Characterization of 3-nitropropionic acid-induced bradycardia in isolated atria.

3-Nitropropionic acid (3-NPA) was identified as the toxic component of several plants and fungi. Recently, it was described that 3-NPA produced hypotension, accompanied by a paradoxical bradycardia. In this study, we identified a possible mechanism of action, explaining the effect of 3-NPA on cardiac tissue. We used isolated, spontaneously beating atria and heart mitochondria to measure electrophysiological properties and mitochondrial oxygen consumption. We also measured Na+/K+ ATPase activity and intracellular ATP levels. In isolated spontaneously beating atria, 3-NPA (10(-4) M) decreased heart rate by 62 +/- 3%. This agent did not affect the amplitude or duration of action potentials. The duration of intervals between two action potentials, however, was prolonged from 530 +/- 285 to 1400 +/- 600 ms after 3-NPA exposure (10(-2) M). Oxygen consumption by heart mitochondria was inhibited by 3-NPA when either malate/glutamate or succinate were used as metabolism substrates. Cytochrome C oxidase activity was not affected by 3-NPA. Finally, atrial ATP content decreased 65 +/- 3% after 3-NPA treatment. In conclusion, we show that 3-NPA decreases atrial rate by increasing the action potential phase 4, probably by inhibition of mitochondrial respiration, thereby decreasing cardiac ATP content. This suggests that 3-NPA-induced bradycardia may be related to intracellular ATP depletion.

Angiotensin II induces TNF production by the thick ascending limb: functional implications.

The effects of angiotensin II (ANG II) on tumor necrosis factor-alpha (TNF) production were determined in freshly isolated tubules from the medullary thick ascending limb (MTAL). ANG II (10(-9) M) increased the accumulation of TNF mRNA associated with enhanced production of TNF by approximately five- to sixfold. ANG II also increased prostaglandin E2 (PGE2) production by the MTAL in a dose-dependent manner and exerted biphasic differential effects on 86Rb uptake, depending on the exposure time of the tubules to the peptide and the doses used. Low-dose ANG II (10(-11) M) increased 86Rb uptake by MTAL tubules after a "short-term" (15 min) challenge, whereas uptake was inhibited after a "long-term" (3 h) incubation period. High-dose ANG II (10(-6) M) inhibited MTAL 86Rb uptake, irrespective of incubation time. Uptake of 86Rb was inhibited by approximately 60% in MTAL tubules that were challenged for 3 h with ANG II. The inhibitory action of ANG II was prevented by eliminating the participation of either TNF with antisera to the cytokine or PGE2 by inhibition of cyclooxygenase with indomethacin. We conclude that ANG II regulates TNF production in the MTAL, an interaction that affects 86Rb uptake via an eicosanoid-dependent mechanism in this nephron segment.

The renal medullary thick ascending limb as a model for understanding lipid mediators in sepsis.

Cytokines are an essential component in those mechanisms that depress renal function in response to inflammatory diseases of the kidney. We have addressed the role of tumor necrosis factor (TNF) in mediating changes in renal function by isolating a nephron segment, the medullary thick ascending limb of Henle's loop (mTAL), and studying the effects of TNF on ion transport via changes in the activity of two epoxygenases, cyclooxygenase (COX) and cytochrome P450 (CYP450) monooxygenase. This nephron segment generates 20-hydroxyeicosatetraenoic acid (20-HETE) as a principal arachidonate metabolite. However, when challenged with lipopolysaccharide (LPS), the mTAL expresses a considerable capacity to metabolize arachidonic acid (AA) via a COX pathway to form PGE2.

TNF production by the medullary thick ascending limb of Henle's loop.

Medullary thick ascending limb of Henle's loop (mTALH) tubules, isolated from kidneys of male Sprague-Dawley rats, expressed the gene for tumor necrosis factor (TNF) and released this cytokine when challenged with lipopolysaccharide (LPS). The TNF produced was biologically active, as determined by cytotoxic activity present in supernatants from LPS-stimulated mTALH, using the TNF-sensitive murine fibrosarcoma cell line, WEHI 164. The amount of TNF produced, approximately 75 nM, has previously been shown to affect ion transport in the mTALH. The TNF-mediated cytotoxicity (and ion transport effects) were completely neutralized with a polyclonal anti-TNF antisera. Further, immunoprecipitation experiments demonstrated that the 17 kDa TNF monomer was formed by de novo protein synthesis. In contrast, the mTALH did not produce the related cytokine, lymphotoxin (LT). Production of TNF was confirmed by demonstrating the accumulation of a 1.6 kb TNF mRNA by Northern blot analysis; mRNA for LT was not detected. Expression of the TNF gene in the mTALH was confirmed by the polymerase chain reaction (PCR). Southern blot analysis and ethidium bromide staining of the resultant PCR products revealed the expected 276 bp sequence of TNF DNA for the mTALH. We have demonstrated that mTALH tubules stimulated with LPS express the gene for TNF, but not LT, and release biologically active TNF. TNF is an important mediator of septic shock and may contribute to changes in renal function associated with endotoxemia. Production of TNF by the mTALH may be an important autocrine regulatory mechanism for this nephron segment.

Cytokines affect ion transport in primary cultured thick ascending limb of Henle's loop cells.

