negative sequelae of alcohol abuse, and may, in part, underlie the distinctive response seen between genders to alcohol use.

Quantification of information transfer via cellular signal transduction pathways.

A conceptual framework is developed for the quantitative analysis of signal transfer through cellular signal transduction pathways and networks. This approach is referred to as signal transfer analysis and is based on formalisms that were first developed for the analysis of metabolic networks. Signal transduction is quantified as the sensitivity, known as the response coefficient of a target (e.g. an ion channel or transcription factor) to a signal (e.g. a hormone, growth factor or neurotransmitter). This response coefficient is defined in terms of the fractional change in the activated target brought about by a small fractional change in the signal. Quantifying the signal transduction in this way makes it possible to prove that for an idealized signaling cascade without feedback loops, the total response equals the product of all the local response coefficients, one for each level of the cascade. We show under which conditions merely having more levels in a cascade can boost the sensitivity of a target to a signal. If a signal propagates to a target through two different routes, these routes contribute independently to the total response, provided there is no feedback from the target. This independence makes the behavior of signaling cascades different from that of metabolic pathways, where different branches are connected through Kirchhoffs law. The relations between the total response and the local kinetics at each level are given for a number of network structures, such as branched signaling pathways and pathways with feedback. The formalism introduced here may provide a general approach to quantify cellular information transfer.

Mitochondrial respiratory chain activity in skeletal muscle from patients with Parkinson's disease.

Different abnormalities in mitochondrial electron transport chain activity have been demonstrated in muscle and other tissues of patients with idiopathic Parkinson's disease (PD). We studied eight Spanish patients with PD to evaluate the functional activity of the electron transport chain in muscle mitochondria from patients of this country. We found lower complex I activity (nmol.min-1.mg-1) in patients (245.8 +/- 42.8) than in controls (331.6 +/- 60.1) (p = 0.004) and lower complex IV activity in patients (46.1 +/- 9) than in controls (144.1 +/- 42.3) (p = 0.00001). Complex V activity was also decreased in two patients and complex II and III activities were normal in all of them. Although these results strongly suggest an alteration in mitochondrial DNA in PD, the various electron transport chain defects in different tissues seem to be nonspecific.

Ethanol-induced stimulation of phosphoinositide turnover and calcium influx in isolated hepatocytes.

Ethanol has been shown to mobilize intracellular calcium in isolated rat hepatocytes by activation of phosphoinositide-specific phospholipase C. However, addition of ethanol to 32P-labeled hepatocytes resulted in a rapid increase in the level of [32P]phosphatidylinositol 4-phosphate over a period of 2 min, concomitant with a small decrease in [32P]phosphatidylinositol 4,5-bisphosphate and an increase in [32P]phosphatidic acid levels. These results indicate that polyphosphatidylinositol metabolism was stimulated by ethanol simultaneously with the activation of phospholipase C. Ethanol also caused a transient increase in the influx of extracellular calcium into quin 2-loaded hepatocytes over a similar period of time. The results demonstrate that ethanol, in common with calcium-mobilizing hormones, directly or indirectly stimulated polyphosphoinositide regeneration and allowed for increased movement of calcium across the hepatocyte plasma membrane.

Effect of ethanol on amylase secretion and cellular calcium homeostasis in pancreatic acini from normal and ethanol-fed rats.

The effects of ethanol on stimulus-secretion coupling were assessed by studying amylase release, Ca2+-homeostasis, and changes in physical properties of membranes in isolated rat pancreatic acini. In acini from normal rats, ethanol (50 mM and above) in vitro caused a dose-dependent stimulation of amylase release and an increase in cytosolic free Ca2+ concentration. Ethanol did not affect amylase secretion stimulated by cholecystokinin-octapeptide (CCK8), a secretagogue that acts by increasing cytosolic free Ca2+ levels, but did potentiate the secretion of amylase induced by vasoactive intestinal peptide (VIP) which raises intracellular cAMP. Ethanol also increased the rate of 45Ca2+ exchange. In acini labeled with the spin-probe 12-doxyl stearic acid, ethanol disordered the pancreatic plasma membranes. By contrast, in acini from animals that had chronically (6-7 weeks) ingested ethanol, the membranes were resistant to this disordering effect of ethanol. Chronic ethanol feeding lowered the total cellular calcium content and ionophore (A23187)-releasable pools of acinar calcium (11 and 24% respectively), and led to a 15-30% decrease in the rate of 45Ca2+ exchange. Chronic ethanol ingestion also lowered the basal rate of amylase secretion, but ethanol in vitro stimulated amylase secretion more than in control preparations. However, these differences in basal and ethanol-induced amylase secretion were not accompanied by corresponding changes in intracellular free Ca2+. The data suggest that ethanol perturbs cell membranes and also disturbs cellular Ca2+ homeostasis. These effects may explain its actions as a weak Ca2+-mediated secretagogue. However, the membrane alterations induced by chronic ethanol feeding do not prevent the ethanol-induced interference with cellular calcium homeostasis.

