Homocysteine remethylation in young broilers fed varying levels of methionine, choline, and betaine.

Methionine is critical in amino acid nutrition for chickens, yet details of the flux of Met metabolites in the avian system are lacking. This study explored the interactions among dietary choline (CHO), betaine (BET), and sulfur amino acid levels on growth and hepatic homocysteine (HCY) remethylation. Graded levels (0, 0.07, 0.11, and 0.24%) of DL-Met were added to diets adequate in CHO and deficient in sulfur amino acids (0.26% digestible Met, 0.26% digestible Cys). Each Met level was tested alone or with the addition of CHO (0.25%) or BET (0.28%). Broilers were reared from 8 to 22 d in raised wire floor battery cages, and the 12 dietary treatments were fed to 3 replicate pens containing 5 birds per pen. Weight gain and feed efficiency were maximized (P < 0.05) with addition of 0.11% supplemental Met, whereas feed intake was maximized (P < 0.05) with addition of 0.07% supplemental Met. Overall, growth parameters were not affected (P > 0.05) by CHO or BET addition. Hepatic tissue primed by the different dietary treatments was subjected to a newly developed stable isotope methodology and HPLC-mass spectrometry to quantify the impact of diet on HCY remethylation. Dietary Met level did not (P > 0.05) affect HCY remethylation, but remethylation through the Met synthase pathway was increased (P < 0.05) by addition of CHO or BET to diets containing deficient or excess levels of Met. Minimal changes in hepatic HCY remethylation through the betaine-homocysteine methyltransferase pathway occurred in response to dietary changes; therefore, data failed to support previous suggestions that BHMT might have a regulatory role when diets containing deficient or excess Met levels are fed. In contrast to previous suppositions based on enzyme activity, under most dietary conditions, the quantity of HCY remethylated by Met synthase appeared to exceed that remethylated by the alternate betaine-homocysteine methyltransferase pathway.

Plasma taurine levels in broilers with pulmonary hypertension syndrome induced by unilateral pulmonary artery occlusion.

Low plasma levels of taurine are associated with losses of cardiac sarcomeric proteins, leading to heart failure in mammals. Recently, it was proposed that cardiac taurine depletion serves to defend the heart against injury caused by regional ischemia in mammals. The role of taurine has not been well documented in broilers, particularly in relation to pulmonary hypertension syndrome (PHS; ascites). Three independent experiments evaluated plasma taurine in male broilers by utilizing the following treatments: unoperated controls (CONTROL; n = 10 in each experiment); sham operated (SHAM; n = 11, 12, and 10); or, unilaterally pulmonary artery clamped (PAC; n = 18, 29, and 24) that did (PAC-ascites) or did not (PAC-normal) develop ascites within 12 d postsurgery. Plasma samples were collected 9 and 11 d postsurgery in Experiments 1 and 2, respectively, and 2 d before and 4, 8, and 12 d after surgery in Experiment 3. Plasma taurine was analyzed by HPLC. Twelve days postsurgery, the birds were euthanatized, and ventricles were weighed for calculating the right:total ventricular weight ratio (RV:TV). The RV:TV of PAC birds (>0.35) consistently was higher (P < 0.01) than that of CONTROL and SHAM birds (<0.27 and 0.25, respectively). In Experiments 1 and 2, plasma taurine was higher (P < 0.05) in PAC-ascites (380 and 370 nmol/mL) than in SHAM broilers (183 and 186 nmol/mL), whereas CONTROL (262 and 278 nmol/mL) and PAC-normal (362 and 300 nmol/mL) broilers tended to have intermediate plasma taurine levels. In Experiment 3, PAC birds had higher (P < 0.05) plasma taurine at 8 and 12 d postsurgery when compared with presurgery levels, whereas plasma taurine was unchanged over time in CONTROL and SHAM birds. These results suggest cardiac taurine may be released into the plasma as a protective mechanism in response to the induction of pulmonary hypertension, hypoxemia, and right-side heart failure, similar to the mechanism reported for protecting cardiac muscle from ischemia in mammals.

Evidence of mitochondrial dysfunction in broilers with pulmonary hypertension syndrome (Ascites): effect of t-butyl hydroperoxide on hepatic mitochondrial function, glutathione, and related thiols.

