Pancreatic Supernatant Research Articles

PANCREATIC PROTEASES ARE NECESSARY FOR INCREASED GUT EPITHELIAL PERMEABILITY DURING TRAUMA/HEMORRHAGIC SHOCK

Shock-induced distant organ injury is associated with increased gut permeability which contributes to gut-origin multiple organ dysfunction syndrome (MODS). Thus, understanding the pathogenesis of trauma- hemorrhagic shock (T/HS)-induced gut injury is important. In this context, we propose the hypothesis that pancreatic proteases play a key role in loss of gut barrier function after T/HS. To test this hypothesis, male Sprague- Dawley rats were subjected to T/HS (MAP 30-35 mmHg × 90 min) with and without pancreatic duct ligation (PDL). After 3 hours of reperfusion, ileal permeability was assessed using FITC-labeled dextran (in-vivo closed loop technique). The magnitude of gut permeability was approximately 3-fold greater in the T/HS rats as compared to T/HS rats with PDL (18.3 ± 4 vs 6.5 ± 5.1 μg/ml P<0.05). Next, we directly tested whether pancreatic proteases increase enterocyte permeability using an in-vitro enterocyte monolayer model. Caco-2 monolayers were incubated for 3 hr or 18 hr with either fetal bovine serum (FBS) or supernatants from homogenized rat pancreas (1:6 w/v PBS). Permeability was assessed using a 10,000 MW rhodamine probe applied to a 3 μm-pore sized transwell cell culture system. We demonstrated a dose-dependent response at both 3hr (r2 = 0.997) and 18 hr (r2 = 0.998) incubation using 5%, 10%, 25% dilutions of initial homogenized pancreas supernatants. Furthermore, we demonstrated that at 18 hr monolayer permeability was approximately 5-fold greater in the cells treated with 25% pancreatic supernatants as opposed to 25 % FBS (16 ± 5 vs 3.3 ± 0.3 % permeability p<0.05). Conclusion: These results support out hypothesis that pancreatic proteases are involved in the pathogenesis of T/HS-induced gut permeability and are able to directly increase enterocyte monolayer permeability.

Manganese Modulates Protein Phosphorylation in the Rat Pancreas

The effects of manganese (Mn2+) on phosphorylation activity in the rat pancreas were examined in an in vitro phosphorylation assay and by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of endogenous proteins. Several histones were phosphorylated in the presence of pancreatic supernatant obtained following a 10-min centrifugation at 15,000 g. The histone preference for this reaction was VII-S greater than V-S or II-S greater than III-S greater than other histones. The Mn2+ (10 mM) enhanced the phosphorylation of some histones (II-A, III-S, VI-S, and VIII-S) but inhibited the phosphorylation of other histones (II-S, V-S, and VII-S). The same concentration of Mn2+ also enhanced the incorporation of 32P into cytosolic proteins in the absence of exogenous histones. This effect was not mimicked by Ca2+, Mg2+, Ba2+, or Zn2+. Analysis of endogenous proteins by SDS-PAGE revealed Mn2(+)-dependent and time-dependent phosphorylation of high (98-200-kDa), intermediate (59-,52-,35-, and 30-kDa), and low-molecular weight proteins. The Mn2+ exerted similar effects in the presence of pancreatic cytosol obtained following a 60-min centrifugation at 100,000 g. However, the 35- and 30-kDa phosphoproteins and the low-molecular weight proteins were not readily visible. In both the 15,000- and the 100,000-g preparations, there was a dose-dependent increase in the phosphorylation of the Mr 98-kDa protein (p98) at concentrations ranging from 0.03 to 1.0 mM Mn2+ but a lesser stimulatory effect at 10.0 mM Mn2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium-calmodulin-stimulated protein kinase in developing pancreas.

To implicate the Ca2+-calmodulin-dependent protein kinase (CDPK) in the secretory response of pancreatic acinar cells to postnatal neurohumoral stimulation, we examined enzymatic protein kinase activity during pancreatic development. CDPK was investigated by measuring the phosphorylation of endogenous proteins and an exogenous substrate, synapsin I. Ca2+-calmodulin (CaM)-stimulated phosphorylation of endogenous proteins as well as synapsin I progressively increased in pancreatic supernatants from rats of increasing age. When compared with embryonic pancreas (1 day before birth), Ca2+-CaM-stimulated protein kinase activity increased two- and sixfold in neonatal (1 day after birth) and adult pancreas (60 days after birth), respectively, when normalized to protein content. To further characterize CDPK activity, soluble fractions prepared from embryonic, neonatal, and adult pancreas were separated by gel filtration chromatography. CDPK activities from embryonic and neonatal pancreas were contained in fractions of relative molecular weights (Mr) less than 200,000. In contrast, kinase activity from adult pancreas was predominantly comprised of a Mr approximately 550,000 species that coeluted with a type II CDPK. Increasing amounts of a Mr = 51,000 CaM-binding protein as a function of increasing age were consistent with the developmental appearance of the CaM-binding subunit of type II Ca2+-CaM-stimulated protein kinase. CaM levels increased in parallel with total Ca2+-CaM-stimulated protein kinase activity. Coordinate enhancement of type II CDPK activity and secretagogue responsiveness during pancreatic maturation may suggest a role for this enzyme in the secretory process.

