A new 90-day drinking water study of sodium cyanide in rats to further evaluate National Toxicology Program findings and inform risk assessment.

BACKGROUND: The National Toxicology Program (NTP, 1993) reported male reproductive effects in a sodium cyanide (NaCN) drinking water study. The critical effect, decreased cauda epididymis weights, was used by U.S. Environmental Protection Agency for their hazard characterization and risk assessment of hydrogen cyanide and cyanide salts. To further investigate potential male reproductive effects, we conducted a new 90-day drinking water study. METHODS: Our study expanded evaluations of testes and thyroid. Male F344 rats received NaCN in drinking water at 0, 0 (water restricted; paired to top dose), 3, 10, 30, 100, and 300 ppm for 13 weeks, followed by 10-weeks recovery. RESULTS: Plasma thiocyanate increased dose-dependently but returned to baseline during recovery. NaCN caused neither effects on survival, body weight, food consumption, hematology, serum chemistry, urinalysis, thyroid hormones, testes or epididymides weights, sperm motility/viability, sperm morphology, or sperm production; nor clinical, ophthalmic, or histopathologic findings. Increased organ weights in thyroid/parathyroid and liver occurred at 300-ppm but were recoverable. No changes occurred in male reproductive organs. CONCLUSIONS: Absent adverse effects, the NOAEL was 300 ppm (21.66 mg/kg/day; highest dose tested). Based on organ weight increases at 300 ppm, the NOEL was 100 ppm (7.46 mg/kg/day).

Cardiovascular responses to putative chemoreceptor stimulation of embryonic common snapping turtles (Chelydra serpentina) chronically incubated in hypoxia (10% O2).

Developmental hypoxia has been shown to result in significant changes in cardiovascular development of American alligators and common snapping turtles. These include similar effects on cardiac mass and aspects of cardiovascular function. However, given the distant phylogenetic relationship between crocodilians and chelonians, we hypothesized that snapping turtles would also exhibit differences in the effects of developmental hypoxia on cardiovascular regulation. This hypothesis was based in part on prior studies that documented differences in plasticity of vagal tone on the heart between alligators and snapping turtles incubated in hypoxic conditions. To test this hypothesis, we investigated how 10% O2 exposure over final 80% of incubation altered the heart rate and blood pressure response to two chemical manipulations of the "chemoreflex" in common snapping turtles at 70% and 90% of incubation. NaCN injections produced a dose dependent bradycardia that was mediated by cholinergic receptor stimulation. This reflex was relatively unaffected by hypoxic incubation conditions in snapping turtle embryos. Injections of the 5-HT3 agonist phenylbiguanide (PBG) caused a pronounced bradycardia that decreased in intensity at 90% of incubation in embryos from the normoxic group while the heart rate response was unchanged in the hypoxic group. This differs from the previously reported diminished heart rate response of embryonic alligators incubated in 10% O2, suggesting plasticity in this chemoreflex response differs between the species. Our data also indicate the cardiovascular response is mediated by a secondary cholinergic receptor stimulation however the inability of ganglionic blockade to inhibit the PBG response leaves the location of the receptors antagonized by PBG in question in embryonic snapping turtles. Primarily, our findings refute the hypothesis that hypoxic incubation decreases the "chemoreflex' response of snapping turtle embryos.

Plasticity of cardiovascular chemoreflexes after prolonged unilateral carotid body denervation: implications for its therapeutic use.