The purpose of this study was to assess mitochondrial function and glutathione (a mitochondrial antioxidant) in response to oxidative stress in mitochondria in vitro obtained from broilers with and without pulmonary hypertension syndrome (PHS). Liver mitochondria from Control and PHS broilers were incubated with 0, 1, and 5-mM tertiary-butyl hydroperoxide (tBH). Indices of mitochondrial function [the respiratory control ratio (RCR) and the adenosine diphosphate to oxygen ratio (ADP:O)], and levels of mitochondrial and extra-mitochondrial reduced (GSH) and oxidized (GSSG) glutathione, cysteine, cystine, glutamate and cysteinyl-glycine were determined following tBH treatment. Lower RCR and ADP:O values were observed in PHS mitochondria than in controls. Whereas control mitochondria remained coupled (RCR > 2.0), only 3 PHS preparations remained coupled after 60 min of incubation with 5 mM tBH, indicating a greater susceptibility to oxidative stress in PHS mitochondria. The lower RCR in PHS mitochondria was due to increased oxygen consumption during State IV respiration. Oxidative stress following tBH treatment (decreased GSH and increased GSSG) was observed, but there were no differences in GSH or GSSG between control and PHS mitochondria. The PHS mitochondria did exhibit elevated mitochondrial and extramitochondrial cystine than controls, however. The results indicate that PHS mitochondria do not lack antioxidant protection from GSH, but lower RCR and ADP:O ratios in PHS mitochondria indicate a dysfunction that may contribute to the pathophysiology of this metabolic disease in broilers.

Research on the probable cause of an outbreak of field rickets in turkeys.

An outbreak of field rickets in turkeys prompted studies on the cause. In Experiment 1, there were four treatments with two replicate pens of 10 poults per pen. The treatments consisted of poults fed newly manufactured feed (control), poults fed a diet containing control feed and 5% clean litter, poults fed control feed and 5% litter from the pens of affected poults, and poults challenged with an intestinal homogenate by gavage. Field rickets did not develop with these treatments. The feed was suspect, and, in Experiment 2, poults were either fed the suspect feed or newly manufactured feed. There were four replicate pens of 25 poults per pen. Poults fed the suspect feed had a decrease (P < or = 0.05) in BW at 1, 2, 3, and 4 wk of age; an increase in the relative weight of the liver, pancreas, kidney, and bursa of Fabricius; and a decrease in bone ash. There were changes in clinical chemistries. In the third study, there were five dietary treatments with two replicate pens of 25 poults per treatment. The treatments consisted of poults fed newly manufactured feed; new feed mixed with 25, 50, or 75% suspect feed; or 100% suspect feed. Body weights of poults fed 100% suspect feed were decreased at 2, 3, and 4 wk as was the relative weight of the liver, pancreas, and bursa of Fabricius. The relative weight of the kidney increased. Lameness, a decrease in bone ash, and changes in hematology and blood chemistry were observed in the poults consuming 100% suspect feed. These data demonstrate that feed from the original outbreak could induce field rickets and was toxic. Because the feed contained adequate vitamin D, calcium, and phosphorus, the cause of this outbreak of field rickets is thought to be a toxic feed contaminant affecting bone development.

Lung lining fluid antioxidants in male broilers: age-related changes under thermoneutral and cold temperature conditions.

