Role of cytosolic calcium balance on cell injury in cultured bovine aortic endothelial cells during hypoxia and reoxygenation]

The integration of pharmacological therapies for comorbid disorders requires an acceptance of independence and interactions of respective addictive and psychiatric disorders. At the same time, alcohol and other drugs induce psychiatric states that are indistinguishable from psychiatric disorders. On the other hand, while psychiatric disorders do not induce addictive use of alcohol and drugs, they do pose vulnerabilities to the development of addictive disorders. Generally, the treatment of comorbid disorders begins with abstinence and evaluation of the effects of alcohol and other drugs in contributing to the psychiatric picture. In the case of comorbid disorders, stabilization and standard treatments can be employed with certain cautions, namely, to avoid the use of addicting medications such as benzodiazepines and opiates beyond the detoxification stage. High potency neuroleptics and antidepressants, particularly selective serotonin reuptake inhibitors (SSRIs) can be used to treat continuing psychiatric states after the exclusionary criteria in DSM-IV for substance-related disorders have been applied to the clinical case. If the psychiatric symptoms clear with sustained abstinence, little or no medications may be required. Specific treatment of the addictive disorders will often determine the extent that addictive disorders are responsible for psychiatric symptomatology. Alternatively, treatment of the psychiatric disorder will enhance compliance with addiction treatment.     

Colon epithelial electrical responses to acute hypoxia and reoxygenation in vitro

Electrogenic epithelial transport depends on oxidative metabolism. Acute hypoxia and subsequent reoxygenation effects on short-circuit current (Isc), transepithelial potential difference (PD) and tissue resistivity (TR) of rat distal colon were assessed. The tissue was mounted in an Ussing chamber filled with Ringer-HCO3-solution at 37 degrees C and bubbled with 95% O2- 5% CO2 which was switched to 95% N2- 5% CO2 for inducing hypoxia; afterwards normal oxygenation was resumed. The effect of 5, 10, 15 and 20 min-hypoxic periods was assessed in isolated mucosa preparations. Recovery was complete after 10- and 15-min hypoxia, but not after 20-min hypoxia. After 5-min hypoxia, an overshoot of Isc and PD was seen on reoxygenation. This effect was further characterized comparatively in mucosa-submucosa and isolated mucosa preparations. In the former (n = 10), control values were Isc = 71.7 +/- 8.6 microA. cm-2, PD = 9.7 +/- 1.6 mV and TR = 134.9 +/- 13.6 omega cm2. A 5-min hypoxia reduced Isc by 47.2 +/- 7.3% and PD by 61.5 +/- 4.9%. Peak values on reoxygenation were 28.1 +/- 4.1% for Isc and 16.8 +/- 5.4% for PD, over controls values. In the isolated mucosa (n = 9), control values were Isc = 52.04 +/- 5.5 microA. cm-2, PD = 5.0 +/- 0.8 mV and TR = 101.04 +/- 10.5 omega. cm2. In hypoxia, Isc decreased by 64.5 +/- 7.6% and PD by 57.2 +/- 7.8%. On reoxygenation peak values of 78.0 +/- 19.0% and 87.5 +/- 17.1%, respectively, were seen. The response to a 5 min-hypoxia was comparable, but that to reoxygenation was weaker and slower, in mucosa-submucosa than in isolated mucosa preparations. This may be explained by a hindrance to oxygen diffusion caused by the submucosal tissue. TR did not change with any period of hypoxia tested, but decreased slightly (8.9 +/- 1.3%) upon reoxygenation in the mucosa-submucosa preparations. Ouabain (10(-3) M) markedly blunted the response to reoxygenation. We conclude that hypoxic periods of 20 min lead to irreversible functional deterioration. Hypoxia decreases electrogenic transepithelial pumping, which may allow sodium to accumulate intracellularly and, if the hypoxia is short enough to prevent damage to the epithelium, increase sodium pump activity when oxygenation is resumed.     

