Calcium-independent modulation of cyclic GMP and activation of guanylate cyclase by nitrosamines.

Nitrosamines markedly increase concentrations of guanosine 3', 5' - monophosphate (cyclic GMP) in several tissues from the rat and in human colonic mucosa. These agents are effective in the absence of extracellular calcium and enhance guanylate cyclase activity in tissue homogenates. Stimulation of cyclic GMP was greatest in liver, where the carcinogenic activity of nitrosamines is also most pronounced.

Protein kinase C is activated in glomeruli from streptozotocin diabetic rats. Possible mediation by glucose.

Glomerular inositol content and the turnover of polyphosphoinositides was reduced by 58% in 1-2 wk streptozotocin diabetic rats. Addition of inositol to the incubation medium increased polyphosphoinositide turnover in glomeruli from diabetic rats to control values. Despite the reduction in inositol content and polyphosphoinositide turnover, protein kinase C was activated in glomeruli from diabetic rats, as assessed by an increase in the percentage of enzyme activity associated with the particulate cell fraction. Total protein kinase C activity was not different between glomeruli from control and diabetic rats. Treatment of diabetic rats with insulin to achieve near euglycemia prevented the increase in particulate protein kinase C. Moreover, incubation of glomeruli from control rats with glucose (100-1,000 mg/dl) resulted in a progressive increase in labeled diacylglycerol production and in the percentage of protein kinase C activity which was associated with the particulate fraction. These results support a role for hyperglycemia per se in the enhanced state of activation of protein kinase C seen in glomeruli from diabetic rats. Glucose did not appear to increase diacylglycerol by stimulating inositol phospholipid hydrolysis in glomeruli. Other pathways for diacylglycerol production, including de novo synthesis and phospholipase C mediated hydrolysis of phosphatidylcholine or phosphatidyl-inositol-glycan are not excluded.

Effects of osmolality and oxygen availability on soluble cyclic AMP-dependent protein kinase activity of rat renal inner medulla.

The renal inner medulla is ordinarily exposed to osmolalities that are much higher and to O2 tensions that are lower than those in other tissues. The effects of media osmolality and O2 availability on basal and arginine vasopressin(AVP)-responsive soluble cyclic (c)AMP-dependent protein kinase activity were examined in slices of rat inner medulla. Increasing total media osmolality from 305 to 750 or 1,650 mosM by addition of urea plas NaCl to standard Krebs-Ringer bicarbonate buffer significantly reduced basal cAMP content and protein kinase activity ratios. This occurred in the presence or absence of O2. Incubation of slices in high osmolality buffer also blunted increases in inner medullary slice cAMP and protein kinase activity ratios induced by O2. These changes reflected predominantly an action of the urea rather than the NaCl content of high osmolality buffers. In contrast to effects on basal activity, high media osmolality significantly enhanced activation of inner medullary protein kinase by AVP. Conversely, increases in media O2 content suppressed AVP stimulation of enzyme activity. This inhibitory effect of O2 was best expressed at low osmolality. Naproxen and ibuprofen, inhibitors of prostaglandin biosynthesis, reduced basal kinase activity ratios and increased AVP responsiveness in the presence, but not in the absence, of O2. Exogenous prostaglandins (PG) modestly increased (PGE2 and PGE1) or did not change (PGF2alpha) cAMP and protein kinase activity ratios in O2-deprived inner medullary slices. Protein kinase activation by PGE2 was not observed in oxygenated inner medulla with high basal activity ratios. The stimulatory effects of PGE2 and PGE1 on protein kinase activity observed in O2-deprived slices were additive with those of submaximal or maximal AVP. PGE2, PGE1, and PGF2alpha all failed to suppress AVP activation of protein kinase. Thus, enhanced endogenous PGE production may contribute to the higher basal protein kinase activity ratios induced by O2. However, the results do not support a role for PGE2, PGE1, or PGF2alpha in O2-mediated inhibition of AVP responsiveness. The present data indicate that both solute content and O2 availability can alter the expression of AVP action on cAMP-dependent protein kinase activity in inner medulla. AVP activation of protein kinase is best expressed when osmolality is high and O2 availability is low, conditions that pertain in inner medulla during hydropenia.

Hormonal modulation of cyclic adenosine 3',5'-monophosphate-dependent protein kinase activity in rat renal cortex. Specificity of enzyme translocation.

