Patricia A. Craven

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)

Calcium-dependent regulation of guanosine 3′,5′-monophosphate in renal cortex: Effects of ionophore A23187 and tetracaine and evidence for independent control of adenosine 3′,5′-monophosphate

The effects of carbamylcholine (Cch), the divalent cation ionophore A23187 and Ca2+ on the cyclic 3,5-guanosine monophosphate (cGMP) and cyclic 3,5-adenosine monophosphate (cAMP) content of rat renal cortical slices were examined. In both the presence and absence of 10 mM theophylline, Cch detectably increased cGMP within 15 sec, with peak responses noted by 2 min. The maximal cGMP response to Cch alone (0.05 mM) was an increase of two- to three-fold over control. Theophylline, which was routinely present in the incubations and which alone increased cGMP of the slices two-fold over basal during 20 min incubations, potentiated the response to Cch (maximal increase, five- to sixfold over theophylline alone). The action of Cch to increase renal cortical cGMP was blocked by prior addition of atropine and was dependent upon the presence of Ca2+ in the incubation media. Exclusion of Ca2+ lowered basal cGMP and abolished increases mediated by Cch, while exclusion of Mg2+ was without detectable effect on cGMP. In slices incubated initially without Ca2+, reexposure to Ca2+ for 1min partially restored the cGMP response to Cch, and reexposure for 3 min completely restored this response. Since prior incubation of tissue in Ca2+-free buffer for only 2 min was sufficient to block the cGMP responses to Cch, depletion of tissue Ca2+ did not appear to be involved. A23187 also increased renal cortical cGMP fivefold in the presence of Ca2+. Its effects were not additive with those of Cch and were not additive with those of Cch and were not expressed by Mg2+ in Ca2+-free media. By contrast, tetracaine, which blocks Ca2+ transport across or binding to biologic membranes, reduced basal cGMP and inhibited the actions of Cch and A23187 to increase cGMP in cortical slices incubated with Ca2+. The action of 1 mM tetracaine to block Cch-mediated increases in cGMP was partially reversed by increasing media Ca2+ from 1.5 to 5 mM, but not by increasing media Mg2+ to 5 mM. In contrast to their effects on cGMP, Cch, A23187, Ca2+ exclusion, and tetracaine did not detectably alter basal renal cortical cAMP or cAMP responses to parathyroid hormone (PTH). Conversely, concentrations of PTH, glucagon, and isoproterenol which maximally increased renal cortical cAMP did not alter cGMP. Furthermore, prior incubation of slices with Cch did not alter their subsequent cAMP response to PTH at a time when cGMP levels were still elevated, while prior incubation with PTH did not affect the subsequent cGMP response to Cch at a time when cAMP was increased. These studies demonstrate modulation of renal cortical cGMP by cholinergic stimuli and Ca2+. They also indicate that cGMP and cAMP in renal cortex can be regulated independently.

Sequential alterations in the hepatic content and metabolism of cyclic AMP and cyclic GMP induced by DL-ethionine: Evidence for malignant transformation of liver with a sustained increase in cyclic AMP