Mitochondrial free calcium regulation in hypoxia and reoxygenation: relation to cellular injury

The role of mitochondria in myocardial ischemic and hypoxic injury is discussed. Increases in mitochondrial Ca content and ionized Ca2+ concentration are observed during and after ischemic and hypoxic exposure and have traditionally been considered to impair mitochondrial function. New data are discussed in which it is shown that increases in mitochondrial [Ca2+] do not necessarily reflect irreversible myocyte injury. Further, it is shown that irreversible cellular injury may occur in hypoxic myocytes in association with increases in mitochondrial [Ca2+] that would ordinarily be considered to fall within a physiologic range. The significance of these observations is considered in context with observations relating to the assessment of post-hypoxic mitochondrial function.     

Mitochondrial calcium transporting pathways during hypoxia and reoxygenation in single rat cardiomyocytes

Remarkable advances have been made in the treatment of cancers that afflict patients of the reproductive age. Many survivors must now face the effects on gonadal function and have concerns about reproductive capacity. The sequelae of different modalities of cancer therapy specifically addressing surgery, chemotherapy, and radiotherapy on reproductive system are reviewed. Assisted reproductive technologies, prenatal diagnosis methods, and contraception counseling are briefly summarized in conclusion.     

Ramiprilat attenuates hypoxia/reoxygenation injury to cardiac myocytes via a bradykinin-dependent mechanism

Isolated rat neonatal cardiac myocytes were subjected to immersion in hypoxic (PO2 < 2 mm Hg), glucose-free Tyrode's solution for 5 h followed by concomitant reoxygenation and staining with the membrane-impermeant fluorophore, propidium iodide, in normoxic (PO2 > 150 mm Hg), serum-free culture media for 15 min in order to assess sarcolemmal damage indicative of myocyte viability due to hypoxia/reoxygenation injury. Prior to hypoxic exposure, cells were pretreated for 90 min with the angiotensin-converting enzyme inhibitor cyclopenta[b]pyrrole-2-carboxylic acid, 1-[2-[(1-carboxy-3-phenylpropyl)amino]-l-oxopropyl]octahydro-[2S-[1[R* (R*)]2 alpha, 3a beta, 6a beta]] (ramiprilat), concomitantly with ramiprilat and H-D-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-D-Tic-Oic-Arg-OH (bradykinin B2 receptor antagonist HOE 140), the bioactive peptide Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg (bradykinin) or concomitantly with bradykinin and HOE 140. Hypoxia/reoxygenation injury to untreated control cardiac myocytes was characterized by a significant loss of sarcolemmal integrity measured at 75 +/- 4% of total cell fluorescence (mean +/- S.E., n = 42 cultures). Compared to propidium iodide staining of the above untreated control myocytes, those pretreated with 30 or 100 microM ramiprilat showed a significant reduction of propidium iodide staining to 45 +/- 9% and 40 +/- 8% (n = 9, P < 0.05) of untreated controls, respectively. Pretreatment with the protective concentrations of ramiprilat concomitant with 10 microM HOE 140 abolished the significant reduction in propidium iodide staining observed with ramiprilat alone. Similarly, pretreatment with 10 or 100 nM bradykinin significantly reduced propidium iodide staining to 35 +/- 5% and 60 +/- 10% (n = 6, P < 0.05) of the untreated hypoxic controls, respectively. In addition, concomitant pretreatment with protective concentrations of bradykinin and 10 microM HOE 140 also abolished the significant reduction in propidium iodide staining observed with bradykinin alone. The results indicate that the angiotensin-converting enzyme inhibitor ramiprilat has a protective effect on isolated cardiac myocytes exposed to hypoxia/reoxygenation and that this effect is most likely related to a local action of bradykinin on the cardiac myocyte via the activation of the kinin B2 receptor.     

