EPR spin-trapping study of nitric oxide formation during bilateral carotid occlusion in the rat

The formation of nitric oxide (NO) radicals was demonstrated by electron paramagnetic resonance spectroscopy in the rat during varying degrees of brain ischemia. Diethyldithiocarbamate and Fe-citrate were used as in vivo spin-trapping reagents. The signal of NO spin adducts increased in accordance with the degree of ischemic insults. The formation of NO radicals was inhibited by N^G-nitro]l-arginine methyl ester.     

Ventilatory response to sustained hypoxia in carotid body denervated rats

Hypoxia stimulates ventilation, but when it is sustained, a decline in the ventilatory response is seen. The mechanism responsible for this decline lies within the CNS, but still remains unknown. In this study, we attempted to elucidate the possible role of hypoxia-induced depression of respiratory neurons by comparing the ventilatory response to hypoxia in intact rats and those with denervated carotid bodies. A whole-body plethysmograph was used to measure tidal volume, frequency of breathing and minute ventilation (VE) in awake and anesthetized intact rats and rats after carotid body denervation during exposure to hypoxia (FIO2 0.1). Fifteen-minute hypoxia induced an initial increase of VE in intact rats (to 248% of control ventilation in awake and to 227% in anesthetized rats) followed by a consistent decline (to 207% and 196% of control VE, respectively). Rats with denervated carotid bodies responded with a smaller increase in VE (to 134% in awake and 114% in anesthetized animals), but without a secondary decline (145% and 129% of control VE in the 15th min of hypoxia). These results suggest that afferentation from the carotid bodies and/or the substantial increase in ventilation are crucial for the biphasicity of the ventilatory response to sustained hypoxia and that a central hypoxic depression cannot fully explain the secondary decline in VE.     

Intermittent hypoxia augments carotid body and ventilatory response to hypoxia in neonatal rat pups.

Carotid bodies are functionally immature at birth and exhibit poor sensitivity to hypoxia. Previous studies have shown that continuous hypoxia at birth impairs hypoxic sensing at the carotid body. Intermittent hypoxia (IH) is more frequently experienced in neonatal life. Previous studies on adult animals have shown that IH facilitates hypoxic sensing at the carotid bodies. On the basis of these studies, in the present study we tested the hypothesis that neonatal IH facilitates hypoxic sensing of the carotid body and augments ventilatory response to hypoxia. Experiments were performed on 2-day-old rat pups that were exposed to 16 h of IH soon after the birth. The IH paradigm consisted of 15 s of 5% O2 (nadir) followed by 5 min of 21% O2 (9 episodes/h). In one group of experiments (IH and control, n = 6 pups each), sensory activity was recorded from ex vivo carotid bodies, and in the other (IH and control, n = 7 pups each) ventilation was monitored in unanesthetized pups by plethysmography. In control pups, sensory response of the carotid body was weak and was slow in onset (approximately 100 s). In contrast, carotid body sensory response to hypoxia was greater and the time course of the response was faster (approximately 30 s) in IH compared with control pups. The magnitude of the hypoxic ventilatory response was greater in IH compared with control pups, whereas changes in O2 consumption and CO2 production during hypoxia were comparable between both groups. The magnitude of ventilatory stimulation by hyperoxic hypercapnia (7% CO2-balance O2), however, was the same between both groups of pups. These results demonstrate that neonatal IH facilitates carotid body sensory response to hypoxia and augments hypoxic ventilatory chemoreflex.     

Disulfiram augments oxidative stress in rat brain following bilateral carotid artery occlusion

We examined the brain oxidative stress which accompanies 30 min of bilateral carotid artery ligation (BCAL) in terms of changes in brain levels of glutathione; reduced (GSH) and oxidized (GSSG) forms and the exacerbation of oxidative stress by disulfiram (DSF). These results indicate that BCAL alone decreases GSH content and limits glutathione reductase (GR) activity, and these changes were enhanced by DSF pretreatment. Similar observations were recorded with DSF alone. GR activity (74.3±4.0 µmol min^–1 mg^–1 tissue; p<0.001) and GSH content (1.23±0.06 µmol min^–1g^–1 tissue; p<0.001) was attenuated in rats subjected to synergistic effect of BCAL and DSF with a concomitant increase of GSSG (0.006±0.006 µmol min^–1 g^–1 tissue; p<0.001). Recovery of GSH/GSSG level and GR activity during reperfusion following 30 min BCAL was considerably delayed (96 h) in the BCAL and DSF group as compared to the recovery time of 24 h in the group subjected to BCAL-reperfusion alone. Perturbation of GSH/GSSG homeostasis as a result of BCAL was augmented by DSF. These findings clearly demonstrate central nervous system oxidative stress due to a BCAL-DSF synergistic effect. Based on the results obtained with this model, we conclude that DSF increases brain oxidative stress and this may be detrimental to alcoholics who might drink and develop an acetaldehyde-induced hypotension while taking DSF.     

