Robert E. Powers

Role of oxygen-derived free radicals in diet-induced hemorrhagic pancreatitis in mice

The role of oxygen-derived free radicals in the pathogenesis of acute pancreatitis was studied by evaluating the effects of catalase, allopurinol, and dimethylsulfoxide on diet-induced acute hemorrhagic pancreatic necrosis in mice. The mortality rate and degree of hyperamylasemia associated with this model of pancreatitis were reduced by catalase but a similar result followed the administration of heat-denatured catalase, suggesting that the apparent protective effect of catalase was not the result of reductions in free radical levels. Neither allopurinol nor dimethylsulfoxide reduced mortality or degree of hyperamylasemia. The increased pancreatic content of amylase and the necrosis that characterize this model of pancreatitis were not altered by any of the agents tested. In contrast, both allopurinol and dimethylsulfoxide reduced peripancreatic edema formation, suggesting that edema, but not the other features that characterize this model of pancreatitis, may result from generation of oxygen-derived free radicals.

Diminished agonist-stimulated inositol trisphosphate generation blocks stimulus-secretion coupling in mouse pancreatic acini during diet-induced experimental pancreatitis.

Young female mice fed a choline-deficient, ethionine-supplemented (CDE) diet rapidly develop acute hemorrhagic pancreatitis. We have observed that pancreatic acini prepared from these mice are unable to secrete amylase in response to addition of the cholinergic agonist carbachol, although they retain the ability to secrete amylase in response to the Ca2+ ionophore A23187. The CDE diet does not alter the binding characteristics (Kd or the maximal number of binding sites) for muscarinic cholinergic receptors as tested using the antagonist [3H]N-methylscopolamine nor the competition for this binding by carbachol. Addition of carbachol to acini prepared from mice fed the CDE diet does not result in as marked an increase in cytosolic free Ca2+ levels as that noted in control samples (evaluated using quin2 fluorescence). These observations indicate that the CDE diet interferes with stimulus-secretion coupling in mouse pancreatic acini at a step subsequent to hormone-receptor binding and prior to Ca2+ release. This conclusion is confirmed by our finding that the hormone-stimulated generation of [3H]inositol phosphates (inositol trisphosphate, inositol bisphosphate, and inositol monophosphate) from acini labeled with [3H]myoinositol is markedly reduced in acini prepared from mice fed the CDE diet. This reduction is not due to a decrease in phosphatidylinositol-4,5-bisphosphate. This communication represents the first report of a system in which a blockade of inositol phosphate generation can be related to a physiologic defect and pathologic lesion.

The role of oxygen-derived free radicals in two models of experimental acute pancreatitis: Effects of catalase, superoxide dismutase, dimethylsulfoxide, and allopurinol

SummaryThe role of oxygen-derived free radicals was evaluated in two models of experimental acute pancreatitis by testing the effects of agents which either reduce oxygen-derived free radical generation or scavenge those free radicals. Those agents (catalase, superoxide dismutase, polyethylene glycol-superoxide dismutase, dimethylsulfoxide, and allopurinol) were evaluated using the choline-deficient ethionine-supplemented diet-induced model of acute hemorrhagic pancreatic necrosis and the supramaximal caerulein stimulation model of acute interstitial edematous pancreatitis. In both models, the only effect associated with administration of the test agents was a reduction in the degree of pancreatic edema. These results suggest that oxygen-derived free radicals may play an important role in the development of pancreatic edema during pancreatitis but that those free radicals do not play an important role in the development of acinar cell injury.

Inositol trisphosphate independent increase of intracellular free calcium and amylase secretion in pancreatic acini

It is generally believed that the activation of various cell surface receptors results in the phospholipase C-catalyzed production of inositol trisphosphate which, in turn, increases the intracellular concentration of free Ca2+ by stimulating its release from nonmitochondrial sources. We have investigated both the production of inositol trisphosphate and changes in intracellular Ca2+ concentration in rat pancreatic acini in response to caerulein and CCK-JMV-180, two analogs of cholecystokinin. Both of these analogs cause comparable increases in the rate of amylase secretion and in intracellular Ca2+ concentration but their effects on inositol phosphate generation are dramatically different; caerulein stimulates significant production of inositol phosphates within 1 min of its addition, whereas no detectable levels of inositol phosphates were generated within the same time after addition of CCK-JMV-180. These results suggest that the CCK-JMV-180 stimulated release of intracellular Ca2+ is not mediated by inositol trisphosphate but some other as yet unidentified messenger.