Many of the intracellular actions of cyclic adenosine 3',5'-monophosphate are expressed through phosphorylation reactions mediated by cAMP-dependent protein kinases, but little is known about hormonal control of endogenous protein kinase activity (PK) in kidney. In the present study, we examined the effects of parathyroid hormone, glucagon, and isoproterenol on cAMP and PK in slices of rat renal cortex. In the presence of 0.5 mM 1-methyl, 3-isobutyl xanthine, all three hormones activated PK in slices, as reflected by an increase in the ratio of enzyme activity assayable in homogenates of the slices without addition of cAMP to the kinase reaction mixture (cAMP-independent activity) over total enzyme activity (+2 uM cAMP in the reaction mixture). When enzyme activity was assayed in whole homogenates prepared from slices, the increase in the enzyme activity ratio (- cAMP/+cAMP) which followed hormonal stimulation was due entirely to an increase in cAMP-independent activity, with no change in total activity. In general, a good correlation existed between the alterations in tissue cAMP levels mediated by the hormones and/or 1-methyl, 3-isobutyl xanthine and concomitant alterations in PK. All three hormones increased PK activity ratios to near unity, suggesting complete enzyme activation. However, the concentrations of parathyroid hormone and glucagon which produced maximal activation of PK were much lower than those required for maximal cAMP responses. Studies with charcoal indicated that these hormonal actions on PK reflected intracellular events rather than representing activation of the enzyme during tissue homogenization, due to release of sequestered cAMP. Thus, homogenization of tissue in charcoal prevented activation of PK by subsequent addition of exogenous cAMP, but did not lower enzyme activity ratios in homogenates of hormone-stimulated cortical slices. When PK was determined in the 20,000 g supernatant fraction of renal cortical slices incubated with the hormones, enzyme activity ratios also increased, but total enzyme activity declined. Lost activity was recovered by extraction of particulate fractions with 500 mM KCl or NaCl, results which implied particulate binding of activated PK. Activated soluble PK from renal cortex was bound equally well by intact, heat- and trypsin-treated renal cortical pellets and by intact and heated hepatic pellets. Accordingly, the apparent translocation of enzyme in hormone stimulated cortex does not necessarily represent binding of the activated PK to specific acceptor sites in the particulate cell fractions or constitute a physiologic hormonal action. Activation of renal cortical PK by increasing concentrations of salts suggests that the enzyme in this tissue resembles the predominant type found in heart.

Bile salt stimulation of colonic epithelial proliferation. Evidence for involvement of lipoxygenase products.

Prostaglandin E2 (PGE2) and several other prostaglandins synthesized by colon suppress the proliferative activity of colonic epithelium. However, bile salts stimulate colonic epithelial proliferation despite the actions of bile salts to enhance the release of arachidonate and consequent colonic synthesis of PGE2. The current study was conducted to assess whether bile salt-induced increases in colonic formation of arachidonate metabolites other than PGE2 were linked to the stimulation of the proliferative activity of colonic epithelium. Within 10 min of addition, deoxycholate markedly stimulated the in vitro release of [14C]arachidonate from prelabeled rat colon. When given in vivo by intracolonic instillation deoxycholate (10 mumol) increased colonic accumulation of immunoreactive prostaglandin E (PGE), thromboxane B2 (TXB2), and the lipoxygenase product 12-hydroxyeicosatetraenoic acid (12-HETE) by two to fourfold over control in 30 min. This effect of intracolonic deoxycholate was followed by a ninefold increase in mucosal ornithine decarboxylase activity (4 h), and a subsequent two to threefold increase in [3H]thymidine [( 3H]Thd) incorporation into DNA of either mucosal scrapings or isolated pools of proliferative colonic epithelial cells (24 h). Intracolonic instillation of indomethacin (50 mumol) suppressed to low or undetectable levels both basal colonic accumulation of PGE and TXB2 and the increases in each parameter induced by subsequent instillation of deoxycholate. By contrast, indomethacin enhanced accumulation of 12-HETE in both control colons and those subsequently exposed to deoxycholate. The increases in 12-HETE induced by indomethacin alone were correlated with stimulation of mucosal ornithine decarboxylase activity and [3H]Thd incorporation into mucosal DNA. Indomethacin also enhanced the increases in these parameters induced by deoxycholate. Intracolonic instillation of phenidone (25-100 mumol) suppressed accumulation of PGE, TXB2, and 12-HETE in control colons and the increases in these parameters induced by a subsequent instillation of deoxycholate. Phenidone alone did not alter mucosal ornithine decarboxylase activity or [3H]thymidine incorporation into mucosal DNA. However, phenidone suppressed or abolished increases in these parameters induced by a subsequent instillation of deoxycholate. 4-(2-[IH-imidazol-1-yl]ethoxy) benzoic acid hydrochloride UK 37,248, which selectively reduced colonic TXB2 to undetectable levels without altering PGE or 12-HETE, had no effect on control or deoxycholate-induced increases in mucosal ornithine decarboxylase activity or [3H]Thd incorporation into DNA. Neither indomethacin nor phenidone altered the increases in [(14)C]arachidonate release induced in vitro by deoxycholate. Chenodeoxycholate and cholate also stimulated [(14)C]arachidonate release from colon in vitro within 10 min, and increased colonic 12-HETE (30 min) and mucosal ornithine decarboxylase activity (4 h) upon intracolonic installation. Prior installation of phenidone inhibited the increases in both 12-HETE and ornithine decarboxylase activity induced by these bile salts. The results support a role for bile salt-induced increases in colonic accumulation of lipoxygenase products, as reflected by 12-HETE, in the subsequent stimulation of the proliferative activity of colonic epithelium.

