Aims/hypothesis A new class of treatments termed bioelectronic medicines are now emerging that aim to target individual nerve fibres or specific brain circuits in pathological conditions to repair lost function and reinstate a healthy balance. Carotid sinus nerve (CSN) denervation has been shown to improve glucose homeostasis in insulin-resistant and glucose-intolerant rats; however, these positive effects from surgery appear to diminish over time and are heavily caveated by the severe adverse effects associated with permanent loss of chemosensory function. Herein we characterise the ability of a novel bioelectronic application, classified as kilohertz frequency alternating current (KHFAC) modulation, to suppress neural signals within the CSN of rodents. Methods Rats were fed either a chow or high-fat/high-sucrose (HFHSu) diet (60% lipid-rich diet plus 35% sucrose drinking water) over 14 weeks. Neural interfaces were bilaterally implanted in the CSNs and attached to an external pulse generator. The rats were then randomised to KHFAC or sham modulation groups. KHFAC modulation variables were defined acutely by respiratory and cardiac responses to hypoxia (10% O₂ + 90% N₂). Insulin sensitivity was evaluated periodically through an ITT and glucose tolerance by an OGTT. Results KHFAC modulation of the CSN, applied over 9 weeks, restored insulin sensitivity (constant of the insulin tolerance test [K_ITT] HFHSu sham, 2.56 ± 0.41% glucose/min; K_ITT HFHSu KHFAC, 5.01 ± 0.52% glucose/min) and glucose tolerance (AUC HFHSu sham, 1278 ± 20.36 mmol/l × min; AUC HFHSu KHFAC, 1054.15 ± 62.64 mmol/l × min) in rat models of type 2 diabetes. Upon cessation of KHFAC, insulin resistance and glucose intolerance returned to normal values within 5 weeks. Conclusions/interpretation KHFAC modulation of the CSN improves metabolic control in rat models of type 2 diabetes. These positive outcomes have significant translational potential as a novel therapeutic modality for the purpose of treating metabolic diseases in humans.

Cigarette smoke (CS) and chronic hypoxia (CH) can produce pulmonary hypertension. Similarities and differences between both exposures and their interaction have not been explored. The aim of the present study was to investigate the effects of CS and CH, as single factors or in combination, on the pulmonary circulation in the guinea pig.51 guinea pigs were exposed to CS for 12 weeks and 32 were sham-exposed. 50% of the animals in each group were additionally exposed to CH for the final 2 weeks. We measured pulmonary artery pressure (P_pa), and the weight ratio between the right ventricle (RV) and left ventricle plus the septum. Pulmonary artery contractility in response to noradrenaline (NA), endothelium-dependent vasodilatation and distensibility were evaluated in organ bath chambers. The number of small intrapulmonary vessels showing immunoreactivity to smooth muscle (SM) α-actin and double elastic laminas was assessed microscopically.CS and CH induced similar increases ofP_paand RV hypertrophy (p<0.05 for both), effects that were further enhanced when both factors were combined. CH increased the contractility to NA (p<0.01) and reduced the distensibility (p<0.05) of pulmonary arteries. Animals exposed to CS showed an increased number of small vessels with positive immunoreactivity to SM α-actin (p<0.01) and those exposed to CH a greater proportion of vessels with double elastic laminas (p<0.05).We conclude that CH amplifies the detrimental effects of CS on the pulmonary circulation by altering the mechanical properties of pulmonary arteries and enhancing the remodelling of pulmonary arterioles.