An increase in putative voltage dependent calcium channel number following reserpine treatment

Rats were treated with reserpine (0.2 mg/kg) on days 1, 3, and 5. On day 6, binding parameters for alpha-1 adrenergic receptors (3H-prazosin) and putative voltage dependent calcium channels, VDCC (3H-nitrendipine), were determined. There was an increase in both the number (2.1 fold) and affinity (1.8 fold) of alpha-1 adrenergic receptors following reserpine treatment. In addition, there was a 2.7 fold increase in the number of VDCCs, but no change in VDCC binding affinity, following reserpine treatment. These data are consistant with the development of smooth muscle supersensitivity following reserpine treatment in a variety of tissues, and suggest that VDCC number may be modulated by the cell in response to tonic levels of catecholamines. Changes in the number of VDCCs may be an important regulatory mechanism for cell function in physiologic and pathologic states.

Inositol trisphosphate production and amylase secretion in mouse pancreatic acini

Dispersed mouse pancreatic acini prelabelled with (3H)-myoinositol generated (3H)-inositol trisphosphate (3H-IP3), (3H)-IP2 and (3H)-IP1 in response to both cholinergic and cholecystokinin analogues. The generation of (3H)-IP3 was very rapid, reaching a maximal value within 5 seconds following hormone stimulation. Stimulation with 10(-3)M carbachol increased (3H)-IP3 to a value which was 13 times that found in unstimulated acini. These results indicate that the mechanism of stimulus-secretion coupling in mouse pancreatic acini may proceed by a mechanism similar to many other systems, including rat pancreatic acini. This sequence includes hormone-stimulated phosphatidylinositol turnover and Ca2+ mobilization, i.e. secretagogue-stimulated generation of IP3 which induces the subsequent release of intracellular Ca2+. These observations differ from those recently reported by Hokin-Neaverson and Sadeghian (J. Biol. Chem. 259: 1346, 1984), in which no hormone stimulated IP3 generation was detected in mouse pancreatic acini.

A Partial Calcium-Free Linker Confers Flexibility to Inner-Ear Protocadherin-15.

Tip links of the inner ear are protein filaments essential for hearing and balance. Two atypical cadherins, cadherin-23 and protocadherin-15, interact in a Ca2+-dependent manner to form tip links. The largely unknown structure and mechanics of these proteins are integral to understanding how tip links pull on ion channels to initiate sensory perception. Protocadherin-15 has 11 extracellular cadherin (EC) repeats. Its EC3-4 linker lacks several of the canonical Ca2+-binding residues, and contains an aspartate-to-alanine polymorphism (D414A) under positive selection in East Asian populations. We present structures of protocadherin-15 EC3-5 featuring two Ca2+-binding linker regions: canonical EC4-5 linker binding three Ca2+ ions, and non-canonical EC3-4 linker binding only two Ca2+ ions. Our structures and biochemical assays reveal little difference between the D414 and D414A variants. Simulations predict that the partial Ca2+-free EC3-4 linker exhibits increased flexural flexibility without compromised mechanical strength, providing insight into the dynamics of tip links and other atypical cadherins.

Lack of oxytocin effect on sperm output in oligospermic males

Abstract Oxytocin when administered intravenously had no detectable effect on sperm count, seminal volume, or total ejaculated sperm number in oligospermic males.

Prostaglandins and the rat seminal vesicle: Effects of mating and adrenergic stimulation

The concentrations of PGE, PGF, and 6-keto-PGF1 alpha were increased in rat seminal vesicle tissue following mating activity. Likewise, synthesis of PGE and PGF was stimulated by epinephrine (3 X 10(-7) to 3 X 10(-6) M) in tissues and media from in vitro incubations of intact rat seminal vesicles. The in vitro stimulation was inhibited by phentolamine, an alpha-adrenoreceptor blocking agent. Carbamylcholine (2 X 10(-6) M) and bradykinin (1 X 10(-6) M) had no effect on PGE or PGF synthesis, even though both compounds stimulated contractility of the rat seminal vesicle at these concentrations. These data suggest that mating and adrenergic stimulation increase prostaglandin synthesis in the rat seminal vesicle, probably through an alpha-adrenergically mediated mechanism.