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Although urea is considered to be a cell stressor even in renal medullary cells perpetually exposed to this solute in vivo by virtue of the renal concentrating mechanism, aspects of urea signaling resemble that of a peptide mitogen. Urea was compared with epidermal growth factor and hypertonic NaCl or hypertonic mannitol using a large-scale expression array-based approach. The expression profile in response to urea stress more closely resembled that of EGF treatment than hypertonic stress, as determined by hierarchical cluster analysis; the effect of urea+NaCl was equidistant from that of either solute applied individually. Among the most highly urea-and hypertonicity-responsive transcripts were genes that had previously been shown to be responsive to these solutes, validating this approach. Increased expression of the activating transcription factor 3 by urea was newly detected via expression array and confirmed via immunoblot analysis. Earlier, we noted an abrogation of tonicity-dependent gene regulation by urea, primarily in a transient transfection-based model (Tian W and Cohen DM. Am J Physiol Renal Physiol 280: F904-F912, 2001). Here we applied K-means cluster analysis to demonstrate that the genes most profoundly up-or downregulated by hypertonic stress were partially restored toward basal levels in the presence of urea pretreatment. These global expression data are consistent with our earlier biochemical studies suggesting that urea affords cytoprotection in this context. In the aggregate, these data strongly support the hypothesis that the urea effect in renal medullary cells resembles that of a peptide mitogen in terms of the adaptive program of gene expression and in terms of cytoprotection from hypertonicity. expression array; tonicity enhancer binding protein; hypertonicity; cytoprotection; activating transcription factor 3; mIMCD3 cells; epidermal growth factor

We have used the patch-clamp technique to study the effect of arachidonic acid (AA) on the basolateral K channels in the medullary thick ascending limb (mTAL) of rat kidney. An inwardly rectifying 50-pS K channel was identified in cell-attached and inside-out patches in the basolateral membrane of the mTAL. The channel open probability ([P.sub.o]) was 0.51 at the spontaneous cell membrane potential and decreased to 0.25 by 30 mV hyperpolarization. The addition of 5 [micro]M AA decreased channel activity, identified as N[P.sub.o], from 0.58 to 0.08 in cell-attached patches. The effect of AA on the 50-pS K channel was specific because 10 [micro]M cis-11,14,17-eicosatrienoic acid had no significant effect on channel activity. To determine whether the effect of AA was mediated by AA per se or by its metabolites, we examined the effect of AA on channel activity in the presence of indomethacin, an inhibitor of cyclooxygenase, or N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), an inhibitor of cytochrome P-450 monooxygenase. Inhibition of cyclooxygenase increased channel activity from 0.54 to 0.9. However, indomethacin did not abolish the inhibitory effect of AA on the 50-pS K channel. In contrast, inhibition of cytochrome P-450 metabolism not only increased channel activity from 0.49 to 0.83 but also completely abolished the effect of AA. Moreover, addition of DDMS can reverse the inhibitory effect of AA on channel activity. The notion that the effect of AA was mediated by cytochrome P-450-dependent metabolites of AA is also supported by the observation that addition of 100 nM of 20-hydroxyeicosatetraenoic acid, a main metabolite of AA in the mTAL, can mimic the effect of AA. We conclude that AA inhibits the 50-pS K channel in the basolateral membrane of the toTAL and that the effect of AA is mainly mediated by cytochrome P-450-dependent metabolites of AA. cyclooxygenase; cytochrome P-450 [omega]-oxidation; 20-hydroxyeicosatetraenoic acid; basolateral K channel

