Frederick R. DeRubertis

Sequential alterations in glomerular prostaglandin and thromboxane synthesis in diabetic rats: Relationship to the hyperfiltration of early diabetes

The present study examined the role of enhanced production of prostaglandin (PG) E2 and 6-keto-PGF1a, the stable metabolite of PGI2, by glomeruli from streptozotocin diabetic rats in the mediation of hyperfiltration. Correlative measurements of insulin clearance (CIn) and glomerular production of PGE2, 6-keto PGF1a and thromboxane (TX) B2 (the stable metabolite of TXA2) were made at two time points, nine to 15 days and 25 to 28 days after streptozotocin. CIn was elevated by 40% to 50% in diabetic rats studied at nine to 15 or 25 to 28 days compared to values in age-matched controls. Basal production of PGE2, 6-keto PGF1a and TXA2 (as reflected by TXB2) and increases in response to A23187 were elevated in glomeruli from nine to 15-day diabetic rats compared to values in control glomeruli. Exogenous arachidonate abolished these differences. Treatment of nine-day diabetic rats with indomethacin (3 mg/kg/d) rapidly (within 24 hours) and reversibly suppressed CIn without altering CIn in control rats. Indomethacin had no effect on plasma glucose in control or diabetic rats. Treatment of nine to 15-day diabetic rats with insulin (10 U/kg/d by osmotic minipump) beginning 24 hours after streptozotocin lowered plasma glucose to values that were not significantly different from control and prevented the rise in CIn. Treatment of diabetic rats with insulin or incubation of glomeruli from untreated diabetic rats with insulin (0.3 mU/mL) for two hours in vitro reduced basal and A23187 induced increases in PGE2, 6-keto PGF1a, and TXB2 to values that were not different from those in control glomeruli.(ABSTRACT TRUNCATED AT 250 WORDS)

Actions of sulfasalazine and 5-aminosalicylic acid as reactive oxygen scavengers in the suppression of bile acid-induced increases in colonic epithelial cell loss and proliferative activity*

Sulfasalazine suppresses mucosal injury in patients with ulcerative colitis, but the mechanism of its therapeutic action is uncertain. In the present study, we examined the mechanism of the protective action of sulfasalazine in a rat model in which colonic epithelial cell loss and subsequent increases in epithelial proliferative activity were induced by intracolonic instillation of sodium deoxycholate. Sulfasalazine or its therapeutically active metabolite 5-aminosalicylic acid suppressed the loss of deoxyribonucleic acid into the colonic lumen and the subsequent increases in mucosal ornithine decarboxylase activity and tritiated thymidine incorporation into deoxyribonucleic acid induced by sodium deoxycholate. Sulfasalazine and 5-aminosalicylic acid also blocked xanthine-xanthine oxidase-induced loss of deoxyribonucleic acid and the subsequent proliferative response. In vitro sodium deoxycholate increased reactive oxygen formation by colonic mucosal scrapings or isolated crypt epithelium. These actions of sodium deoxycholate on reactive oxygen formation were blocked by sulfasalazine or 5-aminosalicylic acid. Sulfapyridine, a therapeutically inactive metabolite of sulfasalazine, had no effect on sodium deoxycholate-induced increases in surface cell sloughing, ornithine decarboxylase, tritiated thymidine incorporation into deoxyribonucleic acid, chemiluminescence, or superoxide production. The ability of sulfasalazine and 5-aminosalicylic acid to scavenge reactive oxygen may play a role in their therapeutic effects of inflammatory bowel disease.

16,16-Dimethyl prostaglandin E2 suppresses the increases in the proliferative activity of rat colonic epithelium induced by indomethacin and aspirin