Renal inner medullary prostaglandin synthesis. A calcium-calmodulin-dependent process suppressed by urea.

Previous studies have demonstrated that hyperosmolar NaCl and mannitol stimulate immunoreactive prostaglandin E (iPGE) production by slices of inner medulla (IM), whereas urea inhibits this process. In the present study, the roles of Ca2+ and calmodulin in the control of PGE synthesis in IM and the basis for the differential actions of solutes were examined. A23187 increased [14C]arachidonate (AA) release and iPGE accumulation in the presence but not in the absence of media Ca2+ whereas stimulation by hypertonic NaCl or mannitol was well expressed with Ca2+ or in Ca2+-free buffer containing 2 mM EGTA. Hypertonic urea and trifluoperazine (TFP), an inhibitor of actions of the Ca2+-CaM complex, suppressed increases in [14C]AA release and iPGE induced by A23187, NaCl, or mannitol. By contrast, increases in iPGE in response to exogenous AA were not altered by urea or TFP. Ca2+ (25-100 microM) increased acyl hydrolase (AH) activity in EGTA washed (4 degrees C) 100,000 g particulate fractions of IM threefold, thereby restoring AH activity to the higher basal values of particulate fractions not washed with EGTA. This action of Ca2+ was blocked by hypertonic urea of TFP, whereas AH activity was not influenced by NaCl or mannitol in the presence or absence of Ca2+. In contrast to their effects on AH activity, hypertonic urea and TFP did not alter conversion of AA to PGE2, PGF2 alpha, or PGD2 by IM microsomal fractions. Ca2+-induced increases in particulate AH were blunted after partial depletion of endogenous CaM-like activity. Ca2+ action was restored by addition of purified exogenous CaM, but not by addition of other small acidic proteins, including troponin C. The findings support a role for CaM in the regulation of PGE synthesis in the IM at the level of Ca2+-responsive AH activity. They further imply that urea suppresses PGE synthesis in IM through inhibition of AH and a reduction in the availability of endogenous AA for conversion to PGE.

Role of local prostaglandin synthesis in the modulation of proliferative activity of rat colonic epithelium.

The role of local prostaglandin (PG) synthesis in the modulation of the proliferative activity of colonic epithelium was examined in rat colon. Experimental rats were given either indomethacin (5 mg/kg s.c. every 8 h for three doses) or aspirin (0.5 g/100 g diet for 3 d). In rats treated with indomethacin or aspirin, the incorporation of [3H]thymidine (dThd) into DNA in vivo was increased approximately twofold over control in mucosal scrapings from distal colon, and approximately threefold over control in the proliferating pool of epithelial cells isolated from distal colon. [3H]dThd incorporation into DNA was also examined ex vivo immediately after distal colonic resection. It was approximately twofold higher in mucosa of colonic segments (1-h incubation) from rats treated with indomethacin or aspirin in vivo, compared with corresponding values of segments from control rats. Immunoreactive (i) prostaglandin E (PGE), the dominant PG product of colon segment incubates by high-performance liquid chromatography analysis of [14C]arachidonate metabolites, was markedly (95%) reduced in the media of 1-h colon incubates from indomethacin- or aspirin-treated rats, compared with control rats. Moreover, the cyclic (c)AMP content of mucosa of segments from indomethacin- or aspirin-treated rats was significantly lower than that of control rats. Prolonged incubation (4-24 h) of colonic segments from indomethacin-treated rats, in the absence of indomethacin in vitro, led to an eventual return of [3H]dThd incorporation into DNA, iPGE, and mucosal cAMP to control values. Conversely, inclusion of indomethacin (0.25 mM) in the incubations (6 h) of colonic segments from indomethacin-treated rats resulted in persistent suppression of iPGE and mucosal cAMP, as well as persistent enhancement of [3H]dThd incorporation into mucosal DNA. However, incubation of colonic segments from control rats (no in vivo drug exposure) with indomethacin or aspirin in vitro for periods up to 24 h failed to alter DNA synthesis, despite marked reduction in media iPGE and lower mucosal cAMP. The latter observations suggested that additional in vivo factors initiated the enhancement of DNA synthesis in indomethacin- or aspirin-treated rats. Exogenous PGE2, D2, I2, or F2 alpha, each of which increased the endogenous mucosal cAMP content of incubated colonic segments from control, indomethacin- or aspirin-treated rats, all suppressed [3H]dThd incorporation into mucosal DNA in vitro. Dibutyryl cAMP, but not dibutyryl cGMP, had an analogous suppressive effect on in vitro [3H]dThd incorporation into DNA. Thus, the present observations are consistent with an inhibitory action of endogenous colonic PG synthesis on the proliferative activity of colonic epithelium. This action may be mediated through cAMP.

Role of activation of protein kinase C in the stimulation of colonic epithelial proliferation and reactive oxygen formation by bile acids.