The purpose of this study was to determine age-related changes in lung lining fluid antioxidants in broilers reared under thermoneutral or cold temperature conditions. Male broilers (Cobb 500) were placed in floor pens within environmental chambers and fed a standard commercial starter diet. The thermoneutral Control chamber was maintained at 32, 30, 27, and 22 to 25 C for Weeks 1, 2, 3, and 4 to 7, respectively, whereas temperature in the Cold chamber was lowered to 18 C during Week 3 and maintained between 15 and 18 C for the rest of the study. At 2, 4, and 7 wk, four to six birds per chamber were selected randomly. The lungs were lavaged with heparinized saline (2 mL/g lung) to obtain lung lining fluid. Antioxidants [reduced (GSH), oxidized (GSSG), and total (TGSH) glutathione, uric acid, ascorbic acid, and alpha- and gamma-tocopherol] in lung lining fluid were determined by HPLC; protein was determined colorimetrically. In Controls, levels of alpha- and -gamma-tocopherol, uric acid, and GSH in lung lining fluid decreased between 2 and 7 wk of age. Birds in the Cold chamber exhibited higher protein, a higher GSSG:TGSH ratio, and a decrease in ascorbic acid (7 wk) in lung lining fluid relative to Controls. Lung lining fluid antioxidants were not correlated with antioxidants in plasma. To determine the effect of vitamin E supplementation on lung lining fluid antioxidants, birds were given a supplement of 200 IU alpha-tocopherol per day for 7 d. Alpha-tocopherol supplementation elevated alpha-tocopherol levels in lung lining fluid, but lowered ascorbic acid, GSH, and GSSG and had no effect on uric acid in lung lining fluid. The results of this study suggest that antioxidant protection in lung lining fluid may diminish with age, that cold conditions in this study produced an oxidative stress in lung lining fluid in broilers, and that oral supplementation of alpha-tocopherol elevated lung lining fluid alpha-tocopherol.

Effects of sodium sulfate on acute N-(3,5-dichlorophenyl)succinimide (NDPS) nephrotoxicity in the Fischer 344 rat.

The agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) induces acute polyuric renal failure in rats. Results of previous studies have suggested that NDPS may induce nephrotoxicity via conjugates of NDPS metabolites. Thus, the purpose of this study was to examine if administered sodium sulfate could alter NDPS nephrotoxicity. Male Fischer 344 rats (four rats per group) were administered a single intraperitoneal (i.p.) injection of sodium sulfate (0.035, 0.07, 0.35 or 3.5 mmol/kg) or sodium chloride (7.0 mmol/kg) 20 min before NDPS (0.2, 0.4 or 0.8 mmol/kg) or NDPS vehicle (sesame oil, 2.5 ml/kg) and renal function monitored at 24 and 48 h. High dose sodium sulfate (3.5 mmol/kg) markedly attenuated NDPS nephrotoxicity, while sodium chloride had no effect on NDPS-induced renal effects. NDPS nephrotoxicity was also attenuated by a pretreatment dose of 0.35 mmol/kg sodium sulfate, while 0.07 mmol/kg sodium sulfate pretreatment potentiated NDPS 0.2 mmol/kg to produce nephrotoxicity without markedly attenuating NDPS 0.4 mmol/kg to induce renal effects. A dose of 0.035 mmol/kg sodium sulfate did not potentiate NDPS 0.2 mmol/kg to induce nephrotoxicity. These results suggest that sulfate conjugates of NDPS metabolites might contribute to NDPS nephrotoxicity.

Effect of dietary dl-alpha-tocopherol on tissue alpha- and gamma-tocopherol and pulmonary hypertension syndrome (ascites) in broilers.

The objectives of this experiment were to determine the effects of high dietary levels of vitamin E on growth performance and pulmonary hypertension syndrome (PHS) mortality. Male broiler chicks (Cobb 500) were randomly assigned to one of four dietary treatments consisting of standard starter and grower diets supplemented with 0, 17, 46, and 87 mg dl-alpha-tocopherol acetate/kg. To encourage the development of PHS, air temperature in the house was 32 and 28 C for Weeks 1 and 2, dropped to 18 C during Week 3, and kept between 10 and 15 C during Weeks 4 through 7. Also, chicks were placed in floor pens on litter used for five previous flocks and ventilation reduced to increase dust and ammonia in the house. Ammonia levels increased from an initial 18 to 36 ppm on Day 42 with the increase in ammonia corresponding to an obvious increase in dust in the air. Lung and liver tissue obtained at 2, 5, and 7 wk of age were analyzed for tissue alpha- and gamma-tocopherol by liquid chromatography. Dietary vitamin E had no effect on body weight, feed intake, or feed efficiency. Cumulative PHS mortality through 7 wk of age was 21% and was also unaffected by dietary treatment. Liver and lung alpha-tocopherol concentrations exhibited a dose-response increase to dietary tocopherol and there was a high correlation between lung and liver tissue alpha-tocopherol (r = 0.72, P < 0.05). Whereas gamma-tocopherol concentrations in lung and liver were unaffected by dietary treatment, liver and lung exhibited age-dependent increases in both alpha- and gamma-tocopherol. Despite dose-dependent increases in tissue alpha-tocopherol, supplementation of diets with up to 87 mg dl-alpha-tocopherol acetate had no effect on growth performance or PHS mortality in broilers under the conditions used in this study.

