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.

Release of prostaglandin by mitogen- and antigen-stimulated leukocytes in culture.

The prostaglandin (PG) content of mitogen- and antigen-stimulated leukocyte cultures was examined by a radioimmunoassay procedure empolying an antiserum reactive with PGB(1) and PGB(2), the alkaline dehydration products of PGE and PGA. At 48 h, mitogen-activated mouse spleen cell cultures showed 2-10-fold increases in the PGE, but not in the PGA, component of immunoreactive PG (iPG) fractionated by silicic acid column chromatography. Increases in iPG were detectable by h 16 in spleen cell cultures incubated with staphylococcal enterotoxin B. Since iPG levels rose only in the culture supernates and not in cells exposed to mitogens for 48 h, increases reflected extracellular release of PG. The validity of the radioimmunoassay determinations of PGE in spleen cell cultures was supported by the results of concomitant assessment of the PGE(2) content of basal and enterotoxin-stimulated cultures by gas chromatography/mass spectrometry. By the latter method, the PGE(2) content was three-fold higher in enterotoxin-activated, compared to basal, cultures at 48 h. Aspirin effectively suppressed increases in both iPG and PGE(2). In spleen cell cultures prepared from mice previously inoculated with an attenuated strain of yellow fever virus in vivo and then incubated with this virus in vitro, iPG levels increased twofold over basal at 48 h. By contrast, iPG content of spleen cell cultures prepared from saline-inoculated mice was not appreciably altered by exposure to the virus in vitro. The enhancement of iPG release from cultured spleen cells by mitogens did not correlate with an ability of these agents to increase cellular cyclic AMP (cAMP) levels. Moreover, epinephrine and cholera toxin markedly increased spleen cell cAMP content but had no demonstrable effect on basal iPG levels, suggesting iPG release from these cells was not mediated by cAMP. Incubation with mitogens also enhanced the iPG content of 72-cultures of human peripheral leukocytes and of human lymphocytes isolated by nylon chromatography. However, the iPG of cultures of human lymphocytes purified by glass bead chromatography and of mouse thymocytes was not appreciably altered when these cells were cultured with mitogens, even though DNA synthesis in both instances was markedly increased. Accordingly, iPG release was not an invariable concomitant of increased DNA synthesis in lymphoid cell cultures. In summary, the results demonstrate that mitogen and antigen stimulation of leukocytes in culture may be accompanied by enhanced release of PGE. The mechanisms mediating this phenomenon and its biologic significance remain to be delineated, but participation of PGE in immunologically induced inflammatory responses seems possible.

Evidence for enhanced cellular uptake and binding of thyroxine in vivo during acute infection with Diplococcus pneumoniae.

Previous work has demonstrated that acute pneumococcal infections in man and in the rhesus monkey are accompanied by accelerated metabolic disposal of L-thyroxine (T(4)). In order to study the influence of acute pneumococcal infection on the kinetics of hormone distribution, the early cellular uptake of T(4) (CT(4)), reflecting the net effect of plasma and cellular binding factors, was assessed in rhesus monkeys from the differences in instantaneous distribution volumes of T(4)-(131)I and albumin-(125)I during the first 60 min after their simultaneous injection. Hepatic and renal uptakes of (131)I were also determined. Plasma binding of T(4) was assessed by measuring the per cent of free T(4) (% FT(4)) in serum. Six monkeys were studied 12 hr (INF-12) and seven 24 hr (INF-24) after intravenous inoculation with Diplococcus pneumoniae; seven controls were inoculated with a heat-killed culture. CT(4) at 60 min as per cent administered dose was 31.5 +/-2.0 (mean +/-SE) in INF-12 and 33.0+/-0.8 in INF-24, values significantly greater than control (22.4+/-1.3). By contrast, mean% FT(4) was identical in control and INF-12 (0.028 +/-0.002 and 0.028 +/-0.001) and variably increased in INF-24 (0.034 +/-0.003). Thus, in the infected monkeys CT(4) and% FT(4) were not significantly correlated. The increased CT(4) in the infected monkeys could not be ascribed to an increase in vascular permeability and did not correlate with the magnitude of fever. Although the increased CT(4) could not be accounted for by increased hepatic or renal uptake of hormone, hepatic and renal T(4) spaces were increased, results consistent with increased binding by these tissues. Our data indicate that the cellular uptake of T(4) is increased early in acute pneumococcal infection and suggest that this results from a primary enhancement of cell-associated binding factors for T(4).