The present studies were performed to determine the contribution of E[P.sub.2] receptors to renal hemodynamics by examining afferent arteriolar responses to PG[E.sub.2], butaprost, sulprostone, and endothelin-1 in E[P.sub.2] receptor-deficient male mice (E[P.sub.2]-/-). Afferent arteriolar diameters averaged 17.8 [+ or -] 0.8 [micro]m in wild-type (E[P.sub.2]+/+) mice and 16.7 [+ or -] 0.7 [micro]m in E[P.sub.2]-/- mice at a renal perfusion pressure of 100 mmHg. Vessels from both groups of mice responded to norepinephrine (0.5 [micro]M) with similar 17-19% decreases in diameter. Diameters of norepinephrine-preconstricted afferent arterioles increased by 7 [+ or -] 2 and 20 [+ or -] 6% in E[P.sub.2]+/+ mice in response to 1 [micro]M PG[E.sub.2] and 1 [micro]M butaprost, respectively. In contrast, afferent arteriolar diameter of E[P.sub.2]-/- mice decreased by 13 [+ or -] 3 and 16 [+ or -] 6% in response to PG[E.sub.2] and butaprost. The afferent arteriolar vasoconstriction to butaprost in E[P.sub.2]-/- mice was eliminated by angiotensin-converting enzyme inhibition. Sulprostone, an E[P.sub.1] and E[P.sub.3] receptor ligand, decreased afferent arteriolar diameter in both groups; however, the vasoconstriction in the E[P.sub.2]-/- mice was greater than in the E[P.sub.2]+/+ mice. Endothelin-1-mediated afferent arteriolar diameter responses were enhanced in E[P.sub.2]-/- mice. Afferent arteriolar diameter decreased by 29 [+ or -] 7% in E[P.sub.2]-/- and 12 [+ or -] 7% in E[P.sub.2]+/+ mice after administration of I nM endothelin-1. These results demonstrate that the E[P.sub.2] receptor mediates a portion of the PG[E.sub.2] afferent arteriolar vasodilation and buffers the renal vasoconstrictor responses elicited by E[P.sub.1] and E[P.sub.3] receptor activation as well as endothelin-1. prostaglandins; endothelin; kidney; microcirculation

Abnormal heart rate characteristics of reduced variability and transient decelerations are present early in the course of neonatal sepsis. To investigate the dynamics, we calculated sample entropy, a similar but less biased measure than the popular approximate entropy. Both calculate the probability that epochs of window length m that are similar within a tolerance r remain similar at the next point. We studied 89 consecutive admissions to a tertiary care neonatal intensive care unit, among whom there were 21 episodes of sepsis, and we performed numerical simulations. We addressed the fundamental issues of optimal selection of m and r and the impact of missing data. The major findings are that entropy falls before clinical signs of neonatal sepsis and that missing points are well tolerated. The major mechanism, surprisingly, is unrelated to the regularity of the data: entropy estimates inevitably fall in any record with spikes. We propose more informed selection of parameters and reexamination of studies where approximate entropy was interpreted solely as a regularity measure. approximate entropy; newborn infant; sepsis

Resting [Ca.sup.2+] absorption by cortical thick ascending limbs (CALs) is passive and proceeds through the paracellular pathway. In contrast, parathyroid hormone (PTH) stimulates active, transcellular [Ca.sup.2+] absorption ([J.sub.Ca]). The [Ca.sup.2+]-sensing receptor (CaSR) is expressed on serosal membranes of CALs. In the present study, we tested the hypothesis that activation of the CAL CaSR indirectly inhibits passive [Ca.sup.2+] transport and directly suppresses PTH-induced cellular [J.sub.Ca]. To test this theory, we measured [J.sub.Ca] and Na absorption ([J.sub.Na]) by single perfused mouse CALs. Net absorption was measured microfluorimetrically in samples collected from tubules perfused and bathed in symmetrical HEPES-buffered solutions or those in which luminal [Na.sup.+] was reduced from 150 to 50 mM. We first confirmed that [Gd.sup.3+] activated the CaSR by measuring intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) in CALs loaded with fura 2. On stepwise addition of [Gd.sup.3+] to the bath, [[[Ca.sup.2+]].sub.i] increased, with a half-maximal rise at 30 [micro]M [Gd.sup.3+] * [J.sub.Ca] and transepithelial voltage ([V.sub.e,]) were measured in symmetrical [Na.sup.+]-containing solutions. PTH increased [J.sub.Ca] by 100%, and 30 [micro]M [Gd.sup.3+] inhibited this effect. [V.sub.e] was unchanged by either PTH or [Gd.sup.3+]. Similarly, NPS R-467, an organic CaSR agonist, inhibited PTH-stimulated [J.sub.Ca] without altering [V.sub.e]. Neither PTH nor [Gd.sup.3+] affected [J.sub.Ca]. Addition of bumetanide to the luminal perfusate abolished [J.sub.Na] and [V.sub.e]. These results show that CaSR activation directly inhibited PTH-induced transcellular [J.sub.Ca] and that cellular [Ca.sup.2+] and [Na.sup.+] transport can be dissociated. To test the effect of CaSR activation on passive paracellular [Ca.sup.2+] transport, [J.sub.Ca] was measured under asymmetrical Na conditions, in which passive [Ca.sup.2+] transport dominates transepithelial absorption. PTH stimulated [J.sub.Ca] by 24% and was suppressed by [Gd.sup.3+]. In this setting, [Gd.sup.3+] reduced [V.sub.e] by 32%, indicating that CaSR activation inhibited both transcellular and paracellular [Ca.sup.2+] transport. We conclude that the CaSR regulates both active transcellular and passive paracellular [Ca.sup.2+] reabsorption but has no effect on [J.sub.Na] by CALs. calcium transport; thick ascending limbs; kidney; parathyroid hormone; transepithelial voltage