Pleiotropic upregulation of Na(+)-dependent cotransporters by retinoic acid in opossum kidney cells.

All-trans-retinoic acid (atRA) is a regulator of cellular growth and differentiation. We investigated whether atRA can upregulate Na(+)-dependent cotransporters in opossum kidney (OK) cells and thus increase uptake from tubular fluid of several solutes needed for growth during early stages of ontogenesis. In OK cells, incubation with atRA for 24 h increased the Na+ gradient-dependent cotransports of phosphate, L-proline, L-glutamic acid, and SO(4)2- by a similar degree (approximately 40%) that was prevented by pretreatment with actinomycin D. In contrast, activities of other Na(+)-dependent transporters, Na(+)-K(+)-adenosinetriphosphatase, gamma-glutamyltranspeptidase, and leucine aminopeptidase, were unchanged by atRA. Cell proliferation determined by [3H]thymidine incorporation was not increased by atRA. The stimulatory effects of atRA and phosphate deprivation on Na(+)-Pi cotransport demonstrated additivity, whereas the combination of atRA and 3,5,3'-triiodothyronine did not. atRA stimulated Na(+)-Pi cotransport in LLC-PK1 cells with an analogous time course and to a similar extent as observed in OK cells. We conclude that atRA stimulates several Na(+)-dependent cotransporters via a genomic mechanism and may represent a synchronous adaptation to nutritional requirements of early phases of ontogenesis.

Opposite paracrine effects of 5-HT and dopamine on Na(+)-Pi cotransport in opossum kidney cells.

Serotonin (5-HT) was recently reported to inhibit cAMP generation in oppossum (OK) cells. We thus investigated the effects of 5-HT upon the Na(+)-Pi cotransport in cultured OK cells and its interactions with dopamine. Incubation of OK cells with 1 nM-10 microM 5-HT resulted in dose-dependent stimulation of Na(+)-Pi contransport (ED50 approximately equal to 8 nM) and also counteracted inhibition of Na(+)-Pi cotransport elicited by dopamine. Pre-incubation with 5-HT decreased cAMP accumulation elicited by forskolin or dopamine and pre-treatment with pertussis toxin abolished both the inhibitory effect of 5-HT upon cAMP levels and stimulation of Na(+)-Pi cotransport. Incubation of OK cells with the 5-HT precursor 5-hydroxytryptophan resulted in time- and dose-dependent accumulation of 5-HT in the medium that also elicited an increase in Na(+)-Pi cotransport. Both the effects of 5-HT and dopamine on Na(+)-Pi cotransport were prevented by carbidopa. The stimulatory effect of 5-HT was specific for the Na(+)-Pi cotransport system since no effects were observed on Na(+)-alanine cotransport. The results indicate that 5-HT stimulates Na(+)-Pi cotransport at least in part via inhibition of cAMP accumulation. We propose that 5-HT and dopamine have opposite actions as paracrine/autocrine regulators of Na(+)-Pi cotransport via opposite effects upon cAMP formation.

Cyclic ADP-ribose metabolism in rat kidney: high capacity for synthesis in glomeruli.

