Chronic hyperhomocysteinemia impairs vascular function in ovariectomized rat carotid arteries

Homocysteine is an independent risk factor for coronary heart disease, as well as for cerebrovascular and peripheral vascular diseases. The purpose of this study was to investigate the effects of hyperhomocysteinemia (HHcy) on vascular reactivity within carotid artery segments isolated from ovariectomized female rats. Treatment with dl-Hcy thiolactone (1 g/kg body weight per day) reduced the phenylephrine-induced contraction of denuded rings. However, the treatment did not alter KCl-induced contractions, or relaxations induced by sodium nitroprusside or acetylcholine. We report elevated expressions of iNOS, eNOS, and nitrotyrosine in homocysteine-treated rat artery sections. Moreover, the inhibition of NOS by l-NAME, 1,400 W, or l-NNA restored phenylephrine-induced vasoconstriction in carotid artery segments from Hcy-treated rats. In conclusion, our findings show that severe HHCy can promote an acute decrease in the endothelium-independent contractile responses of carotid arteries to adrenergic agonists. This effect was restored by nitric oxide synthase inhibitors, which further supports the involvement of nitric oxide in HHcy-derived vascular dysfunction.     

Chronic hypoxia-induced alterations in mitochondrial energy metabolism are not reversible in rat heart ventricles

Chronic hypoxia alters mitochondrial energy metabolism. In the heart, oxidative capacity of both ventricles is decreased after 3 weeks of chronic hypoxia. The aim of this study was to evaluate the reversal of these metabolic changes upon normoxia recovery. Rats were exposed to a hypobaric environment for 3 weeks and then subjected to a normoxic environment for 3 weeks (normoxia-recovery group) and compared with rats maintained in a normoxic environment (control group). Mitochondrial energy metabolism was differentially examined in both left and right ventricles. Oxidative capacity (oxygen consumption and ATP synthesis) was measured in saponin-skinned fibers. Activities of mitochondrial respiratory chain complexes and antioxidant enzymes were measured on ventricle homogenates. Morphometric analysis of mitochondria was performed on electron micrographs. In normoxia-recovery rats, oxidative capacities of right ventricles were decreased in the presence of glutamate or palmitoyl carnitine as substrates. In contrast, oxidation of palmitoyl carnitine was maintained in the left ventricle. Enzyme activities of complexes III and IV were significantly decreased in both ventricles. These functional alterations were associated with a decrease in numerical density and an increase in size of mitochondria. Finally, in the normoxia-recovery group, the antioxidant enzyme activities (catalase and glutathione peroxidase) increased. In conclusion, alterations of mitochondrial energy metabolism induced by chronic hypoxia are not totally reversible. Reactive oxygen species could be involved and should be investigated under such conditions, since they may represent a therapeutic target.     

Effect of the endothelin receptor antagonist bosentan on chronic hypoxia-induced morphological and physiological changes in rat carotid body

Previous experiments have repeatedly demonstrated that exposure to chronic hypoxia (CH) elicits remarkable structural changes and chemosensory hypersensitivity in the mammalian carotid body. Moreover, recent studies have shown that CH upregulates the neuroactive peptide, endothelin (ET), in oxygen-sensitive type I cells. The present study examines the possible involvement of ET in adaptation by concurrently exposing rats to hypobaric CH (B(P) = 380 Torr) and bosentan, a potent nonpeptide antagonist that blocks ET(A) and ET(B) receptors. Carotid body weight indicated that 14 days of CH induced organ enlargement, a response that was blunted in bosentan-treated rats (CH: 2.54 +/- 0.19-fold increase; CH plus bosentan: 1.92 +/- 0.14-fold increase; P < 0.05). Morphometric studies revealed that bosentan substantially eliminated CH-induced hyperplasia of chemosensory cell lobules as well as expansion of the connective tissue matrix. Vascular dilation associated with CH was not altered by the drug. In untreated animals exposed to 3 days of CH, expression of proliferating cell nuclear antigen (PCNA), a marker of mitosis, was increased in lobules of oxygen-sensitive type I cells and in extralobular vascular and connective tissue cells. The incidence of PCNA expression was significantly (P < 0.05) reduced in bosentan-treated animals. In vitro assessments of carotid sinus nerve (CSN) activity showed that enhancement of basal and hypoxia-evoked chemosensory activity following 9 days of CH was significantly (P < 0.001) blunted by concurrent treatment with bosentan. Collectively, our data are consistent with the hypothesis that CH-induced adaptation in the carotid body is at least partially mediated by signaling pathways involving ET receptors.     