Deoxycholate (DOC), chenodeoxycholate, 12-O-tetradecanoyl phorbol-13-acetate (TPA), or 1-oleoyl-2-acetyl-glycerol (OAG) activated colonic epithelial protein kinase C as reflected by translocation from the soluble to the particulate cell fraction. Activation of protein kinase C was correlated with stimulation of enhanced proliferative activity of colonic mucosa and reactive oxygen production. TPA and OAG, but not DOC, directly activated soluble protein kinase C in vitro. However, DOC rapidly increased labeled inositol phosphate and diacylglycerol accumulation in colonic epithelial cells. Retinoic acid inhibited protein kinase C activity and suppressed DOC-, TPA-, and OAG-induced increases in reactive oxygen production. The results support a role for protein kinase C in the stimulation of colonic epithelial proliferative activity and reactive oxygen production induced by bile acids, TPA and OAG. In contrast to TPA and OAG, which activate protein kinase C directly, bile acids appear to activate protein kinase C indirectly by increasing the diacylglycerol content of colonic epithelium.

Role of reactive oxygen in bile salt stimulation of colonic epithelial proliferation.

Our previous studies had suggested a link between bile salt stimulation of colonic epithelial proliferation and the release and oxygenation of arachidonate via the lipoxygenase pathway. In the present study, we examined the role of reactive oxygen versus end products of arachidonate metabolism via the cyclooxygenase and lipoxygenase pathways in bile salt stimulation of rat colonic epithelial proliferation. Intracolonic instillation of 5 mM deoxycholate increased mucosal ornithine decarboxylase activity and [3H]thymidine incorporation into DNA. Responses to deoxycholate were abolished by the superoxide dismutase mimetic CuII (3,5 diisopropylsalicylic acid)2 (CuDIPS), and by phenidone or esculetin, which inhibit both lipoxygenase and cyclooxygenase activities. By contrast, indomethacin potentiated the response. Phenidone and esculetin suppressed deoxycholate-induced increases in prostaglandin E2 (PGE2), leukotriene B4 (LTB4), and 5, 12, and 15-hydroxyeicosatetraenoic acid (HETE), whereas CuDIPS had no effect. Indomethacin suppressed only PGE2. Deoxycholate (0.5-5 mM) increased superoxide dismutase sensitive chemiluminescence 2-10-fold and stimulated superoxide production as measured by cytochrome c reduction in colonic mucosal scrapings or crypt epithelium. Bile salt-induced increases in chemiluminescence were abolished by CuDIPS, phenidone, and esculetin, but not by indomethacin. Intracolonic generation of reactive oxygen by xanthine-xanthine oxidase increased colonic mucosal ornithine decarboxylase activity and [3H]thymidine incorporation into DNA approximately twofold. These effects were abolished by superoxide dismutase. The findings support a key role for reactive oxygen, rather than more distal products of either the lipoxygenase or cyclooxygenase pathways, in the stimulation of colonic mucosal proliferation by bile salts.

Impaired nitric oxide-dependent cyclic guanosine monophosphate generation in glomeruli from diabetic rats. Evidence for protein kinase C-mediated suppression of the cholinergic response.

Nitric oxide (NO)-dependent cyclic guanosine monophosphate (cGMP) generation was examined in glomeruli isolated from 1-2-wk and 2-mo streptozotocin diabetic (D) and control (C) rats. After 1-2 wk of diabetes, ex vivo basal cGMP generation and cGMP responses to carbamylcholine (CCh) were significantly suppressed in glomeruli from D compared with those from C, whereas cGMP responses to the calcium ionophore A23187 and nitroprusside (NP) did not differ in glomeruli from D vs. those from C. After 2 mo, glomeruli from D did not respond to CCh, and responses to A23187 and NP were suppressed compared with those from C. Differences in basal, CCh, and A23187-responsive cGMP between D and C were abolished by the NO synthetase inhibitor NG-monomethyl-L-arginine. Soluble glomerular guanylate cyclase prepared from either D or C responded indistinguishably to NP, suggesting a role for NO quenching in the suppression of cGMP in intact glomeruli from D. Compared with those from C, glomeruli isolated from D demonstrated increased generation of thromboxane A2 (TXA2) and activation of protein kinase C (PKC). Both the TXA2/endoperoxide receptor antagonist Bay U3405 and inhibitors of PKC activity restored a cGMP response to CCh in glomeruli from D. Conversely, in glomeruli from C, the TXA2/endoperoxide analogue U46619 activated PKC and suppressed the cGMP response to CCh. Both of those actions were blocked by inhibitors of PKC. The results indicate a progressive impairment of NO-dependent cGMP generation in glomeruli from D which may be mediated in part by TXA2 and activation of PKC. This impairment may participate in glomerular injury in diabetes.

Calcium-dependent action of osmolality on adenosine 3',5'-monophosphate accumulation in rat renal inner medulla: evidence for a relationship to calcium-responsive arachidonate release and prostaglandin synthesis.