Recent discovery of cyclic ADP-ribose (cADPR) as an agent that triggers Ca2+ release from intracellular stores, through ryanodine receptor channel, is an important new development in the investigation of intracellular signaling mechanisms. We determined the capacity of kidney and its components for synthesis of cADPR from beta-NAD, that is catalyzed by enzyme ADP-ribosyl cyclase, and enzymatic inactivation that is catalyzed by cADPR-glycohydrolase. Little or no activity of ADP-ribosyl cyclase was found in extracts from the whole rat kidney, renal cortex, outer and inner medulla. On the other hand, incubation of beta-NAD with similar extracts from rat liver, spleen, heart, and brain resulted in biosynthesis of cADPR. In addition, extracts from suspension of proximal tubules or microdissected proximal convoluted tubules virtually lacked ADP-ribosyl cyclase activity. In sharp contrast to proximal tubules and cortex, extracts from glomeruli had high ADP-ribosyl cyclase activity, similar to that found in non-renal tissues. Authenticity of cADPR biosynthesized in glomeruli was documented by several criteria such as HPLC analysis, effect of inhibitors and homologous desensitization of Ca(2+)-release bioassay. On the other hand, the activity of cADPR-glycohydrolase was similar in extracts from glomeruli and in extracts from kidney cortex. Mesangial cells and vascular smooth muscle cells grown in primary culture displayed considerable ADPR-ribose cyclase activity. Our results show that extracts from glomeruli, unlike extracts from renal tissue zones and proximal tubules, have a singularly high capacity for synthesis of cADPR. We surmise that cADPR-triggered Ca(2+)-releasing system can serve as an intracellular signaling pathway that may be operant in regulations of glomerular cell functions.

Buthionine sulfoximine (BSO) and N-(3,5-dichlorophenyl)succinimide nephrotoxicity: temporal aspects of BSO administration and BSO effects on renal transport systems.

The agricultural fungicide, N-(3,5-dichlorophenyl)succinimide (NDPS) induces acute polyuric renal failure which is attenuated by pretreatment with the glutathione depletors, diethyl maleate or buthionine sulfoximine (BSO). In the present study, the temporal aspects of BSO attenuation of NDPS nephrotoxicity were investigated. In addition, the ability of BSO to alter the renal accumulation of selected organic ions was examined as a possible mechanism for BSO's ability to attenuate NDPS nephrotoxicity. In the first set of experiments, NDPS (0.2 or 0.4 mmol/kg) or vehicle (sesame oil, 2.5 ml/kg) was administered intraperitoneally (i.p.) to groups of male Fischer 344 rats (4-8 rats/group) 0.25 or 2 h prior to BSO (890 mg/kg, i.p.) and renal function was monitored at 24 and 48 h. NDPS (0.4 mmol/kg) nephrotoxicity was markedly attenuated by administration of BSO at 0.25 h post-NDPS, but was not substantially altered by injection of BSO at 2 h post-NDPS. NDPS (0.2 mmol/kg)-induced renal effects were not potentiated by BSO injected at 0.25 h post-NDPS, and only 1 of 8 rats exhibited marked nephrotoxicity when BSO was administered at 2 h post-NDPS. In the second set of experiments, rats (4/group) were administered BSO (890 mg/kg, i.p.) or vehicle (0.9% saline, 10 ml/kg) and kidneys harvested at 2 or 5 h post-treatment. The ability of renal cortical slices to accumulate organic ions (p-aminohippurate [PAH], alpha-aminoisobutryic acid [AIB] or tetraethylammonium [TEA]) during a 90 min incubation was studied. Only TEA accumulation by renal cortical slices prepared from the 2 h post-treatment group was reduced. Studies were also conducted to examine the in vitro effects of BSO (10(-7)-10(-4) M) on the accumulation of PAH, AIB and TEA by renal cortical slices following 5, 15 or 90 min co-incubations of BSO and an organic ion BSO had no significant effects on the accumulation of any organic ion studied at any time point. These results indicate that BSO can still attenuate NDPS nephrotoxicity when administered at 0.25 h post-NDPS, but BSO loses effectiveness when given 2 h post-NDPS. These results also suggest that BSO is attenuating NDPS nephrotoxicity via glutathione depletion rather than altering renal accumulation of NDPS metabolites via renal PAH, TEA or AIB transporters.

Adenine nucleotide diphosphates: emerging second messengers acting via intracellular Ca2+ release.