Key points: Hypoxic pulmonary vasoconstriction (HPV) is a mechanism by which pulmonary arteries maintain blood oxygenation during alveolar hypoxia. HPV is generally studied using a vasoconstricting co‐stimulus that amplifies the HPV but may also distort its properties; we therefore characterised HPV in isolated rat intrapulmonary arteries during 40 min hypoxic challenges in the absence of any such stimulus. Immediate (phase 1) and sustained (phase 2) components of HPV were unaffected by blocking voltage‐gated Ca²⁺ channels but were abolished by depletion of sarcoplasmic reticulum Ca²⁺. Phase 2 was attenuated by blockade of store‐operated Ca²⁺ entry (SOCE), although it largely persisted in Ca²⁺‐free physiological saline solution. HPV was associated with an increase in the intrapulmonary artery ratio of oxidised to reduced glutathione and was inhibited by antioxidants. HPV resulted primarily from intracellular Ca²⁺ release, with SOCE making a contribution, particularly to phase 2. Sustained HPV involves oxidation of the pulmonary artery redox state. Abstract  Hypoxic pulmonary vasoconstriction (HPV) maintains blood oxygenation during acute hypoxia but contributes to pulmonary hypertension during chronic hypoxia. The mechanisms of HPV remain controversial, in part because HPV is usually studied in the presence of agonist‐induced preconstriction (‘pretone’). This potentiates HPV but may obscure and distort its underlying mechanisms. We therefore carried out an extensive assessment of proposed mechanisms contributing to HPV in isolated intrapulmonary arteries (IPAs) in the absence of pretone by using a conventional small vessel myograph. Hypoxia elicited a biphasic constriction consisting of a small transient (phase 1) superimposed upon a sustained (phase 2) component. Neither phase was affected by the L‐type Ca²⁺ channel antagonists diltiazem (10 and 30 μm) or nifedipine (3 μm). Application of the store‐operated Ca²⁺ entry (SOCE) blockers BTP2 (10 μm) or SKF96365 (50 μm) attenuated phase 2 but not phase 1, whereas a lengthy (30 min) incubation in Ca²⁺‐free physiological saline solution similarly reduced phase 2 but abolished phase 1. No further effect of inhibition of HPV was observed if the sarco/endoplasmic reticulum Ca²⁺‐ATPase inhibitor cyclopiazonic acid (30 μm) was also applied during the 30 min incubation in Ca²⁺‐free physiological saline solution. Pretreatment with 10 μm ryanodine and 15 mm caffeine abolished both phases, whereas treatment with 100 μm ryanodine attenuated both phases. The two‐pore channel blocker NED‐19 (1 μm) and the nicotinic acid adenine dinucleotide phosphate (NAADP) antagonist BZ194 (200 μm) had no effect on either phase of HPV. The lysosomal Ca²⁺‐depleting agent concanamycin (1 μm) enhanced HPV if applied during hypoxia, but had no effect on HPV during a subsequent hypoxic challenge. The cyclic ADP ribose antagonist 8‐bromo‐cyclic ADP ribose (30 μm) had no effect on either phase of HPV. Neither the Ca²⁺‐sensing receptor (CaSR) blocker NPS2390 (0.1 and 10 μm) nor FK506 (10 μm), a drug which displaces FKBP12.6 from ryanodine receptor 2 (RyR2), had any effect on HPV. HPV was virtually abolished by the rho kinase blocker Y‐27632 (1 μm) and attenuated by the protein kinase C inhibitor Gö6983 (3 μm). Hypoxia for 45 min caused a significant increase in the ratio of oxidised to reduced glutathione (GSSG/GSH). HPV was unaffected by the NADPH oxidase inhibitor VAS2870 (10 μm), whereas phase 2 was inhibited but phase 1 was unaffected by the antioxidants ebselen (100 μm) and TEMPOL (3 mm). We conclude that both phases of HPV in this model are mainly dependent on [Ca²⁺]_i release from the sarcoplasmic reticulum. Neither phase of HPV requires voltage‐gated Ca²⁺ entry, but SOCE contributes to phase 2. We can detect no requirement for cyclic ADP ribose, NAADP‐dependent lysosomal Ca²⁺ release, activation of the CaSR, or displacement of FKBP12.6 from RyR2 for either phase of HPV. Sustained HPV is associated with an oxidising shift in the GSSG/GSH redox potential and is inhibited by the antioxidants ebselen and TEMPOL, consistent with the concept that it requires an oxidising shift in the cell redox state or the generation of reactive oxygen species.