Chronic intermittent hypoxia alters NMDA and AMPA-evoked currents in NTS neurons receiving carotid body chemoreceptor inputs

Chronic exposure to intermittent hypoxia (CIH) has been used in animals to mimic the arterial hypoxemia that accompanies sleep apnea. Humans with sleep apnea and animals exposed to CIH have elevated blood pressures and augmented sympathetic nervous system responses to acute exposures to hypoxia. To test the hypothesis that exposure to CIH alters neurons within the nucleus of the solitary tract (NTS) that integrate arterial chemoreceptor afferent inputs, we measured whole cell currents induced by activation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and N-methyl-D-aspartate (NMDA) receptors in enzymatically dispersed NTS neurons from normoxic (NORM) and CIH-exposed rats (alternating cycles of 3 min at 10% O2 followed by 3 min at 21% O2 between 8 AM and 4 PM for 7 days). To identify NTS neurons receiving carotid body afferent inputs the anterograde tracer 4- (4-(dihexadecylamino)styryl-N-methylpyridinum iodide (DiA) was placed onto the carotid body 1 wk before exposure to CIH. AMPA dose-response curves had similar EC50 but maximal responses increased in neurons isolated from DiA-labeled CIH (20.1 +/- 0.8 microM, n = 9) compared with NORM (6.0 +/- 0.3 microM, n = 8) rats. NMDA dose-response curves also had similar EC50 but maximal responses decreased in CIH (8.4 +/- 0.4 microM, n = 8) compared with NORM (19.4 +/- 0.6 microM, n = 9) rats. These results suggest reciprocal changes in the number and/or conductance characteristics of AMPA and NMDA receptors. Enhanced responses to AMPA receptor activation could contribute to enhanced chemoreflex responses observed in animals exposed to CIH and humans with sleep apnea.     

Enhanced glycogen synthase kinase-3beta activity mediates hypoxia-induced apoptosis of vascular smooth muscle cells and is prevented by glucose transport and metabolism

Hypoxia triggers apoptosis in a number of different cell types largely through a mitochondrial cell death pathway, which can be abrogated for the most part by enhanced glucose metabolism. The purpose of the current study was to identify intracellular signaling mechanisms that mediate hypoxia-induced apoptosis and are regulated by glucose metabolism. Hypoxia-induced apoptosis in vascular smooth muscle cells and COS-7 cells was accompanied by a significant reduction in Akt and glycogen synthase kinase-3 (GSK-3) phosphorylation resulting in increased GSK-3 activity. Morphologic features of apoptosis, as well as caspases 3 and 9 activation, were prevented by GSK-3 inhibition with either LiCl or SB216763. Phosphorylation of Akt and GSK-3 was enhanced by glucose metabolism or overexpression of the glucose transporter, GLUT1, and was prevented by glycolytic inhibition. These findings indicate that GSK-3 is an important mediator of hypoxia-induced apoptosis and that GSK-3-mediated apoptotic effects occur via activation of the mitochondrial death pathway. Moreover, the results suggest that prevention of hypoxia-mediated apoptosis by enhanced glucose transport and metabolism results, in part, from inhibition of GSK-3 activation.     

Chronic intermittent stress-induced alterations in the spermatogenesis and antioxidant status of the testis are irreversible in albino rat