Tumor necrosis factor-alpha (TNF) and interleukin-1 (IL-1) affect epithelial cell ion transport. However, the site of action along the nephron has not been elucidated fully for these cytokines. Thus, the effect of TNF and IL-1 on the ion transport function of primary cultured medullary thick ascending limb of Henle's loop (mTALH) cells was determined by measuring rubidium (86Rb) uptake. TNF, IL-1, and lipopolysaccharide (LPS), a known activator of cytokine production, inhibited 86Rb uptake by cultured mTALH cells after a 24-h incubation period but had no effect when incubated with the cells for 1 or 4 h. Furthermore, mTALH cells produced biologically active TNF after stimulation with LPS for 24 h, and the LPS-induced inhibition of 86Rb uptake was abolished in the presence of an anti-TNF antibody, suggesting that TNF produced by the mTALH cells acted in an autocrine manner to inhibit 86Rb uptake. The effects of LPS on 86Rb uptake also were inhibited by the cyclooxygenase inhibitor, indomethacin. As TNF increased prostaglandin E2 synthesis by cultured mTALH cells and as prostaglandin E2 also inhibited 86Rb uptake, LPS presumably inhibited 86Rb uptake by inducing a TNF-mediated increase in prostaglandin synthesis. These data demonstrate that a prostanoid produced by mTALH cells mediates the inhibitory effect of LPS and TNF on 86Rb uptake and imply that endogenous TNF inhibits ion fluxes in the mTALH via a prostaglandin-dependent mechanism.

Renal cytochrome P-450-dependent metabolism of arachidonic acid in cirrhotic rats.

Cirrhosis was induced in Wistar-Kyoto rats by intragastric administration of carbon tetrachloride. Microsomes were obtained from the renal cortex and outer medulla and incubated with [14C]arachidonic acid (AA) (0.2-0.4 microCi) in the presence or absence of indomethacin, NADPH, and SKF-525A. Cytochrome P-450-dependent AA metabolites (those whose formation required NADPH, were inhibited by SKF-525A, but not by indomethacin) were separated by thin-layer chromatography and high-pressure liquid chromatography (HPLC). Compared to controls, total synthesis of cytochrome P-450-dependent AA metabolites was reduced in cirrhotic rats (renal cortex: cirrhotics 380 +/- 52 vs. controls 493 +/- 68 pg/mg protein per 30 min; p less than 0.05; renal outer medulla: cirrhotics 304 +/- 57 vs. controls 387 +/- 53 pg/mg protein per 30 min; p less than 0.05). The cytochrome P-450-dependent AA metabolites were composed of three peaks separated by HPLC. Peak I, which had a retention time of 16.3 +/- 0.3 min and comigrated with 11,12-dihydroxyeicosatrienoic acid, and peak II, which had a retention time of 18.7 +/- 0.4 min and comigrated with 19- and 20-hydroxyeicosatetraenoic acid, were not different in cirrhotics and controls. Peak III, which had a retention time of 26.8 +/- 0.3 min, and comigrated with 11,12-epoxyeicosatrienoic acid, was significantly decreased in the renal cortex of cirrhotic rats compared to controls (cirrhotics 316 +/- 40 vs. controls 473 +/- 89 pg/mg protein per 30 min; p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of synthesis inhibitors of thromboxane A2 and prostaglandin E2 on the regulation of sodium and water.

The aim of this study was to evaluate the response to water deprivation in rats during development in the presence and in the absence of either benzylimidazole or acetaminophen (10 mg/kg of each), inhibitors of the synthesis of thromboxanes and other arachidonic acid metabolites. Whereas water deprivation induced an increment in urine osmolality both in control and vehicle-treated animals, this response was blocked by benzylimidazole and acetaminophen in the 5-day-old rats. No effect was observed in the older rats. Benzylimidazole also increased sodium excretion in the newborn rat. To assess the effect of benzylimidazole on epithelial cells deprived of the influence of hemodynamic factors, sodium transport and water flow were studied in the frog skin and in the toad bladder, respectively. In the frog skin epithelium, benzylimidazole (10(-4) M) inhibited the vasopressin-stimulated (100 mU/ml) sodium transport and in the toad bladder it decreased the vasopressin-stimulated (5 mU/ml) hydroosmotic flow. Our results suggest that thromboxanes are necessary for a full development of the response to vasopressin in sensitive epithelia. In the rat, thromboxanes and other arachidonic acid metabolites appear to play a role in the neonate but not in the older animal.

Development of the receptors to prostaglandin E2 in the rat kidney and neonatal renal functions.

Inhibition of prostaglandins (PGs) synthesis induces responses on renal function that are age-dependent. Indomethacin in the adult rat potentiates the response to vasopressin whereas in the newborn rat blocks the response to dehydration. These differences suggest that the sensitivity of the renal tissue to PGs changes as the animal matures. This study was designed to analyze the characteristics of the receptors to PGE2 in the developing rat and to compare with that of the adult rat. Binding was performed in cortical and medullary slices incubated with tritiated PGE2. It was found that the renal uptake of the radioactive ligand had three components, the first is the synthesis of endogenous prostaglandins, the second is tubular secretion (only in cortex) and the third is binding to receptor sites. We found it necessary to eliminate the first two components (with indomethacin and p-amino-hippurate) to achieve an adequate measurement of the apparent affinity of the binding sites to PGE2. Under these conditions the cortex bound more PGE2 than the medulla (at all ages) and the highest affinity was observed in the neonatal cortex. Synthesis of prostaglandin E2 was higher in newborn than in adult rats. In vivo, indomethacin and acetaminophen blocked the response of the newborn rat to dehydration whereas in the weaning and adult rat the response was not changed by these inhibitors of the synthesis of prostaglandins. Our results suggest a major role of the prostaglandins in the regulation of the water balance in the neonate.