Accelerated cellular uptake and metabolism of L-thyroxine during acute Salmonella typhimurium sepsis.

The effects of acute Salmonella typhimurium sepsis on the kinetics of peripheral L-thyroxine (T(4)) distribution and metabolism and on serum total and free T(4) concentrations were studied in rhesus monkeys inoculated i.v. with either heat-killed or viable organisms. The rate of disappearance of labeled T(4) from serum was increased within 8 h after inoculation of monkeys with either heat-killed or viable Salmonella. The effects of the heat-killed organisms were transient and no longer evident by 16 h postinoculation. The monkeys inoculated with the viable Salmonella experienced a 2-3 day febrile, septic illness that was accompanied by an increase in the absolute rate of T(4) disposal. In the infected monkeys, serum total T(4) and endogenously labeled protein-bound iodine concentrations fell significantly during the period of acute sepsis and then rose during convalescence to values that exceeded the preinoculation values, suggesting that thyroidal secretion of hormone had increased in response to a primary depletion of the peripheral hormonal pool. Total cellular and hepatic uptakes of T(4) were enhanced by 4 h after inoculation of monkeys with either heat-killed or viable Salmonella, but the increase in total cellular uptake persisted for 24 h only in the monkeys inoculated with the viable organisms. These alterations in T(4) kinetics could neither be correlated with changes in the binding of T(4) in plasma nor attributed to an increase in vascular permeability. Moreover, they could not be ascribed to an in vitro product of bacterial growth, suggesting that the presence of the organisms themselves was required. An acceleration of T(4) disappearance was also observed during Escherichia coli and Diplococcus pucumoniae bacteremias. Our findings are consistent with a primary increase in the cellular uptake and metabolism of T(4) during bacterial sepsis, possibly related to phagocytic cell function in the host.

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.

Reduced sensitivity of the hepatic adenylate cyclase-cyclic AMP system to glucagon during sustained hormonal stimulation.

Hormone-induced desensitization of hormonal regulation of cyclic AMP (cAMP) content has been described in a number of tissues. In the present study, we examined responses of rat liver to glucagon after periods of sustained exposure to the hormone in vivo and in vitro. In intact anesthetized rats infused with glucagon (50 ng/min) for 1 h or more and in liver slices incubated with the hormone (10 muM) for this period, hepatic cAMP responsiveness to glucagon was significantly blunted compared with that of tissue exposed to the hormone for shorter periods. The reduction in hepatic cAMP responsiveness to glucagon appeared to be fully expressed by 2 h. With the doses of hormone employed, the sequential alterations in hepatic responsiveness seemed to be limited to the cAMP system, since other parameters of glucagon action did not wane with time. Diminished hepatic cAMP responsiveness during sustained hormonal exposure could not be attributed to decreased glucagon availability, accelerated extracellular release of cAMP, hepatic ATP depletion, or enhanced phosphodiesterase activity. Studies in vitro suggested that modulation of the cAMP response occurred at the level of adenylate cyclase (AC). During sustained exposure of hepatic slices to glucagon, reductions in glucagon-responsive AC correlated temporally with those in cAMP and both changes were reversible. Alterations in glucagon-responsive AC were demonstrated over a wide range of ATP (10 muM-0.1 mM) and glucagon (10 nM-5 MM) concentrations in the cyclase reaction mixture, and appeared to be a noncompetitive phenomenon relative to glucagon. Maximal NaF-responsive AC did not fall concomitantly with time. Thus, the reduction in glucagon-responsive AC was probably not related to a reduction in the catalytic unit of the enzyme, but could have been due to an alteration in glucagon binding to its receptor sites, or in the coupling mechanism involved in transmission of the hormonal signal to the catalytic unit.

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.

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.

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.

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.

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.

The content and metabolism of cyclic adenosine 3', 5'-monophosphate and cyclic guanosine 3', 5'-monophosphate in adenocarcinoma of the human colon.