The study was undertaken to find out whether or not chronic stress-induced alterations in spermatogenesis are accompanied by oxidative damage in the testis and reversibility of these effects. Adult male rats (n?=?10) were subjected to restraint for 1 h and later after a gap of 4 h to forced swimming exercise for 15 min daily for 60 days and controls (n?=?5) were maintained without disturbance. After treatment period, controls and 5 rats in stress group were killed and remaining rats in stress group were maintained without any treatment for 4 months and then autopsied to find out whether effects are reversible or not. The body and testicular weight, total sperm count, and mean number of type A spermatogonia, mid-pachytene spermatocytes, stage 7 spermatids, and elongated spermatids (cellular association in stage VII of spermatogenesis) showed a significant decrease whereas the abnormal sperm count and germ cell apoptosis were increased in stressed and recovery group rats compared to controls. Activities of testicular SOD, CAT, GPx, and GST were significantly decreased whereas MDA levels were significantly increased in stressed rats compared to controls. The SOD, GST, and CAT activities of recovery groups were significantly lower than controls, whereas MDA levels and GPx activity of these rats did not differ from controls. The results, for the first time, reveal that stress-induced loss of germ cells leading to decrease in sperm count may be due to oxidative damage caused by chronic stress and majority of these changes are not reversible.     

Reperfusion-induced arrhythmias are not prevented by ibuprofen in isolated rat heart

Free radicals have been implicated in the genesis of reperfusion-induced arrhythmias and the cyclooxygenase pathways has been suggested as a potential source. We have therefore assessed whether a cyclooxygenase inhibitor, ibuprofen, is able to reduce reperfusion-induced injury in the isolated perfused rat heart. A duration of 10 min of regional ischemia, which resulted in a high (83%) incidence of ventricular fibrillation, was selected and hearts (n = 12/group) were perfused with ibuprofen (2, 20, or 30 mg/L) throughout the experiment. Ibuprofen did not affect heart rate, although it did produce a dose-dependent increase in coronary flow. However, at all doses studied, ibuprofen had no effect upon the time to onset, incidence, or duration of arrhythmias. In subsequent studies with 30 min of regional ischemia, ibuprofen (30 mg/L) again caused vasodilatation but without effect upon heart rate or severity of arrhythmias. In conclusion, we were unable to obtain evidence in support of the concept that cyclooxygenase activity or cyclooxygenase-derived free radicals are involved in the genesis of ischemia- and reperfusion-induced arrhythmias.     

Thiol oxidation-induced embryonic cell death in mice is prevented by the antioxidant dithiothreitol.

The oxidation of cellular sulfhydryl (SH) groups has been implicated in the induction of apoptosis in various types of cells and in the disturbance of the meiotic spindle of murine oocytes during aging. The objective of this study was to determine whether the SH-specific oxidant diamide could inhibit embryo development and induce cell death, and whether the antioxidant dithiothreitol (DTT) could counteract such effects. Exposure of mouse zygotes to diamide for 3 h at 25 or 50 microM (but not 12.5 microM) resulted in cell cycle arrest and cell death with evidence of apoptosis. At higher concentrations (100 or 200 microM), diamide induced necrosis as evidenced by propidium iodide-positive pronuclei within 24 h of treatment. Simultaneous addition of DTT at equimolar concentration prevented these effects. However, when DTT was added later, it was no longer protective. DTT also effectively protected against the thiol-oxidative damage caused by diamide in blastocysts. These results suggest that altering thiol-redox status in zygotes and blastocysts may result in cell cycle arrest, apoptosis, and/or cell death.     

Exposure to cyclic intermittent hypoxia increases expression of functional NMDA receptors in the rat carotid body

Although large quantities of glutamate are found in the carotid body, to date this excitatory neurotransmitter has not been assigned a role in chemoreception. To examine the possibility that glutamate and its N-methyl-d-aspartate (NMDA) receptors play a role in acclimatization after exposure to cyclic intermittent hypoxia (CIH), we exposed male Sprague-Dawley rats to cyclic hypoxia or to room air sham (Sham) for 8 h/day for 3 wk. Using RT-PCR, Western blot analysis, and immunohistochemistry, we found that ionotropic NMDA receptors, including NMDAR1, NMDAR2A, NMDAR2A/2B, are strongly expressed in the carotid body and colocalize with tyrosine hydroxylase in glomus cells. CIH exposure enhanced the expression of NMDAR1 and NMDAR2A/2B but did not substantially change the level of NMDAR2A. We assessed in vivo carotid sinus nerve activity (CSNA) at baseline, in response to acute hypoxia, in response to infused NMDA, and in response to infused endothelin-1 (ET-1) with and without MK-801, an NMDA receptor blocker. Infusion of NMDA augmented CSNA in CIH rats (124.61 +/- 2.64% of baseline) but not in sham-exposed rats. Administration of MK-801 did not alter baseline activity or response to acute hypoxia, in either CIH or sham animals but did reduce the effect of ET-1 infusion on CSNA (CSNA after ET-1 = 160.96 +/- 8.05% of baseline; ET-1 after MK-801 = 118.56 +/- 9.12%). We conclude that 3-wk CIH exposure increases expression of NMDA functional receptors in rats, suggesting glutamate and its receptors may play a role in hypoxic acclimatization to CIH.     