Release of Ca2+ from intracellular stores is a widespread mechanism in regulation of cell function. Two hitherto unknown adenine diphosphonucleotides were recently identified, which trigger Ca2+ release from intracellular stores via channels that are distinct from the well-known receptor/channel controlled by inositol 1,4,5,-trisphosphate (IP3): cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). Here we review synthesis of cADPR from beta-NAD, its hydrolysis to adenosine diphosphoribose (noncyclic) by cADPR glycohydrolase, as well as our knowledge about the metabolism of NAADP. The Ca2+ release triggered by cADPR, NAADP, or IP3 can be distinguished by the action of inhibitors and by desensitization studies. Evidence now emerges that cADPR synthesis from beta-NAD can be stimulated, at least in some cell types by all-trans-retinoic acid as a first messenger. We then review the properties of cADPR and NAADP as potential second messengers in the intracrine regulation of cell functions. Although their exact role in signaling sequences is not yet known, cADPR and NAADP are likely to play important intracellular regulatory functions, as extensively documented for the process of egg fertilization.

beta-Estradiol inhibits Na+-P(i) cotransport across renal brush border membranes from ovarectomized rats.

Estrogens play a major role in mineral homeostasis, however it remains unclear whether they exert regulatory action upon reabsorption of phosphate (P(i)) in proximal tubules of the kidney. We investigated the effect of beta-estradiol E2 injected to thyroparathyroidectomized and ovariectomized rats upon Na+ cotransport of P(i) and other solutes across renal brush border membrane (BBM). In BBM from kidneys of E2-treated rats the capacity for Na+-P(i) cotransport was considerably suppressed (delta% - 42; P<0.01) whereas Na+-cotransports of L-proline D-glucose and SO4 across the same BBM did not differ from controls. We surmise that E2 inhibits selectively Na+-P(i) cotransport by direct interaction with E2 receptors in proximal tubular cells. These results indicate the existence of the inhibitory effect of estrogens upon renal proximal tubular Na+-P(i) cotransport and, by extention, proximal P(i) reabsorption. We suggest that this modulatory action of E2 plays a role in pathophysiology of mineral metabolism due to estrogen deficiency and should be considered when estrogens are used for pharmacotherapy of postmenopausal osteoporosis and some types of cancer.

Nephrotoxic potential of 2-amino-5-chlorophenol and 4-amino-3-chlorophenol in Fischer 344 rats: comparisons with 2- and 4-chloroaniline and 2- and 4-aminophenol.

Nephrotoxicity occurs following intraperitoneal (i.p.) administration of 2-chloroaniline or 4-chloroaniline hydrochloride to Fischer 344 rats, but the nephrotoxicant chemical species and mechanism of nephrotoxicity are unknown. The purpose of this study was to evaluate the in vivo and in vitro nephrotoxic potential of 2-amino-5-chlorophenol and 4-amino-3-chlorophenol, metabolites of 4-chloroaniline and 2-chloroaniline. A comparison was also made between the nephrotoxic potential of the aminochlorophenols and the corresponding aminophenols to examine the effect of adding a chloride group on the nephrotoxic potential of the animophenols. Male Fischer 344 rats (4/group) were given an i.p. injection of a chloroaniline or aminochlorophenol hydrochloride (1.5 mmol/kg), and aminophenol (1.0 or 1.5 mmol/kg), or vehicle, and renal function monitored at 24 and 48 h. Both aminochlorophenols induced smaller and fewer renal effects that the parent chloroanilenes in vivo. Also, 4-aminophenol was markedly more potent as a nephrotoxicant that 4-amino-3-chlorophenol, while 2-aminophenol and 2-amino-5-chlorophenol induced only mild change in renal function. In vitro, the phenolic compounds reduce p-aminohippurate accumulation by renal cortical slices at bath concentrations of 0.01 mM, while a bath concentration of 0.50 mM or greater was required for the chloroanilines. However, all compounds reduced tetraethylammonium accumulation at bath concentrations of 0.1-0.5 mM or greater. These results indicate that extrarenally-produced aminochlorophenol metabolites do not contribute to the mechanism of chloroaniline nephrotoxicity. Also, the reduced nephrotoxic potential of 4-amino-3-chlorophenol compared to 4-aminophenol could result from an altered ability of the aminochlorophenol to redox cycle or form conjugates.

Specific modulation of cyclic ADP-ribose-induced Ca2+ release by polyamines.