When urea and NaCl are employed as the major solutes of high osmolality buffers, the cyclic AMP (cAMP) content of oxygenated slices of rat renal inner medulla increases three- to fivefold as osmolality is decreased from 1,650 to 305 mosM. Incubation of slices in Ca2+-free media containing 2 mM EGTA largely abolished this action of osmolality on cAMP, whereas exclusion of Mg2+ or 5+ from the incubation media was without effect. Addition of Ca2+ to Ca2+-deprived inner medulla incubated at 750 mosM (175 mM Na+, 380 mM urea) significantly increased tissue cAMP and media prostaglandin (PG)E accumulation. Ca2+ also stimulated the release of 14C-fatty acid from Ca2+-deprived slices prelabeled with [14C]arachidonate, but not from those labeled with [14C]palmitate. The divalent cation ionophore A23187 enhanced the actions of Ca2+ to increase tissue cAMP, media PGE accumulation, and the release of [14C]-arachidonate from prelabeled inner medulla. By contrast, when slices were incubated at 1,650 mosM (365 mM Na+, 900 mM urea) in the presence or absence of A23187, all of these actions of Ca2+ were markedly suppressed or abolished. Addition of exogenous arachidonate increased tissue cAMP and media PGE at both 750 and 1,650 mosM, whereas palmitate and stearate had no effect on cAMP at either osmolality. The actions of Ca2+ and arachidonate to increase cAMP and PGE accumulation were abolished by the cyclo-oxygenase inhibitors, indomethacin and meclofenamate. They were also abolished by exclusion of molecular O2, which serves as cosubstrate with arachidonate in prostaglandin synthesis. At maximally effective concentrations, exogenous PGE2 and arachidonate produced similar increases in inner medullary cAMP. The maximal effects of the two agents on cAMP were not additive, but were expressed in the absence of Ca2+ at both 750 and 1,650 mosM. However, in marked contrast to the O2-dependent action of arachidonate, PGE2 increased cAMP in the presence or absence of O2. Comparison of the separate actions of urea and NaCl indicated that suppression of Ca2+-responsive [14C]arachidonate release, PGE, and cAMP accumulation at 1,650 mosM reflected primarily an effect of urea, whereas hypertonic NaCl, mannitol, and sucrose alone stimulated inner medullary cAMP and PGE accumulation by a pathway which did not require extracellular Ca2+. Analogous to the actions of hypertonic urea, tetracaine and mepacrine inhibited the actions of Ca2+ plus A23187 to stimulate [14C]-arachidonate release, PGE, and cAMP accumulation. Inhibition of PGE and cAMP accumulation by tetracaine and mepacrine was also overcome by arachidonate. The results suggest that high osmolaity media with urea as a major solute reduces inner medullary cAMP content, at least in part, through effects on Ca2+-dependent prostaglandin synthesis. Inhibition of PGE synthesis, in turn, may be the result of osmotic suppression of Ca2+-dependent release of arachidonate, the availability of which is often rate limiting to prostaglandin generation.

Modulation of the cyclic AMP content of rat renal inner medulla by oxygen: possible role of local prostaglandins.

The lower O2 tension and more active anerobic metabolism that pertain in the inner medulla (IM) of kidney relative to cortex (C) are well recognized, but there is no evidence that O2 availability constitutes a limiting or regulatory factor in IM metabolism or function. In the present in vitro study, we examined the effects of O2 on adenosine 3',5'-monophosphate (cAMP) metabolism in slices of rat renal C and IM. After a 20-min incubation of slices in Krebs Ringer bicarbonate buffer with 95% O2 + 5% CO2 serving as the gas phase, the cAMP content of IM was 6-10 fold higher than that of C in either the presence or absence of 2 mM 1-methyl-3-isobutylxanthine in the incubation media. In slices of IM incubated for 20 min with 1-methyl-3-isobutylxanthine, cAMP was 22.5+/-SE 2.48 pmol/mg wet weight at 95% O2 and 4.37 without O2. Oxygenation of O2-deprived IM increased cAMP twofold in 2 min, an effect fully expressed in 5 min (fivefold increase). Further, cAMP of IM rose progressively and significantly over a range of atmospheric O2 content from 0 to 50% conditions which should reproduce and encompass O2 tensions that pertain in tissues in vivo. By contrast, basal cAMP content of C varied less than twofold in the presence of 95% versus no O2, implying that O2 modulation of cAMP was specific for IM. Indomethacin and meclofenamate, structurally distinct inhibitors of prostaglandin synthesis, both significantly decreased basal cAMP accumulation in oxygenated slices of IM but not of C. Meclofenamate also reduced basal adenylate cyclase activity determined in homogenates prepared from slices of IM which had been incubated at 95% O2. In slices of IM previously exposed to indomethacin or meclofenamate at 95% O2, a maximally effective concentration of exogenous prostaglandin E1 restored cAMP and adenylate cyclase activity to levels which approximated those observed at 95% O2 in the absence of an inhibitor of prostaglandin synthesis. These results suggest that O2 enhancement of cAMP content in IM may be mediated at least in part by local prostaglandins.

Stimulation of soluble guanylate cyclase activity and cellular accumulation of cyclic guanosine 3',5'-monophosphate by the carcinogen 4-nitroquinoline 1-oxide: brief communication.

