BACKGROUND:The internal milieu of the body is controlled by a system of interoceptors coupled to motor outflows that drive compensatory adaptive responses. These include the arterial chemoreceptors, best known for sensing arterial oxygen. In cardiometabolic diseases, such as essential hypertension, the carotid bodies (CB) exhibit heightened reflex sensitivity and tonic activity without an apparent stimulus. The mechanisms behind CB sensitization in these conditions are not well understood. METHODS:Guided by functional genomics, a range of functional assays is used to interrogate downstream intracellular and interorgan signaling pathways involved in arterial chemosensory function. RESULTS:Here, we report the presence of the MC4R (melanocortin 4 receptor) in the mammalian CB and show its elevated expression in experimental hypertension. We demonstrate that melanocortin agonists activate arterial chemosensory cells, modulating CB chemosensory afferent drive to influence chemoreflex-evoked sympathetic and ventilatory activity. Transcriptional analysis of hypertensive CB implicates the activation of the Mash1 (mammalian achaete-scute homolog 1; Ascl1) regulatory network in driving elevated Mc4r expression. CONCLUSIONS:Collectively, our data indicate a primarily pathophysiological role of melanocortin signaling in arterial chemosensation, contributing to excess sympathetic activity in cardiometabolic disease.

Hypertension is the most important and well-known risk factor for cardiovascular disease (CVD). Recently, acute organophosphate (OP) poisoning has also been pointed as a CVD risk factor. Despite this evidence, no studies have contrasted the acute toxicosis and cardiovascular (CV) effects of OP poisoning under conditions of normotension and hypertension. In this work, adult male normotensive Wistar and Spontaneously Hypertensive rats (SHR) were intraperitoneally injected with saline or chlorpyrifos (CPF), an OP compound, monitored for acute toxicosis signs and 24-h survival. After poisoning, blood pressure, heart rate and ventilation were recorded, the Bezold-Jarisch Reflex (BJR), the Chemoreflex (CR) were chemically activated, as well as the cardiac autonomic tone (AUT) was assessed. Erythrocyte and brainstem acetylcholinesterase and plasmatic butyrylcholinesterase (BuChE) activities were measured as well as lipid peroxidation, advanced oxidation protein products (AOPP), nitrite/nitrate levels, expression of catalase, TNF alpha and angiotensin-I converting enzyme (ACE-1) within the brainstem. CPF induced a much more pronounced acute toxicosis and 33 % lethality in SHR. CPF poisoning impaired ventilation in SHR, the BJR reflex responses in Wistar rats, and the chemoreflex tachypneic response in both strains. CPF inhibited activity of cholinesterases in both strains, increased AOPP and nitrite/nitrate levels and expression of TNF alpha and ACE-1 in the brainstem of Wistar rats. Interestingly, SHR presented a reduced intrinsic BuChE activity, an important bioscavenger. Our findings show that, CPF at sublethal doses in normotensive rats lead to lethality and much more pronounced acute toxicity signs in the SHR. We also showed that cardiorespiratory reflexes were differentially impacted after CPF poisoning in both strains and that the cardiorespiratory disfunction seems to be associated with interference in cholinergic transmission, oxidative stress and inflammation. These results points to an increased susceptibility to acute toxicosis in hypertension, which may impose a significant risk to vulnerable populations.