Nitric oxide dose response during moderate and severe hypoxia in swine

The vectorial nature of redox Bohr effects (redox-linked pK shifts) in cytochrome c oxidase from bovine heart incorporated in liposomes has been analyzed. The Bohr effects linked to oxido-reduction of heme a and CuB display membrane vectorial asymmetry. This provides evidence for involvement of redox Bohr effects in the proton pump of the oxidase.     

Nitric oxide contributes to opioid release from glia during hypoxia

Activation of neuronal nitric oxide (NO) synthase contributes to increased CSF concentrations of the opioids methionine enkephalin and leucine enkephalin during hypoxia in the newborn pig. NO and these opioids, in turn, contribute to hypoxic pial artery dilation. However, the cellular site of origin for opioids detected in CSF cannot be determined using this in vivo model. The present study, therefore, was designed to determine if NO contributes to opioid release from piglet glia grown in primary culture. Glial cell cultures produced more methionine enkephalin than leucine enkephalin under basal conditions. Administration of SNP and 8-Br cGMP to glial cells increased release of both opioids (471+/-58 vs. 1181+/-148 pg/mg protein methionine enkephalin before and after SNP 10-6 M). SNP also increased release of cGMP. Exposure of piglet glial cells to lower than normal O2 increased the release of both opioids (503+/-61 vs. 1488+/-186 pg/mg protein methionine enkephalin before and after hypoxia, (PO2 approximately 15 mmHg). Hypoxia also increased the release of cGMP from glia while the NO synthase inhibitor N-nitro-l-arginine blocked that release. These data show that NO/cGMP and hypoxia release opioids from glia. Additionally, hypoxia releases NO/cGMP from glia. These data therefore suggest that NO contributes to opioid release from glia during hypoxia.     

Inhibition of calcium influx during hypoxia/reoxygenation in primary cultured rat hepatocytes

Calcium has been demonstrated to play an important role in hepatocyte damage during ischemia/reperfusion phases. Calcium influx was determined in primary cultured rat hepatocytes submitted to a succession of warm hypoxia and reoxygenation phases in the presence of diltiazem, gallopamil and a Na+/H+ antiport inhibitor, HOE-694. Only diltiazem significantly inhibited calcium influx with higher potency after reoxygenation than after hypoxia only, suggesting a complex mechanism of action of diltiazem which could act on different physiological functions involved in Ca2+ invasion of hepatocytes after hypoxic insult.     

Cardioprotective effect of angiotensin-converting enzyme inhibition against hypoxia/reoxygenation injury in cultured rat cardiac myocytes

BACKGROUND: Although ACE inhibitors can protect myocardium against ischemia/reperfusion injury, the mechanisms of this effect have not yet been characterized at the cellular level. The present study was designed to examine whether an ACE inhibitor, cilazaprilat, directly protects cardiac myocytes against hypoxia/reoxygenation (H/R) injury. METHODS AND RESULTS: Neonatal rat cardiac myocytes in primary culture were exposed to hypoxia for 5.5 hours and subsequently reoxygenated for 1 hour. Myocyte injury was determined by the release of creatine kinase (CK). Both cilazaprilat and bradykinin significantly inhibited CK release after H/R in a dose-dependent fashion and preserved myocyte ATP content during H/R, whereas CV-11974, an angiotensin II receptor antagonist, and angiotensin II did not. The protective effect of cilazaprilat was significantly inhibited by Hoe 140 (a bradykinin B2 receptor antagonist), NG-monomethyl-L-arginine monoacetate (L-NMMA) (an NO synthase inhibitor), and methylene blue (a soluble guanylate cyclase inhibitor) but not by staurosporine (a protein kinase C inhibitor), aminoguanidine (an inhibitor of inducible NO synthase), or indomethacin (a cyclooxygenase inhibitor). Cilazaprilat significantly enhanced bradykinin production in the culture media of myocytes after 5.5 hours of hypoxia but not in that of nonmyocytes. In addition, cilazaprilat markedly enhanced the cGMP content in myocytes during hypoxia, and this augmentation in cGMP could be blunted by L-NMMA and methylene blue but not by aminoguanidine. CONCLUSIONS: The present study demonstrates that cilazaprilat can directly protect myocytes against H/R injury, primarily as a result of an accumulation of bradykinin and the attendant production of NO induced by constitutive NO synthase in hypoxic myocytes in an autocrine/paracrine fashion. NO modulates guanylate cyclase and cGMP synthesis in myocytes, which may contribute to the preservation of energy metabolism and cardioprotection against H/R injury.     