4-Nitroquinoline 1-oxide (4NQO), a compound that induces tumors in various rat organs, rapidly increased the cellular accumulation of cyclic guanosine 3',5'-monophosphate (cGMP) to peak values fourfold to 13-fold over basal levels in the liver, lung, renal cortex, and gastric and colon mucosa of rats. This action of 4NQO was expressed in the presence or absence of extracellular calcium. When added directly to the broken cell preparations, 4NQO also stimulated guanylate cyclase activity threefold to sixfold over basal levels in the 100,000 X g soluble fractions of each of these tissues. Dicumarol, which blocks the reduction of 4NQO, inhibited 4NQO stimulation of guanylate cyclase and cGMP. Conversely, phenythydrazine, which enhances the reduction of 4NQO, potentiated the actions of 4NQO on guanylate cyclase and cGMP. These results suggested that the activation of the guanylate cyclase-cGMP system may be mediated by reduction products of 4NQO. The activation of the guanylate cyclase system by 4NQO or its derivatives could function in the expression of carcinogenicity.

Subcellular distribution of protein kinase C in rat colonic epithelial cells with different proliferative activities.

Activation of Ca2+ and phospholipid-dependent protein kinase C (PKC) is associated with increased proliferation in several cell types. When activated, PKC is tightly bound to the particulate cell fraction. Accordingly, we examined the subcellular distribution of PKC in superficial (nonproliferative) and proliferative colonic epithelial cells from rat colonic mucosa. PKC was determined in soluble and particulate fractions of these cells following partial purification of enzyme activity of cellular homogenates by DEAE-cellulose chromatography. In the superficial cells, 90% of the PKC was associated with the soluble fraction. By contrast only 42% of the enzyme activity was found in the soluble fraction of proliferative cells. The specific activity of protein kinase C was higher in the particulate fraction of proliferative compared to superficial cells when expressed as a function of either particulate protein or cellular DNA content. Addition of deoxycholate or 12-O-tetradecanoylphorbol-13-acetate induced a translocation of protein kinase C from the soluble to the particulate fraction. [3H]Thymidine incorporation into DNA was higher in colonic epithelial cells isolated from the colons of rats which had been exposed to deoxycholate or 12-O-tetradecanoylphorbol-13-acetate in vivo. Treatment of rats with 1-(5-isoquinolinyl)-2-methylpiperazine (H-7) suppressed basal [3H]thymidine incorporation into DNA and increases in this parameter induced by 12-O-tetradecanoylphorbol-13-acetate and deoxycholate. The results are consistent with a positive role for activation of protein kinase C in the control of colonic epithelial proliferation.

Alterations in protein kinase C in 1,2-dimethylhydrazine induced colonic carcinogenesis.

Protein kinase C activity and the profile of protein kinase C isozymes alpha, beta, and gamma were examined in subcellular fractions of 1,2-dimethylhydrazine induced colonic adenocarcinomas, surrounding uninvolved colonic mucosa and colonic mucosa from age matched control rats. Responsiveness of colonic mucosal protein kinase C to phorbol dibutyrate induced translocation of the enzyme from the soluble to the particulate cell fraction was also assessed. Although total protein kinase C and specific activities of soluble and particulate enzymes were higher in colonic mucosa of carcinogen treated rats which developed tumors than corresponding values of control mucosa, the subcellular distribution of enzyme activity was not different between uninvolved colonic mucosa of 1,2-dimethylhydrazine treated rats and colonic mucosa of age matched control rats. Thus, evidence for activation of the protein kinase C system of mucosa of the carcinogen treated rats was lacking. Exposure of colonic mucosa from control rats to phorbol dibutyrate induced a clear translocation of enzyme activity from the soluble to the particulate fraction. By contrast, no change in subcellular distribution of protein kinase C activity was noted on exposure of colonic mucosa from 1,2-dimethylhydrazine treated rats to phorbol dibutyrate. Immunoblotting of subcellular fractions of colonic mucosa from control and 1,2-dimethylhydrazine treated rats demonstrated the presence of protein kinase C alpha, but no detectable beta and gamma forms. Total protein kinase C activity and the specific activity of protein kinase C in soluble and particulate fractions was significantly lower in adenocarcinomas compared to uninvolved surrounding mucosa. In contrast to results obtained with colonic mucosa from control and 1,2-dimethylhydrazine treated rats, adenocarcinomas expressed predominantly the beta form of protein kinase C. The alpha form represented less than 10% of the total detectable immunoreactivity in adenocarcinomas. The alterations in protein kinase C isoenzyme expression in tumors and loss of responsiveness of premalignant mucosa to phorbol dibutyrate may be involved in the process of malignant transformation.

Fatty acid-stimulated oxidation of methylazoxymethanol by rat colonic mucosa.