Ammonium and potassium chloride share a common taste quality and an amiloride-insensitive route of transduction. An amiloride-sensitive pathway might also be partially activated by these salts, although very few studies have reported effects of amiloride on nonsodium salt perception. This experiment was designed to determine 1) whether rats could discriminate KCl from N[H.sub.4]Cl and, if discrimination was evident, whether performance was impaired with 2) amiloride or 3) gustatory nerve transection. Rats were trained to discriminate KCl from N[H.sub.4]Cl (n = 8) and NaCl from N[H.sub.4]Cl (n = 8). Amiloride (100 [micro]M) impaired NaCl vs. N[H.sub.4]Cl but not KCl vs. N[H.sub.4]Cl performance, whereas both groups showed significant impairments after transection of the chorda tympani (CT) and greater superficial petrosal (GSP) branches of the facial nerve. This suggests that rats can discriminate between KCl and N[H.sub.4]Cl and that this discrimination does not rely on an amiloride-sensitive mechanism but does depend on the CT and/or GSP nerves. This experiment supports the hypothesis that the facial nerve is important for salt taste recognition and discrimination. taste; salt; amiloride; greater superficial petrosal; chorda tympani

This study evaluated possible neuronal mechanisms responsible for the transition from normal breathing (eupnea) to gasping. We hypothesized that a blockade of both inhibitory glycinergic synaptic transmission and potassium channels, combined with an increase in extracellular concentration of potassium, would induce a switch from an eupneic respiratory pattern to gasping. Efferent activities of the phrenic, vagal, and hypoglossal nerves were recorded during eupnea and ischemia-induced gasping in a perfused in situ preparation of the juvenile rat (4-6 wk of age). To block potassium channels, 4-aminopyridine (4-AP, 1-10 [micro]M) was administered. Strychnine (0.2-0.6 [micro]M) was used to block glycinergic neurotransmission. After administrations of 4-AP, excess extracellular potassium (10.25-17.25 mM), and strychnine, the incrementing pattern of eupneic phrenic activity was altered to a decrementing discharge. Hypoglossal and vagal activities became concentrated to the period of the phrenic burst with expiratory activity being reduced or eliminated. These changes in neural activities were similar to those in ischemia-induced gasping. Results are consistent with the concept that the elicitation of gasping represents a switch from a network-based rhythmogenesis for eupnea to a pacemaker-driven mechanism. neural control of breathing; respiratory pattern generation; blockade of inhibition; ionic channels; hypoxia

The roles of oxidative stress and renal superoxide dismutase (SOD) levels and their association with renal damage were studied in Dahl salt-sensitive (S) and salt-resistant (R)/Rapp strain rats during changes in Na intake. After 3 wk of a high (8%)-Na diet in S rats, renal medullary Cu/Zn SOD was 56% lower and Mn SOD was 81% lower than in R high Na-fed rats. After 1, 2, and 3 wk of high Na, urinary excretion of [F.sub.2]-isoprostanes, an index of oxidative stress, was significantly greater in S rats compared with R rats. Plasma [F.sub.2]-isoprostane concentration increased in the 2-wk S high Na-fed group. After 3 wk, renal cortical and medullary superoxide production was significantly increased in Dahl S rats on high Na intake, and urinary protein excretion, an index of renal damage, was 273 [+ or -] 32 mg/d in S high Na-fed rats and 35 [+ or -] 4 mg/d in R high Na-fed rats (P < 0.05). In conclusion, salt-sensitive hypertension in the S rat is accompanied by marked decreases in renal medullary SOD and greater renal oxidative stress and renal damage than in R rats. urinary protein excretion; isoprostane; dietary sodium; renal damage