Treatment of rats with indomethacin rapidly increased ornithine decarboxylase (4 h) of colonic mucosa and [3H]thymidine incorporation into colonic mucosal deoxyribonucleic acid (DNA) (1 or 5 days) when this parameter was examined in vivo and ex vivo. The changes in colonic mucosal ornithine decarboxylase and DNA synthesis induced by indomethacin were correlated temporally with suppression of colonic prostaglandin synthesis, as assessed from ex vivo colonic production of prostaglandin E, the dominant prostaglandin product of colon. Autoradiographic studies indicated that the enhancement of proliferative activity of colonic epithelium after treatment with indomethacin for 1 day was confined to the lower third of the colonic crypt (normal proliferative zone). After 5 days of indomethacin treatment, however, there was an extension of the proliferative zone to the upper third of the colonic crypts. Concurrent treatment of rats with the stable prostaglandin E2 analogue, 16,16-dimethyl prostaglandin E2, suppressed indomethacin-induced increases in colonic mucosal ornithine decarboxylase and DNA synthesis. Concurrent administration of 16,16-dimethyl prostaglandin E2 also prevented the extension of the proliferative zone of colonic epithelium induced by 5 days of indomethacin administration. 16,16-Dimethyl prostaglandin E2 alone for 1-5 days had no detectable effects on colonic mucosal ornithine decarboxylase and DNA synthesis compared with corresponding control values. Increases in colonic mucosal DNA synthesis were also induced by treatment of rats for 5 days with aspirin (ASA). The stimulation of colonic mucosal DNA synthesis induced by ASA was significantly suppressed by concurrent administration of 16,16-dimethyl prostaglandin E2 and was also correlated with the inhibition of colonic prostaglandin synthesis by ASA. The colons of rats treated with indomethacin for 1 day or ASA for 5 days appeared normal by light microscopy. However, treatment of rats for 5 days with indomethacin resulted in mild to moderate inflammation of the lamina propria and some goblet cell depletion at the mucosal surface, but no loss of surface epithelium. The ultrastructure of the surface epithelium of the colons of rats treated with indomethacin or ASA was normal as assessed by electron microscopy. The results thus demonstrate that inhibition of local colonic prostaglandin synthesis is associated with increases in the proliferative activity of colonic epithelium, and that these increases are suppressed by administration of 16,16-dimethyl prostaglandin E2.(ABSTRACT TRUNCATED AT 400 WORDS)

Electron spin resonance study of the role of nitrosyl-heme in the activation of guanylate cyclase by nitrosoguanidine and related agonists

Abstract The responsiveness of soluble rat hepatic guanylate cyclase to N-methyl-N′-nitro-N-nitrosoguanidine,NO, nitrite and nitroprusside is markedly reduced or abolished with partial purification of enzyme activity, and is subsequently restored by addition of free or protein bound heme, plus a reducing agent. Under conditions required for restoration of enzyme responsiveness, formation of paramagnetic nitrosyl-heme complexes from each enzyme agonist was observed by electron spin resonance spectroscopy. Moreover, preformed nitrosylhemoglobin activates purified guanylate cyclase in the absence of both added heme and reducing agents, conditions that do not permit expression of effects of nitrosoguanidine, NO, nitrite or nitroprusside. The capacity of the latter agonists to activate purified enzyme activity correlates with their capacity to generate nitrosyl-heme under different conditions of incubation. These results indicate that formation of nitrosyl-heme may be an obligate step in the activation of guanylate cyclase by nitrosoguanidine, NO, nitrite and nitroprusside.

Fatty acid induced drug and carcinogen metabolism in rat and human colonic mucosa: A possible link to the association of high dietary fat intake and colonic carcinogenesis

Abstract This study examined effects of fatty acids on the metabolism of 1,3 diphenylisobenzofuran (DPBF) and benzo(α) pyrene (BP) by rat or human colonic mucosal microsomes. Arachidonate, linoleate (25 μM) or their hydroperoxides increased oxidation of DPBF or BP 4 to 5-fold, whereas saturated fatty acids and NADPH had no effect. Studies of the influence of O 2 exclusion and indomethacin on DPBF and BP oxidation were consistent with the existence of both cyclooxygenase dependent and independent pathways for fatty acid stimulation of colonic microsomal drug oxidation. These results may have a bearing on the increased prevalence of colon cancer in populations with high fat intakes.

Relationship between calcium stimulation of cyclic GMP and lipid peroxidation in the rat kidney: evidence for involvement of calmodulin and separate pathways of peroxidation in cortex versus inner medulla.