The present study examined fatty acid-initiated metabolism of methylazoxymethanol (MAM) to formaldehyde (HCHO) by the 10,000 X g soluble fraction of rat colonic mucosa, and the role of prostaglandin synthase and lipoxygenase activities in mediating this process. Incubation of MAM with soluble fractions of rat colonic mucosa, in the absence of arachidonate, resulted in significant HCHO production compared to that observed in buffer alone or with the heated tissue fractions. Addition of arachidonate (100 microM), linoleate (100 microM), or arachidonate hydroperoxide, but not palmitate, increased HCHO formation by 50%. Indomethacin (25 to 100 microM) suppressed basal and arachidonate-stimulated HCHO production by 25 to 50%. However, indomethacin did not influence linoleate or arachidonate hydroperoxide-induced increases in HCHO. These data suggested a peroxidative mechanism for MAM oxidation, that was mediated in part by arachidonate metabolism via the prostaglandin synthase system. 5,8,11,14-Eicosatetraynoic acid (25 to 500 microM) suppressed HCHO production by 30 to 80% in the absence of fatty acids, and abolished stimulation by arachidonate or linoleate, but not by arachidonate hydroperoxide. MAM was also oxidized by an NAD+-dependent dehydrogenase, as evidenced by MAM-mediated NAD reduction in 10,000 X g soluble fractions of rat colonic mucosa. On a molar basis, the ability of the soluble fraction of rat colonic mucosa to oxidize MAM by the NAD+-dependent dehydrogenase pathway and the fatty acid-stimulated pathway were similar. However, NADPH did not stimulate HCHO formation by MAM. Moreover, 7,8-naphthoflavone; 2-diethylaminoethyl-2,2-diphenylvalerate; and methimazole, inhibitors of mixed-function oxidase activity, did not suppress HCHO formation, implying that MAM was not metabolized by the colonic mixed-function oxidase activity. MAM metabolism to HCHO was 3 to 4 times greater by soluble fractions of superficial epithelial cells isolated from rat colon compared to those of the isolated proliferative epithelial cell pool. The results are consistent with a role for both the prostaglandin synthase and lipoxygenase systems of colonic mucosa in the oxidative metabolism of MAM. Enhanced oxidation of MAM by superficial cells of colonic epithelium which are preparing to slough may serve to protect the colon against the carcinogenic effect of this drug.

Relationship between loss of rat colonic surface epithelium induced by deoxycholate and initiation of the subsequent proliferative response.

Bile acids increase the proliferative activity of rat colonic epithelium. However, the mechanisms responsible are unknown. The present study examined the relationships between deoxycholate (DOC) induced surface cell sloughing, as measured by loss of DNA into the lumen and by light microscopy, and the subsequent increases in mucosal ornithine decarboxylase activity and [3H]thymidine (dThd) incorporation into mucosal DNA induced by deoxycholate. Intracolonic instillation of DOC (10 mumol; 5 mM) resulted in a progressive increase in luminal DNA content which was significant by 1 min and maximal by 1 h. No further increase in luminal DNA occurred between 1 and 4 h after DOC. Similarly, light microscopy demonstrated a progressive loss of surface epithelium between 10 min and 1 h after DOC instillation. By 4 h after DOC, the colonic mucosal surface was normal histologically. The rapid repair of the epithelial surface occurred without a detectable increase in [3H]dThd incorporation into DNA within 4 h. The latter finding thus suggested that upward migration of nondividing crypt epithelial cells rather than the rapid initiation of new DNA synthesis and new mitotic activity was responsible for surface repair. Enhanced proliferative activity of colonic mucosa, as measured by increased [3H]dThd incorporation into DNA, did occur subsequently (12 to 24 h) after instillation of DOC. The dose response of early surface cell loss and the subsequent proliferative response to DOC were identical, consistent with a link between these two DOC mediated events. However, two observations suggested that surface epithelial loss alone was not sufficient to trigger the proliferative response to DOC: intracolonic instillation of DOC followed by removal of the DOC solution at 1 h, at which time surface epithelial loss was maximal, did not result in an increase in ornithine decarboxylase activity or [3H]dThd incorporation into DNA when these parameters were assessed at 4 h or 12 to 48 h, respectively; phenidone, an antioxidant and radical scavenger, and bis[(3,5-diisopropyl-salicylato) (O,O) copper(II), a lipophilic agent with superoxide dismutase activity, abolished the DOC mediated proliferative response but did not prevent the early loss of surface cells. The results imply that events other than or in addition to surface cell loss are necessary for the expression of the action of DOC to stimulate the proliferative activity of colonic epithelium.

Megabase pair deletions in mutant mammalian cells following exposure to amsacrine, an inhibitor of DNA topoisomerase II.