Unilateral carotid body denervation has been proposed as treatment for sympathetic-related human diseases such as systolic heart failure, hypertension, obstructive sleep apnea, and cardiometabolic diseases. The long-term therapeutic effects of carotid body removal will be maintained if the remnant "buffer nerves," that is, the contralateral carotid nerve and the aortic nerves that innervate second-order neurons at the solitary tract nuclei (NTS), do not modify their contributions to the cardiovascular chemoreflexes. Here, we studied the cardiovascular chemoreflexes 1 mo after unilateral carotid body denervation either by excision of the petrosal ganglion (petrosal ganglionectomy, which eliminates central carotid afferents) or exeresis of a segment of one carotid nerve (carotid neurectomy, which preserves central afferents). Cardiovascular chemoreflexes were induced by intravenous (iv) injections of sodium cyanide in pentobarbitone-anesthetized adult cats. After 1 mo of unilateral petrosal ganglionectomy, without significant changes in basal arterial pressure, the contribution of the contralateral carotid nerve to the chemoreflex increases in arterial pressure was enhanced without changes in the contribution provided by the aortic nerves. By contrast, after 1 mo of unilateral carotid neurectomy, the contribution of remnant buffer nerves to cardiovascular chemoreflexes remained unmodified. These results indicate that a carotid nerve interruption involving denervation of second-order chemosensory neurons at the NTS will trigger cardiovascular chemoreflex plasticity on the contralateral carotid pathway. Then, unilateral carotid body denervation as therapeutic tool should consider the maintenance of the integrity of carotid central chemoafferents to prevent plasticity on remnant buffer nerves.NEW & NOTEWORTHY Unilateral carotid body denervation has been proposed as treatment for sympathetic hyperactivity-related human disorders. Its therapeutic effectiveness for maintaining a persistent decrease in the sympathetic outflow activity will depend on the absence of compensatory chemoreflex plasticity in the remnant carotid and aortic afferents. Here, we suggest that the integrity of central afferents after carotid body denervation is essential to prevent the emergence of plastic functional changes on the contralateral "intact" carotid nerve.

Feasibility of kilohertz frequency alternating current neuromodulation of carotid sinus nerve activity in the pig.

Recent research supports that over-activation of the carotid body plays a key role in metabolic diseases like type 2 diabetes. Supressing carotid body signalling through carotid sinus nerve (CSN) modulation may offer a therapeutic approach for treating such diseases. Here we anatomically and histologically characterised the CSN in the farm pig as a recommended path to translational medicine. We developed an acute in vivo porcine model to assess the application of kilohertz frequency alternating current (KHFAC) to the CSN of evoked chemo-afferent CSN responses. Our results demonstrate the feasibility of this approach in an acute setting, as KHFAC modulation was able to successfully, yet variably, block evoked chemo-afferent responses. The observed variability in blocking response is believed to reflect the complex and diverse anatomy of the porcine CSN, which closely resembles human anatomy, as well as the need for optimisation of electrodes and parameters for a human-sized nerve. Overall, these results demonstrate the feasibility of neuromodulation of the CSN in an anesthetised large animal model, and represent the first steps in driving KHFAC modulation towards clinical translation. Chronic recovery disease models will be required to assess safety and efficacy of this potential therapeutic modality for application in diabetes treatment.

Simple Diffusion as the Mechanism of Okadaic Acid Uptake by the Mussel Digestive Gland.

Okadaic acid (OA) and other toxins of the diarrheic shellfish poisoning (DSP) group are accumulated and transformed mainly in many bivalves, inside the digestive gland cells. In this work the absorption of okadaic acid by those cells has been studied by supplying the toxin dissolved in water and including it in oil droplets given to primary cell cultures, and by checking if the uptake is saturable and/or energy-dependent. Okadaic acid was found to be absorbed preferentially from the dissolved phase, and the uptake from oil droplets was substantially lower. The process did not require energy and was non-saturable, indicating that it involved a simple diffusion across the cellular membrane. Some apparent saturation was found due to the quick biotransformation of OA to 7-O-acyl esters.

Forebrain cellular bioenergetics in neonatal mice.

BACKGROUND: Hypoglycemia occurs frequently in the neonate and may result in neurologic dysfunction. Its impact on the kinetics of cellular respiration and bioenergetics in the neonatal brain remains to be explored. AIMS: Develop murine model to investigate the effects of hypoglycemia on neonatal brain bioenergetics. STUDY DESIGN: Forebrain fragments were excised from euthanized BALB/c pups aged <24 hours to 14 days. We measured cellular respiration (µM O2 min-1.mg-1) in phosphate-buffered saline with and without glucose, using phosphorescence oxygen analyzer, as well as cellular adenosine triphosphate (ATP, nmol.mg-1) using the luciferin-luciferase system. RESULTS: In the presence of glucose, although cellular respiration was 11% lower in pups ≤3 days compared to those 3- 14 days old (0.48 vs. 0.54), that difference was not statistically significant (p = 0.14). Respiration driven by endogenous metabolic fuels (without added glucose) was 16% lower in pups ≤3 days compared to those 3- 14 days (0.35 vs. 0.42, p = 0.03), confirming their increased dependency on exogenous glucose. Although cellular ATP was similar between the two age groups (14.9 vs. 11.2, p = 0.32), the ATP content was more severely depleted without added glucose in the younger pups, especially in the presence of the cytochrome c oxidase inhibitor cyanide. The first-order rate constant of cellular ATP decay (hydrolysis) was 44% lower in 2-day-old pups compared to 14-day-old mice (0.43 vs. 0.77 min-1, p = 0.03). CONCLUSIONS: Forebrain cellular respiration and ATP consumption are lower in young pups than older mice. In the absence of glucose, the support for these processes is reduced in young pups, explaining their brain hypersensitivity to hypoglycemia.