Enhanced pressor response to carotid occlusion in commNTS-lesioned rats: possible efferent mechanisms

Bilateral common carotid occlusion (BCO) over a period of 60 s in conscious rats produces a biphasic pressor response, consisting of an early (peak) and late (plateau) phase. In this study we investigated 1) the effects of lesions of the commissural nucleus of the solitary tract (commNTS) on the cardiovascular responses produced by BCO in conscious rats and 2) the autonomic and humoral mechanisms activated to produce the pressor response to BCO in sham- and commNTS-lesioned rats. Both the peak and plateau of the pressor response produced by BCO increased in commNTS-lesioned rats despite the impairment of chemoreflex responses induced by intravenous potassium cyanide. In sham rats sympathetic blockade with intravenous prazosin and metoprolol, but not vasopressin receptor blockade with the Manning compound, reduced both components of BCO. In commNTS-lesioned rats the sympathetic blockade or vasopressin receptor blockade reduced both components of BCO. The results showed 1) the sympathetic nervous system, but not vasopressin, is important for the pressor response to BCO during 60 s in conscious sham rats; 2) in commNTS-lesioned rats, despite chemoreflex impairment, BCO produces an increased pressor response dependent on sympathetic activity associated with vasopressin release; and 3) the increment in the pressor response to BCO in commNTS-lesioned rats seems to depend only on vasopressin secretion.     

Dihydroceramide-based response to hypoxia

To understand the mechanisms of ceramide-based responses to hypoxia, we performed a mass spectrometry-based survey of ceramide species elicited by a wide range of hypoxic conditions (0.2-5% oxygen). We describe a rapid, time-dependent, marked up-regulation of dihydroceramides (DHCs) in mammalian cells and in the lungs of hypoxic rats. The increase affected all DHC species and was proportional with the depth and duration of hypoxia, ranging from 2- (1 h) to 10-fold (24 h), with complete return to normal after 1 h of reoxygenation at the expense of increased ceramides. We demonstrate that a DHC-based response to hypoxia occurs in a hypoxia-inducible factor-independent fashion and is catalyzed by the DHC desaturase (DEGS) in the de novo ceramide pathway. Both the impact of hypoxia on DHC molecular species and its inhibitory effect on cell proliferation were reproduced by knockdown of DEGS1 or DEGS2 by siRNA during normoxia. Conversely, overexpression of DEGS1 or DEGS2 attenuated the DHC accumulation and increased cell proliferation during hypoxia. Based on the amplitude and kinetics of DHC accumulation, the enzymatic desaturation of DHCs fulfills the criteria of an oxygen sensor across physiological hypoxic conditions, regulating the balance between biologically active components of ceramide metabolism.     

Predominant postglomerular vascular resistance response to reflex renal sympathetic nerve activation during ANG II clamp in rabbits

We have shown previously that a moderate reflex increase in renal sympathetic nerve activity (RSNA) elevated glomerular capillary pressure, whereas a more severe increase in RSNA decreased glomerular capillary pressure. This suggested that the nerves innervating the glomerular afferent and efferent arterioles could be selectively activated, allowing differential control of glomerular capillary pressure. A caveat to this conclusion was that intrarenal actions of neurally stimulated ANG II might have contributed to the increase in postglomerular resistance. This has now been investigated. Anesthetized rabbits were prepared for renal micropuncture and RSNA recording. One group (ANG II clamp) received an infusion of an angiotensin-converting enzyme inhibitor (enalaprilat, 2 mg/kg bolus plus 2 mg.kg(-1).h(-1)) plus ANG II ( approximately 20 ng.kg(-1).min(-1)), the other vehicle. Measurements were made before (room air) and during 14% O(2). Renal blood flow decreased less during ANG II clamp compared with vehicle [9 +/- 1% vs. 20 +/- 4%, interaction term (P(GT)) < 0.05], despite a similar increase in RSNA in response to 14% O(2) in the two groups. Arterial pressure and glomerular filtration rate were unaffected by 14% O(2) in both groups. Glomerular capillary pressure increased from 33 +/- 1 to 37 +/- 1 mmHg during ANG II clamp and from 33 +/- 2 to 35 +/- 1 mmHg in the vehicle group before and during 14% O(2), respectively (P(GT) < 0.05). During ANG II clamp, postglomerular vascular resistance was still increased in response to RSNA during 14% O(2), demonstrating that the action of the renal nerves on the postglomerular vasculature was independent of the renin-angiotensin system. This further supports our hypothesis that increases in RSNA can selectively control pre- and postglomerular vascular resistance and therefore glomerular ultrafiltration.