Hypoxia elicits catecholamine (CA) secretion from the adrenal medulla (AM) in perinatal animals by acting directly on chromaffin cells. However, whether innervation of the AM, which in the rat occurs in the second postnatal week, suppresses this direct hypoxic response is the subject of debate. Opioid peptides have been proposed as mediators of this suppression. To resolve these controversies, we have compared CA-secretory responses with high external concentrations of K⁺ ([K⁺]_e) and hypoxia in the AM of neonatal (1- to 2-day-old) and juvenile (14- or 15- and 30-day-old) rats subjected to superfusion in vitro. In addition, we studied the effect of hypercapnic acidosis on the CA-secretory responses in the AM during postnatal development and the possible interaction between acidic and hypoxic stimuli. Responses to high [K⁺]_e were comparable at all ages, but responses to hypoxia and hypercapnic acidosis were maximal in neonatal animals. Suppression of the hypoxic response in the rat AM was not mediated by opioids, because their agonists did not affect the hypoxic CA response. The association of hypercapnic acidosis and hypoxia, mimicking the episodes of asphyxia occurring during delivery, generates a more than additive secretory response in the neonatal rat AM. Our data confirm the loss of the direct sensitivity to hypoxia of the AM in the initial weeks of life and demonstrate a direct response of neonatal AM to hypercapnic acidosis. The synergistic effect of hypoxia and acidosis would explain the CA outburst crucial for adaptation to extrauterine life observed in naturally delivered mammals.

Glabridin is a major flavonoid in Glycyrrhiza glabra (licorice) root, a traditional Asian medicine. Glabridin is reported to have anti-atherogenic, anti-inflammatory and anti-nephritic properties; however its effects on vascular tone remain unexplored. We examined the effect of glabridin on rat main mesenteric artery using isometric myography and also ELISA to measure cGMP levels. Glabridin (30μM) relaxed arteries pre-constricted with the thromboxane A2 analog U46619 (0.2μM) by ~60% in an endothelium-independent manner. Relaxation to 30μM glabridin was abolished by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (1μM) and by the BKCa channel blocker tetraethyammonium (1mM) but was unaffected by the estrogen receptor antagonist ICI182780. The concentration-response curve to glabridin (0.1 to 30μM) was downshifted by the KATP channel blocker glibenclamide (10μM), the KV channel blocker 4-aminopyridine (300μM), and the KIR blocker BaCl2 (30μM). In U46619-contracted arteries partially relaxed by 0.1μM sodium nitroprusside, application of 10 and 30nM glabridin caused additional vasorelaxation. Glabridin (30μM) approximately doubled tissue [cyclic GMP]. Application of the phosphodiesterase inhibitor isobutylmethylxanthine caused a much larger rise in [cyclic GMP], and glabridin failed to cause vasorelaxation or a further rise in [cGMP] when co-applied with IBMX. Vasorelaxation to glabridin is dependent on the opening of K+ channels, particularly BKCa, probably caused by a rise in cellular [cyclic GMP] owing to phosphodiesterase inhibition. In the presence of sodium nitroprusside an effect of glabridin is observed at nM concentrations, similar those measured in plasma following human ingestion of licorice flavonoid oil.

The association between obstructive sleep apnea (OSA) and metabolic disorders is well-established; however, the underlying mechanisms that elucidate this relationship remain incompletely understood. Since the liver is a major organ in the maintenance of metabolic homeostasis, we hypothesize that liver dysfunction plays a crucial role in the pathogenesis of metabolic dysfunction associated with obstructive sleep apnea (OSA). Herein, we explored the underlying mechanisms of this association within the liver. Experiments were performed in male Wistar rats fed with a control or high fat (HF) diet (60% lipid-rich) for 12 weeks. Half of the groups were exposed to chronic intermittent hypoxia (CIH) (30 hypoxic (5% O₂) cycles, 8 h/day) that mimics OSA, in the last 15 days. Insulin sensitivity and glucose tolerance were assessed. Liver samples were collected for evaluation of lipid deposition, insulin signaling, glucose homeostasis, hypoxia, oxidative stress, antioxidant defenses, mitochondrial biogenesis and inflammation. Both the CIH and HF diet induced dysmetabolism, a state not aggravated in animals submitted to HF plus CIH. CIH aggravates hepatic lipid deposition in obese animals. Hypoxia-inducible factors levels were altered by these stimuli. CIH decreased the levels of oxidative phosphorylation complexes in both groups and the levels of SOD-1. The HF diet reduced mitochondrial density and hepatic antioxidant capacity. The CIH and HF diet produced alterations in cysteine-related thiols and pro-inflammatory markers. The results obtained suggest that hepatic mitochondrial dysfunction and oxidative stress, leading to inflammation, may be significant factors contributing to the development of dysmetabolism associated with OSA.