An iminium ion metabolite hampers the production of the pharmacologically active metabolite of a multikinase inhibitor KW-2449 in primates: irreversible inhibition of aldehyde oxidase and covalent binding with endogenous proteins.

We previously reported that KW-2449, (E)-1-{4-[2-(1H-Indazol-3-yl)vinyl]benzoyl}piperazine, a novel multikinase inhibitor developed for the treatment of leukemia patients, was oxidized to an iminium ion intermediate by monoamine oxidase B (MAO-B) and then converted to its oxo-piperazine form (M1) by aldehyde oxidase (AO). However, it was found that the significant decrease in the pharmacologically active metabolite M1 following repeated administration of KW-2449 in primates might hamper the effectiveness of the drug. The mechanism underlying this phenomenon was investigated and it was found that the AO activity was inhibited in a time-dependent manner in vitro under the co-incubation of KW-2449 and MAO-B, while neither KW-2449 nor M1 strongly inhibited MAO-B or AO activity. These results clearly suggest that MAO-B catalysed iminium ion metabolite inhibited AO, prompting us to investigate whether or not the iminium ion metabolite covalently binds to endogenous proteins, as has been reported with other reactive metabolites as a cause for idiosyncratic toxicity. The association of the radioactivity derived from 14 C-KW-2449 with endogenous proteins both in vivo and in vitro was confirmed and it was verified that this covalent binding was inhibited by the addition of sodium cyanide, an iminium ion-trapping reagent, and pargyline, a MAO-B inhibitor. These findings strongly suggest that the iminium ion metabolite of KW-2449 is highly reactive in inhibiting AO irreversibly and binding to endogenous macromolecules covalently.

Physiologic regulation of heart rate and blood pressure involves connexin 36-containing gap junctions.

Chronically elevated sympathetic nervous activity underlies many cardiovascular diseases. Elucidating the mechanisms contributing to sympathetic nervous system output may reveal new avenues of treatment. The contribution of the gap junctional protein connexin 36 (Cx36) to the regulation of sympathetic activity and thus blood pressure and heart rate was determined using a mouse with specific genetic deletion of Cx36. Ablation of the Cx36 protein was confirmed in sympathetic preganglionic neurons of Cx36-knockout (KO) mice. Telemetric analysis from conscious Cx36 KO mice revealed higher variance in heart rate and blood pressure during rest and activity compared to wild-type (WT) mice, and smaller responses to chemoreceptor activation when anesthetized. In the working heart-brain stem preparation of the Cx36-KO mouse, respiratory-coupled sympathetic nerve discharge was attenuated and responses to chemoreceptor stimulation and noxious stimulation were blunted compared to WT mice. Using whole cell patch recordings, sympathetic preganglionic neurons in spinal cord slices of Cx36-KO mice displayed lower levels of spikelet activity compared to WT mice, indicating reduced gap junction coupling between neurons. Cx36 deletion therefore disrupts normal regulation of sympathetic outflow with effects on cardiovascular parameters.-Lall, V. K., Bruce, G., Voytenko, L., Drinkhill, M., Wellershaus, K., Willecke, K., Deuchars, J., Deuchars, S. A. Physiologic regulation of heart rate and blood pressure involves connexin 36-containing gap junctions.

BDNF and AMPA receptors in the cNTS modulate the hyperglycemic reflex after local carotid body NaCN stimulation.