The cytochrome P-450 eicosanoid 20-hydroxyeicosatetraenoic acid (20-HETE) is a potent vasoconstrictor that is implicated in the regulation of blood pressure. The identification of selective inhibitors of renal 20-HETE formation for use in vivo would facilitate studies to determine the systemic effects of this eicosanoid. We characterized the acetylenic fatty acid sodium 10-undecynyl sulfate (10-SUYS) as a potent and selective mechanism-based inhibitor of renal 20-HETE formation. A single dose of 10-SUYS caused an acute reduction in mean arterial blood pressure in 8-wk-old spontaneously hypertensive rats. The decrease in mean arterial pressure was maximal 6 h after 10-SUYS treatment (17.9 [+ or -] 3.2 mmHg; P < 0.05), and blood pressure returned to baseline levels within 24 h after treatment. Treatment with 10-SUYS was associated with a decrease in urinary 20-HETE formation in vivo and attenuation of the vasoconstrictor response of renal interlobar arteries to ANG II in vitro. These results provide further evidence that 20-HETE plays an important role in the regulation of blood pressure in the spontaneously hypertensive rat. spontaneously hypertensive rat; cytochrome P-450; sodium 10-undecynyl sulfate; 20-hydroxyeicosatetraenoic acid

We compared the effect of hyperammonemia on NADH levels in brain slices and on the rate of oxygen consumption from isolated nonsynaptic brain mitochondria in ammonia-sensitive Wistar rats with that in ammonia-tolerant gulf toadfish (Opsanus beta). The NADH content was significantly decreased (12% less than control after 45 min with 1 mM N[H.sub.4]Cl) in rat brain slices, but it was not affected in brain slices from toadfish (with both 1 and 6 mM N[H.sub.4]C1). The rates of oxygen consumption of different sets of enzymes of the electron transport chain (ETC; complexes I, II, III, and IV; II, III, and IV; and IV alone) were unaltered by hyperammonemic conditions in isolated nonsynaptic mitochondria from either rats or toadfish. These results lead us to conclude that the differing effects of ammonia on NADH levels in rat and toadfish brain slices must be due to aspects other than the direct effects of ammonia on enzymes of the ETC. Additionally, because these effects were seen in vitro, our studies enabled us to rule out the possibility that effects of ammonia on metabolism were via indirect systemic effects. These results are discussed in the context of current views on mechanisms of central nervous system damage in hyperammonemic states. hepatic encephalopathy; hyperammonemia; NADH; mitochondria; Opsanus beta; glutamine metabolism

It has been proposed that fetal adaptations to intrauterine nutrient deprivation permanently reprogram the cardiovascular system. We investigated the impact of restricted periconceptional nutrition and/or restricted gestational nutrition on fetal arterial blood pressure (BP), heart rate, rate pressure product, and the fetal BP responses to ANG II and the angiotensin-converting enzyme inhibitor captopril during late gestation. Restricted periconceptional nutrition resulted in an increase in fetal mean arterial BP between 115 and 125 days gestation (restricted 41.5 [+ or -] 2.8 mmHg, n = 12; control 38.5 [+ or -] 1.5 mmHg, n = 13) and between 135 and 147 days gestation (restricted 50.5 [+ or -] 2.2 mmHg, n = 8; control 42.5 [+ or -] 1.9 mmHg, n = 10) as well as an increase in the rate pressure product in twin, but not singleton, fetuses between 115 and 147 days gestation. Mean BP and fetal plasma ACTH were also positively correlated in twin, but not singleton, fetuses. This is the first demonstration that maternal undernutrition during the periconceptional period results in an increase in fetal arterial BP. This increase occurs concomitantly with an increase in fetal ACTH but is not dependent on activation of the fetal renin-angiotensin system. fetus; blood pressure; renin-angiotensin system