Nitric oxide regulates cell proliferation during Drosophila development

Cell division and subsequent programmed cell death in imaginal discs of Drosophila larvae determine the final size of organs and structures of the adult fly. We show here that nitric oxide (NO) is involved in controlling the size of body structures during Drosophila development. We have found that NO synthase (NOS) is expressed at high levels in developing imaginal discs. Inhibition of NOS in larvae causes hypertrophy of organs and their segments in adult flies, whereas ectopic expression of NOS in larvae has the opposite effect. Blocking apoptosis in eye imaginal discs unmasks surplus cell proliferation and results in an increase in the number of ommatidia and component cells of individual ommatidia. These results argue that NO acts as an antiproliferative agent during Drosophila development, controlling the balance between cell proliferation and cell differentiation.     

Characterization of an ochratoxin-A-dedifferentiated and cloned renal epithelial cell line

Ochratoxin A (OTA) is a ubiquitous fungal metabolite with predominant nephrotoxic action. OTA impairs postproximal renal electrolyte handling and increases the incidence of renal adenoma and carcinoma. Furthermore, it is supposed to be involved in the pathogenesis of different forms of human renal diseases. Previously we have shown that OTA activates extracellular signal-regulated kinase 1 (ERK1) and ERK2 in the C7 clone but not in the C11 clone of renal epithelial MDCK cells. Here we show that nanomolar concentrations of OTA lead to stable and irreversible phenotypical and genotypical alterations, resulting in sustained dedifferentiation of MDCK-C7 cells but not of MDCK-C11 cells. Dedifferentiated MDCK-C7 cells (OTA-C7 cells) display a distinct morphology from the parent cell line (spindle-shape, pleiomorphic, narrow intercellular spaces, increased cell size) and show a reduced proliferation rate and numerical chromosomal aberrations. Functionally, OTA-C7 cells are characterized by a dramatic reduction of transepithelial electrolyte transport and the complete loss of responsiveness to the mineralocorticoid hormone aldosterone. Our data provide further evidence that OTA can lead to cell dedifferentiation and eventually to transformation of cloned quiescent cells. The changes in phenotype due to this dedifferentiation could explain some of the OTA-induced changes in renal function.     

Epithelial cell shedding in acute renal injury

1. To address the postulate that sublethally injured tubular cells may be shed from renal epithelium while still viable, studies were undertaken in vivo in human 'acute tubular necrosis' and in rabbit models of renal tubular injury. 2. Substantial numbers of viable tubular cells were voided in the urine. When placed in culture, these cells gave rise to monolayers, confirming viability. The majority of intact cells demonstrated markers of proximal tubule. 3. In vitro studies of human renal proximal tubular cells exposed to hypoxia/anoxia showed rounding and retraction associated with disruption of actin microfilaments. Phalloidin stabilized the filaments and prevented the changes in cell shape indicative of altered adherence.     