The application of sodium cyanide (NaCN) to the carotid body receptors (CBR) (CBR stimulation) induces rapid blood hyperglycemia and an increase in brain glucose retention. The commissural nucleus tractus solitarius (cNTS) is an essential relay nucleus in this hyperglycemic reflex; it receives glutamatergic afferents (that also release brain derived neurotrophic factor, BDNF) from the nodose-petrosal ganglia that relays CBR information. Previous work showed that AMPA in NTS blocks hyperglycemia and brain glucose retention after CBR stimulation. In contrast, BDNF, which attenuates glutamatergic AMPA currents in NTS, enhances these glycemic responses. Here we investigated the combined effects of BDNF and AMPA (and their antagonists) in NTS on the glycemic responses to CBR stimulation. Microinjections of BDNF plus AMPA into the cNTS before CBR stimulation in anesthetized rats, induced blood hyperglycemia and an increase in brain arteriovenous (a-v) of blood glucose concentration difference, which we infer is due to increased brain glucose retention. By contrast, the microinjection of the TrkB antagonist K252a plus AMPA abolished the glycemic responses to CBR stimulation similar to what is observed after AMPA pretreatments. In BDNF plus AMPA microinjections preceding CBR stimulation, the number of c-fos immunoreactive cNTS neurons increased. In contrast, in the rats microinjected with K252a plus AMPA in NTS, before CBR stimulation, c-fos expression in cNTS decreased. The expression of AMPA receptors GluR2/3 did not change in any of the studied groups. These results indicate that BDNF in cNTS plays a key role in the modulation of the hyperglycemic reflex initiated by CBR stimulation.

Voltage- and receptor-mediated activation of a non-selective cation channel in rat carotid body glomus cells.

A recent study showed that hypoxia activates a Ca2+-sensitive, Na+-permeable non-selective cation channel (NSC) in carotid body glomus cells. We studied the effects of mitochondrial inhibitors that increase Ca2+ influx via Ca2+ channel (Cav), and receptor agonists that release Ca2+ from endoplasmic reticulum (ER) on NSC. Mitochondrial inhibitors (NaCN, FCCP, H2S, NO) elevated [Ca2+]i and activated NSC. Angiotensin II and acetylcholine that elevate [Ca2+]i via the Gq-IP3 pathway activated NSC. However, endothelin-1 (Gq) and 5-HT (Gq) showed little or no effect on [Ca2+]i and did not activate NSC. Adenosine (Gs) caused a weak rise in [Ca2+]i but did not activate NSC. Dopamine (Gs) and γ-aminobytyric acid (Gi) were ineffective in raising [Ca2+]i and failed to activate NSC. Store-operated Ca2+ entry (SOCE) produced by depletion of Ca2+ stores with cyclopiazonic acid activated NSC. Our results show that Ca2+ entry via Cav, ER Ca2+ release and SOCE can activate NSC. Thus, NSC contributes to both voltage- and receptor-mediated excitation of glomus cells.

Deficiency of Parkinson's disease-related gene Fbxo7 is associated with impaired mitochondrial metabolism by PARP activation.

The Parkinson's disease (PD)-related protein F-box only protein 7 (Fbxo7) is the substrate-recognition component of the Skp1-Cullin-F-box protein E3 ubiquitin ligase complex. We have recently shown that PD-associated mutations in Fbxo7 disrupt mitochondrial autophagy (mitophagy), suggesting a role for Fbxo7 in modulating mitochondrial homeostasis. Here we report that Fbxo7 deficiency is associated with reduced cellular NAD+ levels, which results in increased mitochondrial NADH redox index and impaired activity of complex I in the electron transport chain. Under these conditions of compromised respiration, mitochondrial membrane potential and ATP contents are reduced, and cytosolic reactive oxygen species (ROS) production is increased. ROS activates poly (ADP-ribose) polymerase (PARP) activity in Fbxo7-deficient cells. PARP inhibitor restores cellular NAD+ content and redox index and ATP pool, suggesting that PARP overactivation is cause of decreased complex I-driven respiration. These findings bring new insight into the mechanism of Fbxo7 deficiency, emphasising the importance of mitochondrial dysfunction in PD.