Ethanol inhibits the peak of muscarinic receptor-stimulated formation of inositol 1,4,5-trisphosphate in neuroblastoma SH-SY5Y cells.

The effect of ethanol on muscarinic receptor-stimulated formation of inositol 1,4,5-trisphosphate was studied in human neuroblastoma SH-SY5Y cells. Stimulation with carbachol induced a biphasic increase of inositol 1,4,5-triphosphate with an initial peak after 10 sec declining to a plateau phase of elevation above basal levels, which was sustained for at least 5 min in the presence of agonist. The peak, but not the plateau phase, was concentration-dependently decreased by exposure to ethanol. Maximal inhibition was obtained within 30 sec of exposure to ethanol. Ethanol caused an increase in the EC50 value of carbachol for the initial rate of inositol 1,4,5-trisphosphate formation, measured after 10 sec of stimulation, from 98 microM in the absence to 196 microM in the presence of 100 mM ethanol. The potencies of pirenzepine and hexahydro-sila-difenidol hydrochloride for inhibiting [3H]quinuclidinyl benzilate binding and inositol 1,4,5-trisphosphate formation suggest that both phases are mediated via the muscarinic M1 receptor. Phorbol 12-myristate 13-acetate inhibited both phases of inositol 1,4,5-trisphosphate formation, whereas okadaic acid and modulators of cAMP-dependent protein kinase were without any effect. There was no inhibitory effect of ethanol when protein kinase C was inhibited by H7 and calphostin C, indicating that the ethanol effect is dependent on protein kinase C activity.

Kinetics and mechanisms of glutamate transport across the mitochondrial membrane.

The kinetics of glutamate influx and efflux on the glutamate-hydroxyl carrier have been measured and compared in rat liver mitochondria. At pH 7.4 and 25 degrees C, the Michaelis constants and V'max values were in agreement with the Haldane relationship when the alpha pH was accounted for. The Km values for glutamate influx and aspartate efflux on the glutamate-aspartate translocator are also reported. Extrapolation of the maximum velocities to 37 degrees and the intact liver provide values of 5.6 and 2.4 mmol/g dry wt/hr for glutamate influx and efflux, respectively, on the glutamate-aspartate translocator. Both translocators operate by a sequential mechanism with formation of a ternary complex. Their possible regulatory role in urea synthesis by liver is assessed.

Ethanol consumption and susceptibility of the pancreas to cerulein-induced pancreatitis.

Despite the fact that alcoholism is one of the major causes of pancreatitis, the pathogenesis of this disorder remains obscure. Factors such as the pattern of ethanol consumption, diet, and genetic predisposition may be contributing factors. The failure to produce alcoholic pancreatitis in experimental animals suggests that experimental provision of ethanol may only increase the predisposition to pancreatitis. To test this possibility, we developed an assay system using the in vitro model of cerulein-induced pancreatitis. In this system, pancreatic lobules were first exposed to a supraphysiologic concentration (10(-6) M) of the cholecystokinin analogue, cerulein, after which homogenates were incubated for up to 6 h. Activation of trypsinogen and chymotrypsinogen was observed only in cerulein-treated preparations. We then investigated the effects of the duration of ethanol feeding on cerulein-induced changes in rat pancreas. The pancreata from rats fed ethanol for 9-12 months were more susceptible to cerulein-induced activation of chymotrypsinogen compared to the pancreata from pair-fed control animals. This susceptibility also paralleled morphologic changes, such as dilatation of endoplasmic reticulum, only in the ethanol-fed group. In contrast, during the early stages (up to 3 months) of ethanol consumption, there was resistance (p < 0.01) to cerulein-induced changes. These results suggest that long-term ethanol consumption increases susceptibility to pancreatitis and raises the possibility that a similar mechanism may operate in human alcoholics.

Inhibition of ethanol-induced platelet activation by agents that elevate cAMP.

Ethanol activates phosphoinositide-specific phospholipase C in human platelets resulting in the mobilization of intracellular calcium and shape change (Arch. Biochem. Biophys. 260, 480-492, 1988). Preincubation of platelets with agents that increase levels of cAMP (i.e., forskolin, prostacyclin) inhibited these responses to ethanol in a concentration- and time-dependent manner. The inhibitory effect was potentiated by the phosphodiesterase inhibitor, isomethybutylxanthine. When added after ethanol, these agents also reversed platelet shape change and lowered cytosolic free calcium to basal levels. The results demonstrate a direct inhibitory effect of cAMP on the ethanol-induced activation of phospholipase C.