Activation of ecto-5'-nucleotidase by protein kinase C attenuates irreversible cellular injury due to hypoxia and reoxygenation in rat cardiomyocytes

Adenosine, synthesized by ecto-5'-nucleotidase, is cardioprotective against ischemia and reperfusion injury. We have previously reported that activation of protein kinase C increases ecto-5'-nucleotidase activity of the rat cardiomyocytes, raising the possibility that activation of protein kinase C protects cardiomyocytes from the irreversible cellular injury via activation of ecto-5'-nucleotidase. To test this hypothesis, cardiomyocytes were isolated from adult male Wistar rats and suspended in modified HEPES-Tyrode buffer solution. The cardiomyocytes were incubated with and without exposure to methoxamine (1 x 10(-6) mol/l) or phorbol 12-myristate 13-acetate (PMA. 1 x 10(-8) mol/l). Ecto-5'-nucleotidase activity increased 15 min after the onset of an exposure to either methoxamine or PMA. Adenosine release during hypoxia and reperfusion was augmented in the methoxamine- and PMA-pretreated cardiomyocytes compared with the untreated cardiomyocytes, which was inhibited by alpha, beta-methyleneadenosine 5'-diphosphate (AOPCP), an inhibitor of ecto-5'-nucleotidase. Irreversible cellular injury assessed by the extent of release of lactate dehydrogenase and the trypan blue exclusion test following 60 min of hypoxia and 60 min of reoxygenation was attenuated in the methoxamine- and PMA-pretreated cardiomyocytes compared with the untreated group, which was also blunted by AOPCP and 8-sulfophenyltheophylline, an adenosine receptor antagonist. An adenosine A1 receptor agonist, N6-cyclohexyladenosine, restored the cardioprotection under the treatment with PMA and AOPCP. We conclude that activation of ecto-5'-nucleotidase via protein kinase C contributes to the attenuation of the irreversible injury of the rat cardiomyocytes due to hypoxia and reoxygenation.     

Relationship between glomerular epithelial cell injury and proteinuria in IgA nephropathy

The cost of in-hospital percutaneous transluminal coronary angioplasty (PTCA) has risen since the introduction of the coronary stent. Increased attention is now being given to the PTCA charges in Japan and a multicenter study is necessary with regard to in-hospital charges. To clarify the differences in in-hospital charges for PTCA with and without coronary stent [Stent Group and plain old balloon angioplasty (POBA) Group, respectively], we studied the PTCA charges of 352 patients in 6 hospitals. Age, male gender and extent of coronary artery disease were not different. The ratio of acute myocardial infarction ranged from 16% to 64% and that of coronary stenting ranged from 24% to 65% (p < 0.001). In-hospital charge ranged from 1.4 +/- 0.8 to 2.2 +/- 1.0 million yen (p < 0.0001). The procedural charge accounted for 53% to 75% of the in-hospital charge (p < 0.01). The in-hospital charge ranged from 1.6 +/- 0.7 to 3.3 +/- 1.6 million yen in the Stent Group, higher than the charge of 1.1 +/- 0.8 to 1.9 +/- 0.7 million yen in the POBA Group (p < 0.0001). There was a statistical difference in the number of balloon catheters used (1.1 +/- 0.4 to 2.1 +/- 0.9, p < 0.005) but not in the mean number of stents used (1.1 +/- 0.3 to 1.4 +/- 0.7). The procedural charge of the institutes with higher stenting rate (> 45%) seemed to be lower than that of the institutes with lower stenting rate (p < 0.02). In conclusion, there are large variation between institutions in PTCA charges, and in-hospital charges increased with the use of stents on introduction of the Diagnosis Related Group used in the United States. We should charge separately for coronary stenting and POBA. Despite any initial increase in the in-hospital charge for coronary stenting compared to POBA, successful stent implantation will result in a superior saving in procedural charges.     