Novel Metabolic Abnormalities in the Tricarboxylic Acid Cycle in Peripheral Cells From Huntington's Disease Patients.

Metabolic dysfunction is well-documented in Huntington's disease (HD). However, the link between the mutant huntingtin (mHTT) gene and the pathology is unknown. The tricarboxylic acid (TCA) cycle is the main metabolic pathway for the production of NADH for conversion to ATP via the electron transport chain (ETC). The objective of this study was to test for differences in enzyme activities, mRNAs and protein levels related to the TCA cycle between lymphoblasts from healthy subjects and from patients with HD. The experiments utilize the advantages of lymphoblasts to reveal new insights about HD. The large quantity of homogeneous cell populations permits multiple dynamic measures to be made on exactly comparable tissues. The activities of nine enzymes related to the TCA cycle and the expression of twenty-nine mRNAs encoding for these enzymes and enzyme complexes were measured. Cells were studied under baseline conditions and during metabolic stress. The results support our recent findings that the activities of the pyruvate dehydrogenase complex (PDHC) and succinate dehydrogenase (SDH) are elevated in HD. The data also show a large unexpected depression in MDH activities. Furthermore, message levels for isocitrate dehydrogenase 1 (IDH1) were markedly increased in in HD lymphoblasts and were responsive to treatments. The use of lymphoblasts allowed us to clarify that the reported decrease in aconitase activity in HD autopsy brains is likely due to secondary hypoxic effects. These results demonstrate the mRNA and enzymes of the TCA cycle are critical therapeutic targets that have been understudied in HD.

Aerobic metabolism on muscle contraction in porcine iris sphincter.

Eyes are supplied O2 through the cornea and vessels of the retina and iris, which are tissues characterized by aerobic metabolism. Meanwhile, there are no reports on the association between iris sphincter contraction and aerobic metabolism. In this paper, we studied the aforementioned association. Eyes from adult pigs of either sex were obtained from a local abattoir. A muscle strip was connected to a transducer to isometrically record the tension. O2 consumption was measured using a Clark-type polarograph connected to a biological oxygen monitor. Creatine phosphate (PCr) and adenosine triphosphate (ATP) contents were measured in the muscle strips by high-performance liquid chromatography (HPLC). Iris sphincter muscles were measured in resting, contractile or hypoxic phases. Contraction was induced by hyperosmotic 65 mM KCl (H-65K+) or carbachol (CCh), and hypoxia was induced by aeration with N2 instead of O2 or by addition of sodium cyanide (NaCN). H-65K+- and CCh-induced muscle contraction, involved increasing O2 consumption. Hypoxia and NaCN significantly decreased H-65K+- and CCh-induced muscle contraction and/or O2 consumption and PCr contents. Our results suggest that the contractile behavior in porcine iris sphincter highly depends on mitogen oxidative metabolism.

Blockade of dorsolateral pontine 5HT1A receptors destabilizes the respiratory rhythm in C57BL6/J wild-type mice.

The neurotransmitter serotonin (5HT) acting via 5HT1a receptors (5HT1aR) is a potent determinant of respiratory rhythm variability. Here, we address the 5HT1aR-dependent control of respiratory rhythm variability in C57BL6/J mice. Using the in situ perfused preparation, we compared the effects of systemic versus focal blockade of 5HT1aRs. Blocking 5HT1aRs in the Kölliker-Fuse nucleus (KFn) increased the occurrence of spontaneous apneas and accounted for the systemic effects of 5HT1aR antagonists. Further, 5HT1aRs of the KFn stabilized the respiratory rhythm's response to arterial chemoreflex perturbations; reducing the recovering time, e.g., the latency to return to the baseline pattern. Together, these results suggest that the KFn regulates both intrinsic and sensory determinants of respiratory rhythm variability.

The role of the Kölliker-Fuse nuclei in the determination of abdominal motor output in a perfused brainstem preparation of juvenile rat.