To characterize and study the variations of IGF-I binding during the development of trout muscle cells, in vitro experiments were conducted using myocyte cultures, and IGF-I binding assays were performed in three stages of cell development: mononuclear cells (day 1), small myotubes (day 4), and large myotubes (day 10). Binding experiments were done by incubating cells with IGF-I for 12 h at 4[degrees]C. Specific IGF-I binding increased with the concentration of labeled IGF-I and reached a plateau at 32 pM. The displacement of cold human and trout IGF-I showed a very similar curve (E[C.sub.50] = 1.19 [+ or -] 0.05 and 0.95 [+ or -] 0.05 nM, respectively). IGF binding proteins did not interfere significantly because displacement of labeled IGF-I by either cold trout recombinant IGF-I or Des (1-3) IGF-I resulted in similar curves. Insulin did not displace labeled IGF-I even at very high concentrations (>1 [micro]M), which indicates the specificity of IGF-I binding. The amount of receptor ([R.sub.0]) increased from 253 [+ or -] 51 fmol/mg DNA on day I to 766 [+ or -] 107 fmol/mg DNA on day 10. However, the affinity ([K.sub.d]) of IGF-I receptors did not change significantly during this development (from 1.29 [+ or -] 0.19 to 0.79 [+ or -] 0.13 nM). On the basis of our results, we conclude that rainbow trout muscle cells in culture express specific IGF-I receptors, which increase their number with development from mononuclear cells to large myotubes. insulin-like growth factor-I; receptor

The existence of endothelin binding sites on the catecholaminergic neurons of the hypothalamus suggests that endothelins (ETs) participate in the regulation of noradrenergic transmission modulating various hypothalamic-controlled processes such as blood pressure, cardiovascular activity, etc. The effects of ET-1 and ET-3 on the neuronal release of norepinephrine (NE) as well as the receptors and intracellular pathway involved were studied in the rat anterior hypothalamus. ET-1 (10 nM) and ET-3 (10 nM) diminished neuronal NE release and the effect blocked by the selective ET type B receptor antagonist BQ-788 (100 nM). [N.sup.[omega]]-nitro-L-arginine methyl ester (10 [micro]M), methylene blue (10 [micro]M), and KT5823 (2 [micro]M), inhibitors of nitric oxide synthase activity, guanylate cyclase, and protein kinase G, respectively, prevented the inhibitory effects of both ETs on neuronal NE release. In addition, both ETs increased nitric oxide synthase activity. Furthermore, 100 [micro]M picrotoxin, a GAB[A.sub.A]-receptor antagonist, inhibited ET-1 and ET-3 response. Our results show that ET-1 as well as ET-3 has an inhibitory neuromodulatory effect on NE release in the anterior hypothalamus mediated by the ET type B receptor and the involvement of a nitric oxide-dependent pathway and GAB[A.sub.A] receptors. ET-1 and ET-3 may thus diminish available NE in the synaptic gap leading to decreased noradrenergic activity. endothelin receptor; soluble guanylyl cyclase; BQ-610; BQ-788; GAB[A.sub.A] receptor, nitric oxide, norepinephrine

Several conditions that inhibit female sexual behavior are thought to be associated with altered corticotropin-releasing hormone (CRH) activity in the brain. The present experiments examined the hypothesis that endogenous CRH receptor signaling mediates the inhibition of estrous behavior by undernutrition and in other instances of sexual dysfunction. Intracerebroventricular (ICV) infusion of CRH or urocortin inhibited estrous behavior in ovariectomized steroid-primed hamsters. Conversely, ICV infusion of the CRH receptor antagonist astressin prevented the suppression of estrous behavior by food deprivation or by ICV administration of neuropeptide Y. Astressin treatment also induced sexual receptivity in nonresponders, animals that do not normally come into heat when treated with hormones, and this effect persisted in subsequent weekly tests in the absence of any further astressin treatment. Activation of the hypothalamo-pituitary-adrenocortical axis was neither necessary nor sufficient to inhibit estrous behavior, indicating that this phenomenon is due to other central actions of CRH receptor agonists. This is the first direct evidence that CRH receptor signaling may be a final common pathway by which undernutrition and other conditions inhibit female sexual behavior. urocortin; astressin; neuropeptide Y; nutritional infertility; estrous behavior; cortisol; food intake