Abstract The relationship between Ca 2+ stimulation of renal cGMP accumulation, release of endogenous arachidonic acid (AA) from lipid stores, lipid peroxidation and prostaglandin (PG) synthesis were examined in rat renal cortex and inner medulla. In slice incubates of each tissue, increases in slice cGMP induced by Ca 2+ plus ionophore A23187 were preceded by or occurred concurrently with Ca 2+ induced increases in (1) release of [ 14 C] AA from prelabeled lipid stores, (2) lipid peroxidation, as monitored by accumulation of malondialdehyde (MDA) in the media, and (3) inner medullary slice PGE content. Ca 2+ induced increases in cGMP, MDA and PGE required O 2 . Exogenous AA also increased MDA, PGE and cGMP in the presence but not in the absence of O 2 . In inner medulla, the cyclooxygenase inhibitors indomethacin or meclofenamate suppressed or abolished the actions of Ca 2+ , Ca 2+ plus A23187 or exogenous AA to increase MDA, PGE and cGMP, thus implicating products of the prostaglandin synthetic pathway as potential mediators of Ca 2+ effects on cGMP in this tissue. By contrast, in renal cortex, the cyclooxygenase inhibitors did not alter Ca 2+ , A23187 or AA induced increases in MDA or cGMP. However, preformed AA hydroperoxide significantly stimulated soluble and particulate guanylate cyclase activities from both regions of the kidney, suggesting that oxygenation of AA by the lipoxygenase pathway could result in generation of products capable of enhancing cGMP accumulation in cortex. Trifluoperazine (TFP), a phenothiazine that binds to and inhibits many of the biologic actions of the Ca 2+ -calmodulin complex, suppressed increases in [ 14 C] AA release, MDA and cGMP induced by Ca 2+ or Ca 2+ plus A23187 in both cortex and medulla. By contrast, TFP did not alter increases in MDA or cGMP in response to exogenous AA or the increase in cGMP induced by nitroprusside. Promethazine, a phenothiazine which binds poorly to Ca 2+ -calmodulin, had no effect on Ca 2+ induced increases in MDA or cGMP in cortex or medulla, TFP, but not promethazine, also suppressed Ca 2+ induced increases in acyl hydrolase activities in the 100,000 xg particulate fractions from cortex and medulla. Reduction of the endogenous calmodulin-like activity of particulate fractions from inner medulla by extraction with EGTA was associated with loss of Ca 2+ responsive acyl hydrolase activity. Ca 2+ -responsiveness was restored by addition of purified exogenous calmodulin. The data are consistent with the proposal that Ca 2+ induced increases in cGMP involve (1) Ca 2+ stimulation of Ca 2+ -calmodulin responsive acyl hydrolase activity with liberation of AA from lipid stores, and (2) oxygenation of AA by cyclooxygenase (medulla) or lipoxygenase (cortex) pathways to products which activate guanylate cyclase.

Calcium and O2-dependent control of inner medullary cGMP: Possible role for Ca2+-dependent arachidonate release and prostaglandin synthesis in expression of the action of osmolality on renal inner medullary guanosine 3′5′ monophosphate

The present study examined the effects of osmolality on basal cGMP metabolism and cGMP responses to carbamylcholine in rat inner medulla. The basal cGMP content of inner medullary slices and cGMP responses to carbamylcholine fell as media osmolality was increased from 305 to 1650 mosmole/liter by the addition of urea plus NaCl to standard Krebs bicarbonate buffer. Exclusion of extracellular Ca2+ or addition of tetracaine abolished the effects of both a reduction in osmolality and carbamylcholine to increase cGMP. Readdition of Ca2+ to Ca2+-deprived slices restored the actions of media osmolality and carbamylcholine on cGMP. Ionophore A23187 enhanced the effects of Ca2+ to increase slice cGMP content. Analogous to the effects of Ca2+ alone, increases in cGMP accumulation in response to Ca2+ plus A23187 were significantly suppressed at high osmolality. In slices prelabeled with [14C]-arachidonate, the stimulatory effects of Ca2+ plus A23187 on cGMP were correlated with enhanced release of [14C]-arachidonate into the media and with increased accumulation of prostaglandin E in the media, all of which were depressed in slices incubated at 1650 compared to 750 mosmole/liter. Exogenous arachidonate increased cGMP 50% to twofold in the absence of Ca2+. However, the effects of exogenous arachidonate on cGMP were clearly less than those of either Ca2+ or carbamylcholine. Addition of indomethacin or exclusion of O2 abolished effects of exogenous arachidonate, Ca2+ plus A23187, reduced osmolality, and carbamylcholine to increase cGMP. In the presence of indomethacin or in the absence of O2, Ca2+, and arachidonate-induced prostaglandin E accumulation was also not detectable. By contrast, addition of indomethacin or exclusion of O2 had no effect on Ca2+-induced [14C]-arachidonate release. Changes in cGMP accumulation in inner medulla in response to changes in media osmolality, Ca2+ plus A23187, and exogenous arachidonate were accompanied by directionally similar alterations in cAMP. Moreover, increases in cAMP induced by Ca2+ or arachidonate were inhibited by indomethacin and O2 deprivation. These data suggest that Ca2+-dependent fatty acid release and oxygenation by fatty acid release and oxygenation by cyclooxygenase may participate in the control of the metabolism of both cGMP and cAMP in inner medulla. However, PGE2 and PGI2 increased cAMP but not cGMP, whereas carbamylcholine increased cGMP but not cAMP. Thus, to the extent that arachidonate oxygenation products mediate changes in cyclic nucleotide metabolism in inner medulla, it is likely that more than one product is involved or that changes in cGMP and cAMP occur in separate compartments of inner medulla.