The abdominal muscles are largely quiescent during normal breathing but may exhibit tonic activity or subtle respiratory modulation. The origin of baseline abdominal motor nerve activity (AbNA) if present remains uncharacterised. The contribution of the Kölliker-Fuse nucleus (KF) in the dorsolateral pons in the patterning and amplitude of AbNA was investigated using in situ perfused brainstem preparations of juvenile rats (n=12). Two types of AbNA were observed: Type I - expiratory-modulated (n=7), and Type II - weakly inspiratory/post-inspiratory-modulated (n=5). Despite this, all preparations exhibited the same bi-phasic late expiratory/postinspiratory bursts upon elicitation of the peripheral chemoreflex. Interestingly, the type of AbNA exhibited correlated with postinspiratory duration. Targeted microinjections of GABA-A receptor agonist isoguvacine (10mM; 70nl) into KF however did not significantly modify pattern or amplitude of baseline AbNA in either Type besides the selective abolition of the postinspiratory phase and, consequently, postinspiratory modulation in AbNAwhen present. In sum, the KF is not a major contributorin setting baseline abdominal motor output.

Meclizine Preconditioning Protects the Kidney Against Ischemia-Reperfusion Injury.

Global or local ischemia contributes to the pathogenesis of acute kidney injury (AKI). Currently there are no specific therapies to prevent AKI. Potentiation of glycolytic metabolism and attenuation of mitochondrial respiration may decrease cell injury and reduce reactive oxygen species generation from the mitochondria. Meclizine, an over-the-counter anti-nausea and -dizziness drug, was identified in a 'nutrient-sensitized' chemical screen. Pretreatment with 100 mg/kg of meclizine, 17 h prior to ischemia protected mice from IRI. Serum creatinine levels at 24 h after IRI were 0.13 ± 0.06 mg/dl (sham, n = 3), 1.59 ± 0.10 mg/dl (vehicle, n = 8) and 0.89 ± 0.11 mg/dl (meclizine, n = 8). Kidney injury was significantly decreased in meclizine treated mice compared with vehicle group (p < 0.001). Protection was also seen when meclizine was administered 24 h prior to ischemia. Meclizine reduced inflammation, mitochondrial oxygen consumption, oxidative stress, mitochondrial fragmentation, and tubular injury. Meclizine preconditioned kidney tubular epithelial cells, exposed to blockade of glycolytic and oxidative metabolism with 2-deoxyglucose and NaCN, had reduced LDH and cytochrome c release. Meclizine upregulated glycolysis in glucose-containing media and reduced cellular ATP levels in galactose-containing media. Meclizine inhibited the Kennedy pathway and caused rapid accumulation of phosphoethanolamine. Phosphoethanolamine recapitulated meclizine-induced protection both in vitro and in vivo.

Pericytes contribute to the disruption of the cerebral endothelial barrier via increasing VEGF expression: implications for stroke.

Disruption of the blood-brain barrier (BBB) integrity occurring during the early onset of stroke is not only a consequence of, but also contributes to the further progression of stroke. Although it has been well documented that brain microvascular endothelial cells and astrocytes play a critical role in the maintenance of BBB integrity, pericytes, sandwiched between endothelial cells and astrocytes, remain poorly studied in the pathogenesis of stroke. Our findings demonstrated that treatment of human brain microvascular pericytes with sodium cyanide (NaCN) and glucose deprivation resulted in increased expression of vascular endothelial growth factor (VEGF) via the activation of tyrosine kinase Src, with downstream activation of mitogen activated protein kinase and PI3K/Akt pathways and subsequent translocation of NF-κB into the nucleus. Conditioned medium from NaCN-treated pericytes led to increased permeability of endothelial cells, and this effect was significantly inhibited by VEGF-neutralizing antibody. The in vivo relevance of these findings was further corroborated in the stroke model of mice wherein the mice, demonstrated disruption of the BBB integrity and concomitant increase in the expression of VEGF in the brain tissue as well as in the isolated microvessel. These findings thus suggest the role of pericyte-derived VEGF in modulating increased permeability of BBB during stroke. Understanding the regulation of VEGF expression could open new avenues for the development of potential therapeutic targets for stroke and other neurological disease.

Evidence for a carotid body homolog in the lizard Tupinambis merianae.