Data from cultured cells have suggested that cyclic AMP and cyclic GMP may be important determinants of cell growth and transformation. However, few studies have examined cyclic nucleotide content and metabolism in naturally occurring tumors of man. Accordingly, in the present study we compared cAMP and cGMP levels and metabolism in carcinomas of the human colon to those of the adjacent uninvolved mucosa after therapeutic resection of these tissues. The cAMP content of the tumors, determined in samples frozen 30 min after excision, was significantly lower than that of the adjacent mucosa, when expressed on the basis of tissue wet weight, protein, or DNA content. By contrast, the cGMP content of the tumors was higher than that of the surrounding mucosa if calculated on the basis of tissue wet weight, but this difference did not persist when correction was made for the higher protein or DNA content of the tumors. Incubation of slices of mucosa or tumor with or without theophylline in vitro increased tissue cAMP and cGMP content above levels observed in frozen samples of the same tissue. However, after such incubations cAMP levels in the tumors remained clearly below that of the mucosa, while cGMP content of the two tissues did not differ. The failure of theophylline to abolish differences in cAMP content and the comparable activities of high and low Km cAMP-phosphodiesterase in homogenates of the two tissues suggested that the lower cAMP content of the tumors was a consequence of diminished cAMP synthesis rather than enhanced degradation. This possibility was supported by the reduction in basal and maximal prostaglandin E1 (PGE1)-responsive adenylate cyclase activity found in tumor homogenates relative to those of mucosa, and the lower levels of cAMP in tumor slices after incubation of the tissues with a maximal dose of PGE1 and theophylline. Since NaF-responsive adenylate cyclase activity was not significantly reduced in the tumors, the lower basal and PGE1 activities may not be related to a deficiency of the catalytic unit of the cyclase complex in this tissue. The role of reduced activity of the adenylate cyclase-cAMP system and/or reduced tissue cAMP-to-cGMP ratios in the pathogenesis of colonic carcinoma is uncertain, but these changes might favor unregulated cellular proliferation.

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.

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.

"Essential" hypernatremia due to ineffective osmotic and intact volume regulation of vasopressin secretion.

A physiological explanation for sustained hyperosmolality was sought in a patient with histiocytosis. During 23 days of observation with only sodium intake regulated at 100 mEq daily, elevation (mean 310 mOsm/kg of water) and fluctuation (range 298-323) of the fasting plasma osmolality were recorded. The presence of endogenous vasopressin was indicated by the patient's ability to concentrate the urine to as high as 710 mOsm/kg of water with a creatinine clearance of 84 cc/min, and by dilution of the urine in response to alcohol. The failure of increasing fluid intake to as high as 6.2 liters daily to lower the plasma osmolality indicated that deficient fluid intake was not solely responsible for the elevated plasma osmolality. Hypertonic saline infusion during water diuresis resulted in the excretion of an increased volume of dilute urine. The water diuresis continued despite a rise in plasma osmolality from 287 to 339. An isotonic saline infusion initiated during hydropenia resulted in a water diuresis which continued despite a rise in the plasma osmolality from 303 to 320. Stable water diuresis induced during recumbency by either oral ingestion of water or intravenous infusion of normal saline was terminated by orthostasis and resumed with the return to the recumbent position. Antecedent alcohol ingestion blocked the antidiuresis of orthostasis. The data are interpreted as indicating impairment of the osmoreceptor mechanism as the primary cause of the hyperosmolar syndrome. They also indicate that vasopressin secretion was regulated primarily by changes in effective blood volume. Chlorpropamide was found to be an effective treatment for the syndrome.

Effect of prostaglandin E 1 on certain renal actions of parathyroid hormone.

Parathyroid hormone increased basal adenyl cyclase activity and that increase was inhibited by prostaglandin E(1) (PGE(1)). Tissue cyclic 3',5'-adenosine monophosphate (cyclic AMP) concentrations were increased by parathyroid hormone and that increase was likewise inhibited by PGE(1). Both parathyroid hormone and dibutyryl cyclic AMP increased (32)P incorporation into renal cortical phospholipids. PGE(1) diminished the effect of parathyroid hormone but not dibutyryl cyclic AMP to influence that parameter. PGE(1) likewise modulated the effect of parathyroid hormone but not dibutyryl cyclic AMP to decrease fractional phosphate reabsorption by the renal tubule. It is suggested that PGE(1) inhibits the effect of parathyroid hormone by decreasing its effect on adenyl cyclase. Such interaction may be important in modulating the intracellular action of parathyroid hormone on kidney cortex.

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.