Reperfusion-induced arrhythmias are not prevented by uric acid in the isolated rat heart

Oxygen-derived free radicals have been implicated in ventricular arrhythmogenesis during coronary reperfusion following an acute ischemic event. We have investigated the possibility that uric acid, a potentially important physiological antioxidant (inhibits lipid peroxidation and scavenges various radical species during oxidation to allantoin), or oxonic acid (inhibitor of uricase enzyme), are able to prevent reperfusion-induced ventricular dysrhythmias in isolated buffer-perfused rat hearts. Rat hearts (n = 12/group) underwent 15 minutes occlusion; arrhythmias were monitored during ischemia and for 10 minutes of reperfusion. There was no difference in the incidence of ventricular fibrillation or ventricular tachycardia in either uric acid or oxonic acid treated hearts compared to untreated controls. Mean duration of ventricular fibrillation appeared to be reduced in hearts treated with 10(-3) and 10(-4) M oxonic acid compared to controls but these data did not achieve a level of statistical significance. These results demonstrate that uric acid and oxonic acid failed to prevent reperfusion-mediated ventricular dysrhythmias in this experimental preparation. Although oxygen-derived free radicals may contribute to the initiation of either ischemia- or reperfusion-induced arrhythmogenesis, our findings provide little support for this hypothesis.     

Nitric oxide synthase in the rat carotid body and carotid sinus

The participation of nitric oxide synthase (NOS) in the innervation of the rat carotid body and carotid sinus was investigated by means of NADPH-diaphorase histochemistry and NOS immunohistochemistry using antisera raised against purified neuronal NOS and a synthetic tridecapeptide. NOS was detected in 23% of neurons at the periphery of the carotid bodies. Some negative neurons were surrounded by NOS-positive terminals. NOS-containing varicose nerve fibres innervated the arterial vascular bed and, to a lesser extent, the islands of glomus cells. These fibres persisted after transection of the carotid sinus nerve and are probably derived from intrinsic neurons. Large NOS-positive axonal swellings in the wall of the carotid sinus were absent after transection of the sinus nerve, indicating their sensory origin. The results suggest a neuronal nitrergic control of blood flow, neuronal activity and chemoreception in the carotid body, and an intrinsic role of NO in the process of arterial baroreception.     

Comparative analysis of neonatal and adult rat carotid body responses to chronic intermittent hypoxia.

Previous studies suggest that carotid body responses to long-term changes in environmental oxygen differ between neonates and adults. In the present study we tested the hypothesis that the effects of chronic intermittent hypoxia (CIH) on the carotid body differ between neonates and adult rats. Experiments were performed on neonatal (1-10 days) and adult (6-8 wk) males exposed either to CIH (9 episodes/h; 8 h/day) or to normoxia. Sensory activity was recorded from ex vivo carotid bodies. CIH augmented the hypoxic sensory response (HSR) in both groups. The magnitude of CIH-evoked hypoxic sensitization was significantly greater in neonates than in adults. Seventy-two episodes of CIH were sufficient to evoke hypoxic sensitization in neonates, whereas as many as 720 CIH episodes were required in adults, suggesting that neonatal carotid bodies are more sensitive to CIH than adult carotid bodies. CIH-induced hypoxic sensitization was reversed in adult rats after reexposure to 10 days of normoxia, whereas the effects of neonatal CIH persisted into adult life (2 mo). Acute intermittent hypoxia (IH) evoked sensory long-term facilitation of the carotid body activity (sensory LTF, i.e., increased baseline neural activity following acute IH) in CIH-exposed adults but not in neonates. The effects of CIH were associated with hyperplasia of glomus cells in neonatal but not in adult carotid bodies. These observations demonstrate that responses to CIH differ between neonates and adults with regard to the magnitude of sensitization of HSR, susceptibility to CIH, induction of sensory LTF, reversibility of the responses, and morphological remodeling of the chemoreceptor tissue.     