Two-step high-performance liquid chromatography method for the determination of myo-inositol and sorbitol

A simple two-step HPLC method for the separation and quantitation of myo-inositol and sorbitol in extracts of glomeruli from rat kidneys is described. The limit of detection is 2 ng. The procedure involves fractionation of the sugar alcohols on a Waters Sugar Pak column, preparation of the p-nitrobenzoate derivatives, and further purification with quantitation by absorbance at 254 nm using a Waters mu Porasil column. The applicability of the procedure to determination of sorbitol and myo-inositol in biological samples was demonstrated by the finding of marked alterations in sorbitol and myo-inositol content of glomeruli isolated from diabetic compared to that from normal rat kidneys.

Effects of reduced ATP content on hepatic responses to glucagon

Expression of the glycogenolytic action of glucagon in liver requires ATP for cAMP formation and for several subsequent phosphorylation reactions. To assess the extent to which ATP availability is rate-limiting to this hormonal action, responses to glucagon of intact liver and of liver with marked reductions in ATP content induced by ethionine was examined in female Wistar rats in vivo and in vitro. Compared to values in quick-frozen liver samples from control rats, basal hepatic ATP was 75% lower and cAMP, two fold higher in rats treated with ethionine. Activation of glycogen phosphorylase and inactivation of glycogen synthetase, phosphorylation reactions which require ATP and are initiated by cAMP, were also evident in basal liver samples from ethionine-treated rats. These hepatic alterations were associated with portal glucose and insulin levels which were significantly lower and portal glucagon levels which were four fold higher than values in controls. In ethionine-treated rats, glucose infusion decreased hepatic cAMP content and phosphorylase activity and increased synthetase activity. This and other observation suggested that the higher cAMP and the altered enzyme activities seen in vivo after ethionine administration were mediated by the hyperglucagonemia and/or by other endogenous glycogenolytic stimuli, and accordingly implied that liver remained responsive to such stimuli despite reduced ATP. Pharmacologic doses of exogenous glucagon clearly increased cAMP in vivo and in vitro in livers with decreased ATP. However, the lower ATP of liver exposed to ethionine was associated with a significantly blunted cAMP response to maximal glucagon stimulation. By contrast, alterations in phosphorylase and synthetase activities were not similarly blunted, suggesting that the smaller increases in cAMP seen in liver with reduced ATP content were adequate for the expression of these actions of the hormone. It is concluded that the actions of glucagon to increase cAMP and to activate phosphorylase and inactivate synthetase are not abolished by marked reductions in hepatic APT.

Activation of guanylate cyclase during the oxidation of arylamine carcinogens

Abstract When added alone, the arylamine procarcinogens N-acetyl-aminofluorene, 4-acetyl-aminobiphenyl or their N-hydroxy derivatives failed to alter partially purified soluble guanylate cyclase from rat liver or particulate guanylate cyclase activity from colonic mucosa. However, addition of linoleic acid hydroperoxide to the enzyme preparation in the presence N-OH-acetyl-aminofluorene or N-OH-acetyl-aminobiphenyl significantly increased guanylate cyclase activity. With linoleic acid hydroperoxide plus N-OH-acetyl-aminofluorene, both the activation of hepatic guanylate cyclase and the formation of the carcinogen oxidation product 2-nitrosofluorene required hematin but not molecular O 2 . Both processes were inhibited by ascorbic acid. These data strongly imply that guanylate cyclase activation was dependent upon hematin catalyzed oxidation of N-OH-acetyl-aminofluorene by the lipid peroxide. The results provide the first evidence that guanylate cyclase activation can occur during the conversion of a procarcinogen to a more reactive chemical species, and thereby emphasize the importance of examining carcinogen interaction with the GC system under conditions which permit such chemical conversion.