Amsacrine, [4'-(9-acridinylamino)-methanesulfon-m-auisidide], belongs to the class of cancer chemotherapeutic agents that target DNA topoisomerase II. We show that, over its cytotoxic range, amsacrine is a potent mutagen of the S1 phenotype in the AL (human x hamster) hybrid cell line. By contrast, amsacrine induction of the HPRT- phenotype in AL cells is at least two decades less frequent and is not concentration dependent. Such differential mutation frequencies are hypothesized to reflect the concomitant loss of essential genes neighboring the hprt locus. It may be that some amsacrine cytotoxicity is due to the inactivation of essential genes by large deletions. The AL mutation system is well suited for the detection and mapping of mutations which are large deletions because its MIC1 locus, which controls the expression of the selectable cell surface antigen S1, is on a single human chromosome. This human chromosome 11 is in addition to the genome of the Chinese hamster ovary cell and is basically nonessential. Since there are no sister human chromosomes in AL cells, deletions which extend beyond the MIC1 locus may be conveniently and unambiguously mapped. We have detected the presence or absence of 9 different chromosome 11 markers in 48 S1- mutants cloned from amsacrine-treated cultures. We find that almost all (92%) of the mutants have deletions of at least 1.5-2 megabase pairs in length. The distribution of marker loss frequencies flanking the MIC1 locus does not appear symmetric with respect to distance from that locus. We speculate that amsacrine-induced deletions are mediated by a series of subunit exchanges between overlapping topoisomerase II dimers at the bases of replicons or larger chromosomal structures such as replicon clusters or chromosome minibands.

Cyclic nucleotide metabolism in rat colonic epithelial cells with different proliferative activities.

Cyclic nucleotide metabolism was examined in rat distal colonic epithelial cells with different proliferative activities. Lower crypt cells had DNA synthetic rates 7-10-fold higher than surface cells. Without a phosphodiesterase inhibitor proliferative cells had reduced basal cyclic AMP-, cyclic GMP-, and cyclic AMP-dependent protein kinase activity ratios, as well as blunted cyclic AMP responses to prostaglandin E2 and vasoactive intestinal peptide compared to superficial cells. In the presence of 3-isobutyl-1-methylxanthine, basal cyclic AMP and responses to prostaglandin E2 and vasoactive intestinal peptide of proliferative cells exceeded values in superficial cells. This correlated with higher membrane adenylate cyclase activity in the proliferative cells. By contrast, particulate and soluble guanylate cyclase activities of superficial cells were higher than in proliferative cells. The apparent high Km soluble and particulate cyclic AMP and cyclic GMP phosphodiesterase activities of proliferative cells were 4-7-fold higher than those in superficial cells. Moreover, the apparent low Km soluble activity was absent in superficial cells. Thus, an altered rate of nucleotide degradation may mediate reduced cyclic AMP and cyclic GMP in proliferative versus superficial cells. Dibutyryl cyclic AMP, prostaglandin E2 or vasoactive intestinal peptide inhibited [3H]thymidine incorporation into DNA of colonic segments. Thus, reduced cyclic AMP in lower crypt cells may be a determinant of their greater proliferative activity.

Requirement for heme in the activation of purified guanylate cyclase by nitric oxide.

Guanylate cyclase activity was purified to apparent homogeneity from rat liver (7700-fold) and bovine lung (8600-fold) soluble fractions by ammonium sulfate precipitation, DEAE-cellulose chromatography, agarose gel filtration and isoelectric focussing. The purified enzymes did not contain heme and did not respond to NO, nitroprusside or NO-cysteine in the absence of exogenous hematin. By contrast, preformed NO-hemoglobin increased enzyme activity 10-12-fold or 60-80-fold when 4 mM MnCl2 or 4 mM MgCl2, respectively, were employed as the metal ion co-factor. Addition of hematin to the enzyme preparations restored responsiveness to NO, nitroprusside or NO-cysteine to levels seen with NO-hemoglobin. Partial purification of guanylate cyclase from the soluble fraction of bovine lung (2400-fold) by ammonium sulfate precipitation, DEAE-cellulose chromatography, agarose gel filtration and high pressure liquid chromatography (HPLC) resulted in a preparation which contained endogenous heme as indicated by absorbance at 436 nm and responded to NO, nitroprusside and NO-cysteine in the absence of added hematin. By contrast, guanylate cyclase purified from the hepatic supernatant by the identical procedure, did not contain detectable absorption due to heme and did not respond or responded poorly to NO, nitroprusside or NO-cysteine in the absence of exogenous hematin. Analogous to hepatic guanylate cyclase purified by isoelectric focussing, the HPLC purified hepatic enzyme was activated 14-fold by NO-hemoglobin in assays which contained 4 mM MnCl2 and 60-fold in assays with 4 mM MgCl2. Further, addition of hematin to the HPLC purified enzyme restored responsiveness to NO, nitroprusside and NO-cysteine to levels seen with NO-hemoglobin. These effects of hematin were specific for hematin and were not mimicked by albumin, sucrose or dithiothreitol. Moreover, the failure to observe stimulation of purified hepatic guanylate cyclase was not explained by a shift in the concentration response relationship between NO and guanylate cyclase activity. Several observations indicated that neither NO-thiol complexes nor [Fe(CN)5NO]-3 were the proximate moieties responsible for activation of guanylate cyclase by nitroprusside and related agents, as has been previously suggested. These results strongly support the proposal that activation of guanylate cyclase by NO and related agents specifically requires formation of an NO-heme complex.