Renal hemodynamics and renal O2 uptake during hypoxia in the anesthetized rabbit

The effects of hypoxic hypoxia on renal hemodynamics and metabolism have been studied in anaesthetized mechanically ventilated rabbits. Acute hypoxa (FIO2 = 0.10, PaO2 = 35 torr) induces at constant mean arterial pressure a 45% decrease in RBF, GFR, TNa and RVO2 whereas free water clearance increases. These alterations were still apparent 50 min after resuming normal arterial oxygenation. In order to assess the role of the stimulation of catecholamine release in these observations, two other sets of experiments were performed: 1) the animals were ventilated with the same hypoxic gas mixture but after alpha adrenergic blockade (phentolamine: 0.2 mg - kg - min-1 i.v.), 2) hypoxia was induced by ventilating the animals with CO (FICO = 0.002) at constnat PaO2. Increase in renal vascular resistance and reduction of renal O2 uptake were still observed. This indicates that adrenergic stimulation cannot fully explain the renal vasoconstriction encountered in hypoxia. The role of a local vasoactive factor, especially that of the renin angiotensin system is discussed. The apparent O2 cost of Na reabsorption was not greatly modified by any type of hypoxia and the Na: O2 ratio remained close to the value observed in normoxic animals. This indicates that the kidney may adapt to hypoxia by reducing its O2 demand keeping unaltered its tubular function and basal O2 needs.     

Nitric oxide inhibited peroxyl and alkoxyl radical formation with concomitant protection against oxidant injury in intestinal epithelial cells

Respiratory sinus arrhythmia (RSA) was examined in aerobically trained (AT) and untrained (NT) college-aged males during 12 periods consisting of a 3-min sitting baseline, six common 3-min absolute exercise stages, and five 3-min recovery stages that followed voluntary exhaustion to determine the relationship of work and training status to parasympathetic influence upon the heart. RSA systematically decreased during absolute exercise, was observed at heart rates (HR) above 100 beats x min(-1), and progressively increased during recovery. Additionally, independent of work stages, comparative regression analyses were conducted for both the exercise and recovery phases, separately, in which HR was regressed on RSA, as well as RSA on % VO2max, to contrast the obtained relationships for the AT and NT. No differences were revealed as a function of endurance training status as the slopes and intercepts obtained for the two groups from each of these analyses were similar. The within-subject correlations between RSA and % VO2max, calculated for each of the individuals across all 12 periods, were consistently negative. Between-subjects correlations of RSA with RR and tidal volume were predominantly nonsignificant, indicating that RSA, as measured here, is independent of individual differences in ventilatory activity and, as such, can be compared between groups during exercise. The findings demonstrate that RSA is detectable during both exercise and recovery, even at HR beyond 100 beats x min(-1), and reveals a similar relationship to HR and metabolic state in both aerobically trained and untrained populations.     

Nitric oxide in renal health and disease

Nitric oxide (NO) is a labile radical gas that is widely acclaimed as one of the most important molecules in biology. Through covalent modifications of target proteins and redox reactions with oxygen and superoxide radical and transition metal prosthetic groups, NO plays a critical role in many vital biological processes, including the control of vascular tone, neurotransmission, ventilation, hormone secretion, inflammation, and immunity. Moreover, NO has been shown to influence a host of fundamental cellular functions, such as RNA synthesis, mitochondrial respiration, glycolysis, and iron metabolism. NO is formed from L-arginine by NO synthases (NOSs), a family of related enzymes encoded by separate unlinked genes. The different NOS isozymes exhibit disparate tissue and intrarenal distributions and are governed by unique regulatory mechanisms. In the kidney, NO participates in several vital processes, including the regulation of glomerular and medullary hemodynamics, the tubuloglomerular feedback response, renin release, and the extracellular fluid volume. While NO serves beneficial roles as a messenger and host defense molecule, excessive NO production can be cytotoxic, the result of NO's reaction with reactive oxygen and nitrogen species, leading to peroxynitrite anion formation, protein tyrosine nitration, and hydroxyl radical production. Indeed, NO may contribute to the evolution of several commonly encountered renal diseases, including immune-mediated glomerulonephritis, postischemic renal failure, radiocontrast nephropathy, obstructive nephropathy, and acute and chronic renal allograft rejection. Moreover, impaired NO production has been implicated in the pathogenesis of volume-dependent hypertension. This duality of NO's beneficial and detrimental effects has created extraordinary interest in this molecule and the need for a detailed understanding of NO biosynthesis.