Cyclic ADP-ribose (cADPR) is a potent mediator of Ca2+ mobilization from intracellular stores in sea urchin eggs. However, the regulation of the cADPR-induced Ca2+ release system is not yet fully elucidated. We now report that spermine and related polyamines, in physiological concentrations, were able to inhibit the Ca2+ release induced by cADPR in sea urchin egg homogenate bioassays, as measured using the Ca2+ indicator fluo 3, but had no effect on the Ca2+ release induced by D-myo-inositol 1,4,5-trisphosphate (IP3) or by nicotinate adenine dinucleotide phosphate (NAADP). Spermine was a more potent inhibitor of the cADPR-induced Ca2+ release than spermidine and putrescine. Spermine inhibited not only the release induced by cADPR but also the Ca2+ release induced by caffeine and ryanodine. Finally, pretreatment of the sea urchin egg homogenates with caffeine or Sr2+ and Ca2+ prevented the inhibitory effect of spermine on cADPR-induced Ca2+ release. We propose that polyamines, which are present in millimolar concentrations in fertilized eggs, are specific inhibitors of the ryanodine channel and perhaps may serve as endogenous regulators of the cADPR-induced Ca2+ release system.

Effect of dimethyl sulfoxide on N-(3,5-dichlorophenyl)succinimide (NDPS) and NDPS metabolite nephrotoxicity.

Dimethyl sulfoxide (DMSO) is frequently used as a solvent to assist in dissolving compounds which are not readily soluble in other injection vehicles. The purpose of this study was to determine the suitability of DMSO as a vehicle for administering the nephrotoxicant, N-(3,5-dichlorophenyl)succinimide, (NDPS) and two nephrotoxicant NDPS metabolites, N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS) and N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (NDHSA). Male Fischer 344 rats (4/group) were administered a single intraperitoneal injection of NDPS (0.4 or 0.8 mmol/kg), NDHS (0.1 or 0.2 mmol/kg), or NDHSA (0.1 or 0.2 mmol/kg) dissolved in 25% DMSO in sesame oil or 100% sesame oil (2.5 ml/kg), while control rats received vehicle only. Renal function was then monitored at 24 and 48 h. Including DMSO in the vehicle markedly attenuated NDPS 0.4 mmol/kg-induced nephrotoxicity and reduced NDPS 0.8 mmol/kg-induced renal effects. Thus, the magnitude of the attenuating effect of DMSO depended in part on the nephrotoxicant dose of NDPS. In addition, NDHS nephrotoxicity was not altered by DMSO and only slight effects on NDHSA nephrotoxicity were observed. These results suggest that DMSO is capable of attenuating NDPS nephrotoxicity, and that the primary mechanism of this interaction might be due to an inhibition of the biotransformation of NDPS to NDHS.

All-trans-retinoic acid stimulates synthesis of cyclic ADP-ribose in renal LLC-PK1 cells.

Cyclic adenosine diphospho-ribose (cADPR) triggers Ca2+ release from intracellular stores and is therefore proposed to function as a second messenger in cellular signaling; however, an extracellular stimulus, i.e., first messenger (hormone or autacoid) that modulates cADPR metabolism has not been identified. We discovered that all-trans-retinoic acid (atRA) is a potent stimulus to increase cADPR synthesis by cultured LLC-PK1 cells. The stimulation of cADPR synthesis by atRA is dose dependent between 0.1 nM and 1 microM (maximum increase approximately delta + 600%), while atRA does not alter the rate of cADPR hydrolysis by LLC-PK1 cells. The activity of other intrinsic apical membrane enzymes was not significantly altered. The stimulation of cADPR synthesis by atRA occurs after a lag period of 6-8 h, and the stimulation is inhibited by actinomycin D and by cycloheximide. Our results therefore demonstrate that atRA in physiological concentrations is a potent extracellular stimulus, first messenger, that enhances cADPR synthesis, and the effect of atRA requires de novo protein synthesis. We suggest that some of the diverse biologic actions of atRA such as morphogenetic and cell differentiation may be mediated via cADPR.

Metabolism of cyclic ADP-ribose in opossum kidney renal epithelial cells.