There is evidence than adenosine 3,5-monophosphate (cAMP) and guanosine 3,5-monophosphate (cGMP) may have antagonistic actions on cell growth, with cAMP inhibiting and cGMP stimulating this process. However, reductions in cAMP and increases in cGMP are not charactersitic of all neoplastic tissues. Thus, benign and malignant tissues from hepatoma-bearing rats exposed to the hepatic carcinogen DL-ethionine have elevated rather than depressed cAMP, compared to control liver, and parenteral administration of this drug increases hepatic cAMP within hours. In the present study, the effects of ethionine ingestion on the hepatic content and metabolism of both cAMP and cGMP were examined sequentially in rats at 2 and then 6 wk intervals, from the initiation of drug administration until the development of hepatomas. After 2 wk, cAMP content of quick-frozen liver from rats receiving ethionine (E) was significantly increased (826 +/- 91 pmole/g wet weight) above that of liver from pair-fed controls (C, 415 +/- 44), whether calculated by tissue wet weight, protein, or DNA content. In benign tissue from E, higher cAMP was still evident after in vitro incubations of slices with 2 mM 1-methyl-3-iso-butylxanthine (MIX) and was associated with enhanced adenylate cyclase and unchanged high or low Km cAMP-phosphodiesterase activities. These findings are compatible with accelerated cAMP generation in liver from E. Protein kinase activity ratios were significantly increased in frozen liver from E (0.52 +/- 0.04 versus 0.36 +/- 0.03 in C), and the percent glycogen synthetase in the I form was clearly reduced (19% +/- 2% in E versus 47% +/- 5% in c). incubation of hepatic slices from E or C with MIX and/or 10 muM glucagon further increased cAMP and protein kinase activity ratios, data which imply higher effective, as well as total, cellular cAMP in E. Changes in cAMP metabolism and action observed at 2 wk persisted throughout the 38-wk period of drug ingestion. Adenylate cyclase activity, cAMP content, and protein kinase activity ratios of ethionine-induced hepatomas exceeded those of both the surrounding liver from tumor-bearing rats and that of control liver, but alterations in these parameters were qualitatively similar in both tissues from E. By contrast, while cGMP in quick-frozen surrounding liver from tumor-bearing rats (36 +/- 4 pmole/g wet weight) did not differ from that of control liver (30 +/- 3), cGMP in the hepatomas was increased. This change was evident in both frozen tumor (89 +/- 10) and in tumor slices incubated in vitro with MIX (C, 90 +/- 11; surrounding liver, 85 +/- 10; hepatoma 231 +/- 29). These results indicate that malignant conversion can occur in liver with a sustained elevation of both total and effective cAMP during the premalignant phase. The increase in cGMP detected in ethionine-induced hepatomas could also be a key determinant of malignant transformation in the model, although premalignant changes in cGMP were not apparent.

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.

Contribution of platelet thromboxane production to enhanced urinary excretion and glomerular production of thromboxane and to the pathogenesis of albuminuria in the streptozotocin-diabetic rat

Previous studies have demonstrated that urinary thromboxane B2 (TXB2) excretion (UTXB2) and glomerular production of TXB2 are enhanced in experimental diabetes and that selective inhibitors of TX synthesis prevent or delay the development of albuminuria. The present study was conducted to examine the contribution of platelet TXB2 production to the enhancement of UTXB2 and glomerular TXB2 production and to the pathogenesis of albuminuria in the partially insulin-treated moderately hyperglycemic (blood glucose, 200 to 400 mg/dL) streptozotocin-diabetic rat (SDR). Treatment of control rats or of SDR with diabetes of 5 months duration with antiplatelet serum for 4 consecutive days reduced circulating platelet counts and serum TXB2 generation, an index of platelet cyclooxygenase activity, by 80% or greater, but reduced UTXB2 excretion by only 30%. UTXB2 and glomerular production of TXB2 of thrombocytopenic SDR remained markedly elevated compared with corresponding values from age-matched thrombocytopenic or platelet-replete, nondiabetic controls. Similarly, treatment of rats for 180 days with a dose of aspirin (ASA), which selectively inhibited platelet versus renal cyclooxygenase activity, reduced UTXB2 of both SDR and controls by 25% to 35%. The absolute reductions in UTXB2 induced by either ASA or thrombocytopenia in SDR were significantly greater than the absolute decrements in corresponding controls, suggesting that increased platelet TXB2 production in SDR may contribute to the enhanced UTXB2. However, as in the thrombocytopenic SDR, UTXB2 and glomerular production of TXB2 of SDR treated with ASA remained clearly above corresponding control values. Moreover, chronic ASA treatment failed to prevent the development of albuminuria in SDR.(ABSTRACT TRUNCATED AT 250 WORDS)