The homolog to the mammalian carotid body has not yet been identified in lizards. Observational studies and evolutionary history provide indirect evidence for the existence of a chemoreceptor population at the first major bifurcation of the common carotid artery in lizards, but a chemoreceptive role for this area has not yet been definitively demonstrated. We explored this possibility by measuring changes in cardiorespiratory variables in response to focal arterial injections of the hypoxia mimic sodium cyanide (NaCN) into the carotid artery of 12 unanesthetized specimens of Tupinambis merianae. These injections elicited increases in heart rate (f(H); 101±35% increase) and respiratory rate (f(R); 620±119% increase), but not mean arterial blood pressure (MAP). These responses were eliminated by vagal denervation. Similar responses were elicited by injections of the neurotransmitters acetylcholine (ACh) and serotonin (5-HT) but not norepinephrine. Heart rate and respiratory rate increases in response to NaCN could be blocked or reduced by antagonists to ACh (atropine) and/or 5-HT (methysergide). Finally, using immunohistochemistry, we demonstrate the presence of putative chemoreceptive cells immunopositive for the cholinergic cell marker vesicular ACh transporter (VAChT) and 5-HT on internal lattice-like structures at the carotid bifurcation. These results provide evidence in lizards for the existence of dispersed chemoreceptor cells at the first carotid bifurcation in the central cardiovascular area that have similar properties to known carotid body homologs, adding to the picture of chemoreceptor evolution in vertebrates.

Intrinsic chemosensitivity of rostral ventrolateral medullary sympathetic premotor neurons in the in situ arterially perfused preparation of rats.

Brainstem hypoperfusion is a major excitant of sympathetic activity triggering hypertension, but the exact mechanisms involved remain incompletely understood. A major source of excitatory drive to preganglionic sympathetic neurons originates from the ongoing activity of premotor neurons in the rostral ventrolateral medulla (RVLM sympathetic premotor neurons). The chemosensitivity profile of physiologically characterized RVLM sympathetic premotor neurons during hypoxia and hypercapnia remains unclear. We examined whether physiologically characterized RVLM sympathetic premotor neurons can sense brainstem ischaemia intrinsically. We addressed this issue in a unique in situ arterially perfused preparation before and after a complete blockade of fast excitatory and inhibitory synaptic transmission. During hypercapnic hypoxia, respiratory modulation of RVLM sympathetic premotor neurons was lost, but tonic firing of most RVLM sympathetic premotor neurons was elevated. After blockade of fast excitatory and inhibitory synaptic transmission, RVLM sympathetic premotor neurons continued to fire and exhibited an excitatory firing response to hypoxia but not hypercapnia. This study suggests that RVLM sympathetic premotor neurons can sustain high levels of neuronal discharge when oxygen is scarce. The intrinsic ability of RVLM sympathetic premotor neurons to maintain responsivity to brainstem hypoxia is an important mechanism ensuring adequate arterial pressure, essential for maintaining cerebral perfusion in the face of depressed ventilation and/or high cerebral vascular resistance.

Tight coupling of Na+/K+-ATPase with glycolysis demonstrated in permeabilized rat cardiomyocytes.

The effective integrated organization of processes in cardiac cells is achieved, in part, by the functional compartmentation of energy transfer processes. Earlier, using permeabilized cardiomyocytes, we demonstrated the existence of tight coupling between some of cardiomyocyte ATPases and glycolysis in rat. In this work, we studied contribution of two membrane ATPases and whether they are coupled to glycolysis--sarcoplasmic reticulum Ca2+ ATPase (SERCA) and plasmalemma Na+/K+-ATPase (NKA). While SERCA activity was minor in this preparation in the absence of calcium, major role of NKA was revealed accounting to ∼30% of the total ATPase activity which demonstrates that permeabilized cell preparation can be used to study this pump. To elucidate the contribution of NKA in the pool of ATPases, a series of kinetic measurements was performed in cells where NKA had been inhibited by 2 mM ouabain. In these cells, we recorded: ADP- and ATP-kinetics of respiration, competition for ADP between mitochondria and pyruvate kinase (PK), ADP-kinetics of endogenous PK, and ATP-kinetics of total ATPases. The experimental data was analyzed using a series of mathematical models with varying compartmentation levels. The results show that NKA is tightly coupled to glycolysis with undetectable flux of ATP between mitochondria and NKA. Such tight coupling of NKA to PK is in line with its increased importance in the pathological states of the heart when the substrate preference shifts to glucose.