Chronic hypoxia upregulates connexin43 expression in rat carotid body and petrosal ganglion.

Recent studies have demonstrated that oxygen-sensitive type I cells in the carotid body express the gap junction-forming protein connexin43 (Cx43). In the present study, we examined the hypothesis that chronic exposure to hypoxia increases Cx43 expression in type I cells as well as in chemoafferent neurons in the petrosal ganglion. Immunocytochemical studies in tissues from normal rats revealed diffuse and granular Cx43-like immunoreactivity in the cytoplasm of type I cells and dense punctate spots of immunoreactive product at the margins of type I cells and near the borders of chemosensory cell lobules. Cx43-like immunoreactivity was not detectable in petrosal ganglion neurons from normal animals. After a 2-wk exposure to hypobaric (380 Torr) hypoxia, Cx43 immunostaining was substantially enhanced in and around type I cells. Moreover, chronic hypoxia elicited the expression of Cx43-like immunoreactivity in the cytoplasm of afferent neurons throughout the petrosal ganglion. Quantitative RT-PCR studies indicate that chronic hypoxia evokes a substantial increase in Cx43 mRNA levels in the carotid body, along with a marked elevation of Cx43 expression in the petrosal ganglion. Increased Cx43 expression and gap junction formation in type I cells and sensory neurons may contribute to carotid body adaptation during sustained stimulation in extreme physiological conditions.     

Chronic hypoxia-induced spontaneous and rhythmic contractions in the rat main pulmonary artery

The effect of chronic hypoxia (CH; 1-4 wk) on the electromechanical properties of the rat main pulmonary artery (MPA) was investigated. MPA rings obtained from rats exposed for 14 days to hypobaric (50.5 kPa) CH exhibited spontaneous and rhythmic contractions (SRCs) that were never observed in control (normoxic) rats. SRCs were unaffected by tetrodotoxin, phentolamine, BQ-123 and BQ-788, N-nitro-L-arginine methyl ester, or endothelium removal. CH depolarized smooth muscle cells from -58.8 +/- 9 to -38.6 +/- 5.4 mV and increased the resting cytosolic Ca2+ concentration from 67.3 +/- 11.9 to 112.5 +/- 16.4 nM. CH also induced spontaneous spikelike depolarizations. All of these effects were inhibited by external Ca2+ removal or nifedipine (1 microM). Moreover, depletion of intracellular Ca2+ stores with ryanodine (1-5 microM) or cyclopiazonic acid (3 microM) progressively attenuated SRCs. This study demonstrates that CH switches the MPA from a quiescent to a spontaneously active mechanical state. Finally, the fact that SRCs precede the development of right ventricle hypertrophy and disappear when this hypertrophy reaches a maximal value (after 3-4 wk of CH) suggests that SRCs may play a role in the adaptive process of the pulmonary circulation to CH.     

Neurodegeneration in mnd2 mutant mice is not prevented by parkin transgene

Parkinson’s disease (PD) is a common neurodegenerative disorder. The motor neuron degeneration 2 mutant (mnd2) mouse is considered to be an animal model of PD, and exhibits striatal neuron loss, severe muscle wasting, weight loss and death before 40 days of age. We found for the first time that parkin expression was decreased in the mnd2 mouse brain. Since parkin is a crucial protein for PD, the neurodegenerative disorder in mnd2 mice may be caused by parkin protein loss. We therefore examined whether compensation of parkin protein prevents neurodegenerative disorders in mnd2 mice by generating parkin-transgenic (parkin-Tg) mnd2 mice. However, both parkin-Tg mnd2 mice and mnd2 mice were smaller than wild type mice. In muscle strength and survival rate, parkin-Tg mnd2 mice showed similar values to mnd2 mice. Our data suggest that repression of parkin protein does not play a major role in neurodegeneration of mnd2 mice and administration of parkin protein does not rescue mnd2 mice.