We have previously shown that NAD+ inhibits renal Na(+)-Pi symport; however, the biochemical mechanism of NAD+ in this action is not clarified. We now propose that NAD+ acts indirectly by first being converted to cyclic ADP-ribose (cADPR), a potent stimulator of intracellular Ca2+ mobilization. In permeabilized opossum kidney (OK) cells, a cell line often employed as a model for study of proximal tubular epithelial transport, cADPR is synthesized from beta-NAD+ in a substrate concentration (0.01-1 mM) and time-dependent manner. That cADPR was generated from beta-NAD+ by OK cells was verified by coelution with authentic cADPR on anion exchange high-performance liquid chromatography and by homologous desensitization of the Ca2+ release bioassay to authentic cADPR. cADPR synthesized by permeabilized OK cells was not influenced by the addition of parathyroid hormone. The OK cell also contains the enzyme activity necessary to catalyze catabolism of cADPR. Identification of these two key enzyme activities of cADPR metabolism in OK cells is consistent with a possible role of cADPR in regulation of the Na(+)-Pi symporter by NAD+ in response to metabolic stimuli.

Nicotinate adenine dinucleotide phosphate (NAADP) triggers a specific calcium release system in sea urchin eggs.

Transient fluxes of intracellular ionized calcium (Ca2+) from intracellular stores are integral components of regulatory signaling pathways operating in numerous biological regulations, including in early stages of egg fertilization. Therefore, we explored whether NADP, which is rapidly generated by phosphorylation of NAD upon fertilization may, directly or indirectly, exert a regulatory role as a trigger of Ca2+ release from intracellular stores in sea urchin eggs. NADP had no effect, but we found that the deamidated derivative of NADP, nicotinate adenine dinucleotide phosphate (beta-NAADP), is a potent and specific stimulus (ED50 16 nM) for Ca2+ release in sea urchin egg homogenates. NAADP triggers the Ca2+ release via a mechanism which is distinct from the well-known Ca2+ release systems triggered either by inositol-1,4,5-triphosphate (IP3) or by cyclic adenosine diphospho-ribose (cADPR). The NAADP-induced release of Ca2+ is not blocked by heparin, an antagonist of IP3, or by procaine or ruthenium red, antagonists of cADPR. However, it is selectively blocked by thionicotinamide-NADP which does not inhibit the actions of IP3 or cADPR. NAADP produced by heating of NADP in alkaline (pH = 12) medium or synthetized enzymatically by nicotinic acid-NADP reaction catalyzed by NAD glycohydrolase have identical properties. The results presented herein thus describe a novel endocellular Ca(2+)-releasing system controlled by NAADP as a specific stimulus. The NAADP-controlled Ca2+ release system may be an integral component of multiple intracellular regulations occurring in fertilized sea urchin eggs, which are mediated by intracellular Ca2+ release, and may also have similar role(s) in other tissues.

CNP is present in canine renal tubular cells and secreted by cultured opossum kidney cells.

C-type natriuretic peptide (CNP) is a vasoactive and antimitogenic peptide that is structurally similar but genetically distinct from atrial natriuretic peptide. While first discovered in the brain, CNP has been shown to be produced by endothelial cells and may function in a paracrine and autocrine fashion in the control of vascular tone. Recently, CNP immunoreactivity and B-type natriuretic peptide receptors (NPR-B), for which CNP is a specific ligand, have been identified in the kidney. The present study was designed to determine whether renal epithelial cells produce and secrete CNP and whether CNP immunoreactivity is present in canine kidney. Opossum kidney (OK) cells that express proximal tubular cell characteristics were incubated for 6 h in fetal calf serum-free Dulbecco's modified Eagle's medium (DMEM). CNP immunoreactivity was measured in the preincubation and 6-h conditioned media by radioimmunoassay (RIA) using a specific antibody to CNP-22. Furthermore the molecular form of this CNP-like protein was determined by reverse-phase high-performance liquid chromatography (HPLC), and intracellular localization of the CNP immunoreactivity was determined by immunohistochemical staining. CNP immunoreactivity was also determined in renal tissue from dogs subjected to saline or endothelin infusion. Six-hour incubation in DMEM resulted in accumulation of CNP immunoreactivity (baseline below detection level vs. 6 h = 117.3 +/- 8.3 pg/ml, P < 0.001). Intracellular CNP concentration determined after sonication was 1.9 +/- 0.2 micrograms/g protein, and immunohistochemical staining for CNP was markedly positive in the cytoplasm.(ABSTRACT TRUNCATED AT 250 WORDS)