Effectiveness of aprotinin in blocking a hypotensive factor of pancreatic origin from the pig, dog and monkey

Minced pancreas or a collagenase solution alone had no effect on blood pressure when infused into experimental animals. However, collagenase digested fragments or the digested pancreatic supernatant (PSF) produces profound hypotension. Previously we have shown that aprotinin, a proteolytic enzyme inhibitor, successfully blocked PSF-induced hypotension in the pig but did not alter this hypotension in the dog or monkey. Species variability of aprotinin blockade was further investigated by infusing PSF from either the pig, dog, or monkey into five animals of the other two species (“across species”). PSF alone produced hypotension in every animal “across species” but PSF “across species” and in combination with aprotinin did not produce a hypotensive reaction in each “across species” combination. These species differences of the aprotinin blockade should be considered when seeking reasons why a protinin has not proven effective in preventing the morbidity and mortality of human pancreatitis.

Ribonuclease and ribonuclease inhibitor in the human pancreas.

Previous investigations have failed to confirm any specificity of elevated serum ribonuclease (RNase) in the diagnosis of pancreatic cancer. Although RNase had been known to be present in two forms (free and inhibitor-bound) in various rat tissues, little was known about its presence in the human pancreas. This report investigates the presence of RNase inhibitor in the human pancreas through the assay of both active (=free) and total (=sum of free and inhibitor-bound) RNases. Inhibitor-bound RNase was also named as latent RNase. RNase was classified into three types according to pH (acid, neutral, and alkaline RNases) in the pancreatic supernatant fraction. An inhibitor was separated from latent RNase by p-chloromercuribenzoic acid (PCMB), and the latent RNase was changed to an active form. Latent RNase was more active on the alkaline side with a maximum at pH 7.5. Hence, the presence of RNase inhibitor was highly suggested in the pancreatic supernatant fraction. RNase inhibitor is most likely a protein, which binds with both neutral and alkaline RNases. The presence of RNase inhibitor has not yet been demonstrated in the normal human serum.

Ribonuclease and ribonuclease inhibitor in the human pancreas

Several investigators have failed to confirm any specificity of elevated serum ribonuclease (RNase) in the diagnosis of pancreatic cancer. Although RNase had been known to be present in two forms, free and inhibitor-bound, in various tissues of the rat, little was known about it in the human pancreas. The object of this report was to explore the presence of RNase inhibitor in the human pancreas through the assay of both active (= free) and total (= sum of free and inhibitor-bound) RNases. Inhibitor-bound RNase is also referred to as latent RNase. RNase was classified into three types according to pH (acid, neutral, and alkaline RNases) in the pancreatic supernatant fraction. An inhibitor was separated from latent RNase by p-chloromercuribenzoic acid (PCMB), and the latent RNase was changed to an active form. Latent RNase was more active on the alkaline side with a maximum at pH 7.5. Hence, the presence of RNase inhibitor was highly probable in the pancreatic supernatant fraction. RNase inhibitor is most likely a protein, which is bound with both neutral and alkaline RNases. RNase inhibitor may be a cause of nonspecificity and/or low sensitivity of RNase in the serum as a diagnostic marker for pancreatic cancer.

The identification of proinsulin-synthesizing polysomes in fetal bovine pancreas.

Antiserum against bovine pancreatic RNase A was produced in the rabbit. (Fab′)2 fragments were prepared from the antiserum and were found to inhibit the degradation of rat liver polysomes by fetal bovine pancreatic supernatant which contained RNase activity. Using this anti-RNase preparation, we were able to obtain fetal bovine pancreatic polysomes which appeared intact. Radioimmunoassay of the polysomal fractions after sucrose density gradient centrifugation showed that insulin immunoreactivity was associated mainly with tetrasomes, pentasomes, and some larger polysomal aggregates, suggesting the participation of these polysomes in the synthesis of proinsulin. The possibility that a precursor larger than proinsulin might be involved in the synthesis of insulin was discussed.

Studies on in vitro activation of high molecular weight pancreatic lipase

Summary Effect of protein kinase, cAMP, Mg, Ca and ATP on the activation of high molecular weight purified pancreatic lipase and adipose tissue homogenate has been studied. A 60% stimulation of lipase activity was observed in pancreatic supernatant fraction by the addition of Mg, ATP, cAMP and with or without protein kinase. With purified high molecular weight pancreatic lipase, a 40% stimulation was observed by the addition of protein kinase, Mg, ATP and cAMP. Both cAMP and Ca were found to be essential for maximum activation. A 120% stimulation of lipase activity was observed in homogenates of epididymal fat pads by the addition of various cofactors.

Composition of cellular membranes in the pancreas of the guinea pig. II. Lipids.

The lipid composition of rough and smooth microsomal membranes, zymogen granule membranes, and a plasmalemmal fraction from the guinea pig pancreatic exocrine cell has been determined. As a group, membranes of the smooth variety (i.e., smooth microsomes, zymogen granule membranes, and the plasmalemma) were similar in their content of phospholipids, cholesterol and neutral lipids, and in the ratio of total lipids to membrane proteins. In contrast, rough microsomal membranes contained much less sphingomyelin and cholesterol and possessed a smaller lipid/protein ratio. All membrane fractions were unusually high in their content of lysolecithin (up to approximately 20% of the total phospholipids) and of neutral lipids, especially fatty acids. The lysolecithin content was shown to be due to the hydrolysis of membrane lecithin by pancreatic lipase; the fatty acids, liberated by the action of lipase on endogenous triglyceride stores, are apparently scavenged by the membranes from the suspending media. Similar artifactually high levels of lysolecithin and fatty acids were noted in hepatic microsomes incubated with pancreatic postmicrosomal supernatant. E 600, an inhibitor of lipase, largely prevented the appearance of lysolecithin and fatty acids in pancreatic microsomes and in liver microsomes treated with pancreatic supernatant.