Dexamethasone (DM) was administered to pregnant ewes as three weekly courses of four injections of 2 mg at 12-h intervals. DM (n = 7) or saline (n = 7) was given starting at 103 days of gestation (dGA; term ~149 days). Fetal femoral arteries (~300-[micro]m internal diameter) were evaluated using wire myography at 119 dGA. DM-exposed fetuses were significantly smaller than saline-exposed fetuses. DM exposure increased maximal contraction to 125 mM KCl, and maximum tension developed along with sensitivity to endothelin-1 and relaxation to bradykinin. Preincubation with the nitric oxide synthase inhibitor [N.sup.G]-nitro-L-arginine methyl ester shifted the dose-response curves to endothelin-1 and acetylcholine to the right in controls but not in the DM-exposed group. Relaxation to acetylcholine and to the nitric oxide donor sodium nitroprusside was similar in both groups. The combination of enhanced endothelin-induced vasoconstriction, abnormal endothelium-dependent relaxation, and normal endothelium-independent relaxation indicates microvessel dysfunction following antenatal DM administration. Because such dysfunction is associated with several forms of adult hypertension, our results indicate the potential for consequences of antenatal glucocorticoid exposure on adult cardiovascular health. wire myography; endothelin-1; nitric oxide; [N.sup.G]-nitro-L-arginine methyl ester; bradykinin; acetylcholine

There is a close relationship between inflammatory bowel disease (IBD) and various hepatobiliary disorders. The objective of this study was to determine whether hepatic leukocyte recruitment occurs in experimental colitis. We used the murine model of colitis induced by 2,4-dinitrobenezenesulfonic acid (DNBS). Male C57Bl/6 mice received an intrarectal injection of 4 mg DNBS in 100 [micro]l 50% ethanol. Controls received 100 [micro]l 50% ethanol. The hepatic microcirculation was examined at 3 and 14 days post-DNBS by intravital video microscopy. Three days post-DNBS, when mice had developed acute colitis, there was associated hepatic leukocyte recruitment. Within the postsinusoidal venules there was a fourfold increase in the flux of rolling leukocytes that was P-selectin dependent but not [[alpha].sub.4]-integrin dependent. There was also an increase in stationary leukocytes within the sinusoids, although this was not associated with an increase in serum alanine transaminase. By 14 days post-DNBS when macroscopic evidence of colonic inflammation was resolved, rolling within the postsinusoidal venules had returned to control levels. In this murine model of colitis, we describe a link between acute colonic inflammation and remote hepatic leukocyte recruitment that is P-selectin dependent. Active IBD may lead to remote hepatic inflammation. inflammatory bowel disease; intravital microscopy; microcirculation; P-selectin; integrins

In inflamed colonic mucosa, the equilibrium between absorptive and secretory functions for electrolyte and salt transport is disturbed. We compared the expression of three major mediators of the intestinal salt transport between healthy and inflamed colonic mucosa to understand the pathophysiology of diarrhea in inflammatory bowel disease. Expression levels of the cystic fibrosis transmembrane regulator (CFTR) ([Cl.sup.-] channel), SLC26A3 ([Cl.sup.-]/HC[O.sup.-.sub.3] exchanger) and SLC9A3 ([Na.sup.+]/[H.sup.+] exchanger) mRNAs were measured by real-time quantitative RT-PCR in peroperative colonic samples from controls (n = 4) and patients with ulcerative colitis (n = 10). Several samples were obtained from each individual. Tissue samples were divided into three subgroups according to their histological degree of inflammation. Expression of CFTR and SLC26A3 proteins were determined by immunohistochemistry and Western blotting from the same samples, respectively. Increased expression of CFTR mRNA was observed in all three groups of affected tissue samples, most pronounced in mildly inflamed colonic mucosa (5-fold increase in expression; P < 0.001). The expression of the CFTR protein was detected from health and inflamed colon tissue. Although the expression of the SLC26A3 mRNA was significantly decreased in severe ulcerative colitis (P < 0.05), the SLC26A3 protein levels remained unchanged in all groups. The expression of SLC9A3 mRNA was significantly changed between the mild and severe groups. Intestinal inflammation modulates the expression of three major mediators of intestinal salt transport and may contribute to diarrhea in ulcerative colitis both by increasing transepithelial [Cl.sup.-] secretion and by inhibiting the epithelial NaCl absorption. inflammation; anion transport; diarrhea; inflammatory bowel disease; cystic fibrosis transmembrane regulator

Activation of poly(ADP-ribose) polymerase (PARP) mediates oxidative stress-induced cell injury. We tested the hypothesis that PARP contributes to ischemia-reperfusion (I/R) damage of the liver by triggering the mechanisms of microcirculatory failure. Leukocyte- and platelet-endothelial cell interactions as well as sinusoidal perfusion were analyzed by intravital fluorescence microscopy after lobar hepatic I/R (90 min/30 min) in C57BL/6 X 129/Sv wild-type (PARP+/+) and PARP-deficient (PARP-/-) mice. Hepatic I/R induced leukocyte/platelet-endothelial cell interactions and tissue injury in PARP+/+ mice, as indicated by impaired sinusoidal perfusion and increased alanine aminotransferase (ALT)/aspartate aminotransferase (AST) serum activities. In PARP-/- mice, however, the postischemic increase in the numbers of rolling/adherent leukocytes and platelets was significantly lower. In addition, I/R-induced translocation of CD62P as well as mRNA expression of CD62E, CD54, and CD106 were attenuated. The degree of perfusion failure was reduced and the increase in the ALT/AST activities was lower in PARP-/- mice compared with PARP+/+ mice. We conclude that PARP contributes to hepatic microvascular injury by triggering the expression/translocation of adhesion molecules and modulating leukocyte/platelet-endothelial cell interactions. liver; microcirculation; leukocytes; platelets; adhesion molecules

Mitochondria in saponin-skinned cardiac fiber bundles were reported to have an order of magnitude lower apparent affinity to ADP than isolated mitochondria. Although ADP was measured outside the bundles, it was thought that the low affinity was not caused by diffusion gradients because of relatively short diffusion distances. Here we test the hypothesis that considerable ADP diffusion gradients exist and can be diminished by increasing the intrafiber ADP production rate. We increased the ADP-producing activity in rat heart skinned fiber bundles by incubating with 100 IU/ml yeast hexokinase and glucose. Consequently, we observed a significant decrease of the apparent Michaelis constant ([K.sub.m]) to ADP of the respiration rate of bundles from 216 [+ or -] 59 to 50 [+ or -] 9 [micro]M. Fitting the results with a mathematical model, we estimated the [K.sub.m] of mitochondria in the bundles to be 25 [micro]M. We conclude that the affinity to ADP of in situ mitochondria in heart is of the same order of magnitude as that of isolated mitochondria. mitochondria; heart; soleus; adenosine triphosphatase; diffusion; Michaelis constant; adenosine 5'-triphosphate

Experiments were carried out to study in vitro the effects of 17[beta]-estradiol ([E.sub.2]), homologous pituitary homogenate (HPH), and recombinant red sea bream growth hormone (sbGH) on vitellogenin (VTG) secretion from cultured sea bream liver fragments. Basal secretion of VTG was found to be significantly higher in the prespawning period, compared with sea bream liver in the spawning and postspawning periods. Similarly, the sea bream liver obtained during the prespawning period responded more significantly to treatments with [E.sub.2], HPH, or sbGH compared with sea bream liver during spawning. In the postspawning period, treatments with [E.sub.2], HPH, or sbGH were without significant effect on VTG secretion level in sea bream liver. The level of [E.sub.2] receptors was also analyzed by Western blot analysis. The result demonstrates a significantly higher level of [E.sub.2] receptors in the sea bream liver at the prespawning stage compared with those at the spawning and postspawning stages. The findings support the hypothesis that homologous upregulation of estrogen receptors plays an important role in the estrogen-sensitive control of VTG synthesis in the sea bream liver. vitellogenin induction; hormones; estradiol receptor

Evaluation of ovarian POMC mRNA through quantitative RT-PCR analysis in Rana esculenta. Am J Physiol Cell Physiol 280: C1038-C1044, 2001.--The evaluation of changes in the expression of specific genes requires accurate measurement of the corresponding mRNA concentration, especially when the gene is expressed at a very low level. We previously showed that the proopiomelanocortin (POMC) gene is expressed in the ovary of the frog Rana esculenta, and, to evaluate its mRNA content in frog ovary, we have now developed a sensitive quantitative RT-PCR method. This study provides evidence for the validation of this method and for the effects of captivity and hypophysectomy on POMC gene expression in the ovary of this anuran. Our data indicate that ovarian POMC gene is involved in short-term captivity stress response and seems not influenced by pituitary. These results are discussed taking into account the knowledge of the role played by opioids in stress response; moreover, a local control of POMC gene expression is also suggested. frog; ovary; proopiomelanocortin gene expression; captivity; hypophysectomy