ATP acting on P2X2/3 receptors within carotid bodies (CBs) underpins chemoreflex hyperreflexia and hypertonicity of sympathetic activity in Spontaneously Hypertensive rats (SHR). As the exact mechanisms remain elusive, we hypothesized either a greater release and/or decreased breakdown of ATP in CBs of SHR versus Wistar rats, and that high concentrations of exogenous ATP in the CB of normotensive rats would produce the sensitised motor responses portrayed by SHRs. Three experiments were performed to investigate the generation, breakdown and physiological responses of ATP in the CB: First, we sought to quantify the amount of ATP released from CBs of male Wistar and SHRs (N=10 for each strain; 4-5 weeks old) using an in vitro colorimetric technique; second, in both strains (Wistar, N=10; SHR, N=12), we used digital droplet polymerase chain reaction (ddPCR) for quantitative analysis of gene expression of six enzymes, which are all involved with the extracellular metabolism of ATP (Enpp1-3, Entpd 2-3, and Nt5e); we also quantified the levels of gene expression for tyrosine hydroxylase (TH) and Panx-1 channel, which are markers for CB type I (or glomus cells) and type-II cells, respectively. Third, we used the working heart-brainstem preparation (WHBP) for analysis of the CB chemoreflex responses evoked by ATP stimulation in Wistar rats (N= 25, 60-80g). Stimulation of CBs was carried out with local intra-arterial injection of potassium cyanide (KCN 20-100 μL, 0.04%; i.a.), and direct intra-CB injection of ATP (10-100 μL, 50 μM - 5 mM) or α, β-methylene ATP (10 μM, 450 μM, and 5mM).Although no rat strain differences in Enpp1 and Entpd2-3 were detected, Enpp2-3 and Nt5e expression were increased in SHR (p<0.05) versus Wistar rats suggesting greater breakdown of ATP in SHR CBs. However, ATP released from the CB at baseline and with KCN stimulation was two-fold greater in SHR (P<0.05). KCN stimulation of the CB, evoked hyperpnoea, bradycardia and sympathoexcitation in Wistar rats, whereas ATP produced hypopnoea/apnoea, repetitive burst discharge of post-inspiratory activity and elevations in both abdominal motor activity and SNA. We found that the ATP-induced hyponoea/apnoea was prevented by ipsilateral nodosectomy suggesting activation of non-CB afferent/s. Microinjecting either ATP or α, β-methylene ATP directly into the CB produced hyperpnoea and sympathoexcitation. Despite upregulation of some ATP degrading enzymes in the SHR, CB release of ATP is higher in this strain and produces activation of respiratory and sympathetic systems. Health Research Council of New Zealand This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Hypertension is the most important and well-known risk factor for cardiovascular disease (CVD). Recently, acute organophosphate (OP) poisoning has also been pointed as a CVD risk factor. Despite these evidence, no studies have contrasted the cardiovascular (CV) effects of OP poisoning under conditions of normotension and hypertension. We hypothesized that hypertensive rats would have a greater impairment of CV function after acute exposure to chlorpyrifos (CPF), an OP compound. 12-14 week-old male normotensive Wistar and Spontaneously Hypertensive rats (SHRs) were intraperitoneally (i.p.) injected with saline (NaCl 0.9%) or 20 mg.kg-1 of CPF, a previously shown sublethal dose in normotensive rats. 24-hour survival was recorded and through cannulation of the femoral artery and vein, we assessed blood pressure (BP) and heart rate (HR) recordings and chemically activated the Bezold-Jarisch Reflex (BJR), the Chemoreflex (CR), as well as assessed the cardiac autonomic tone (AUT). BJR was activated by phenylbiguanide (PBG; 1.5; 3; 6; 12; 24 μg/kg; i.v.) and the CR by potassium cyanide (KCN, 10, 20, 40 and 80 μg/ rat; i.v.) injections, while the AUT was assessed by pharmacological blockade with atenolol (4mg.kg-1, i.v) and atropine (2mg.kg-1, i.v). CPF acute exposure induced 33% mortality in SHR without leading to lethality in Wistar rats. CPF poisoning impaired the BJR hypotensive response in Wistar rats for all PBG doses tested (ΔDBP: - 33.83±4.88 vs. -14.16±2.68; -44.58±3.48 vs. -30.58±2.16; -66.16±2.50 vs. -36.16±2.54; -72.66±2.66 vs. -47.91±2.50; -78.66±2.35 vs.-61.58±2.40; p<0.05 Wistar saline vs Wistar CPF) and the bradycardic response at the dose of 6 and 12μg/kg, respectively (ΔHR: -268.66±14.77 vs. -216.66±11.11; -304.58±13.42 vs. -241.66±11.01; p<0.05 Wistar saline vs Wistar CPF), without affecting the SHR strain. No CPF-induced changes were observed for the CR and AUT between strains. Our findings show that, despite being sublethal in normotensive rats, CPF lead to mortality in hypertensive rats. However, contrary to our hypothesis, CPF poisoning did not further impaired CV function in SHR, but significantly worsen BJR function in Wistar rats. Despite not producing greater impairment in CV function, CPF poisoning leads to greater mortality under hypertension, which may impose a significant risk in individuals with this comorbidity. Aitken AV, was recipeint of Fapes Foundation scholarship. Minassa VS, Batista TJ were recipients of Capes Foundation scholarship. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

AbstractAimsThe carotid bodies (CBs) of spontaneously hypertensive (SH) rats exhibit hypertonicity and hyperreflexia contributing to heightened peripheral sympathetic outflow. We hypothesized that CB hyperexcitability is driven by its own sympathetic innervation.Methods and resultsTo test this, the chemoreflex was activated (NaCN 50–100 µL, 0.4 µg/µL) in SH and Wistar rats in situ before and after: (i) electrical stimulation (ES; 30 Hz, 2 ms, 10 V) of the superior cervical ganglion (SCG), which innervates the CB; (ii) unilateral resection of the SCG (SCGx); (iii) CB injections of an α1-adrenergic receptor agonist (phenylephrine, 50 µL, 1 mmol/L), and (iv) α1-adrenergic receptor antagonist prazosin (40 µL, 1 mmol/L) or tamsulosin (50 µL, 1 mmol/L). ES of the SCG enhanced CB-evoked sympathoexcitation by 40–50% (P < 0.05) with no difference between rat strains. Unilateral SCGx attenuated the CB-evoked sympathoexcitation in SH (62%; P < 0.01) but was without effect in Wistar rats; it also abolished the ongoing firing of chemoreceptive petrosal neurones of SH rats, which became hyperpolarized. In Wistar rats, CB injections of phenylephrine enhanced CB-evoked sympathoexcitation (33%; P < 0.05), which was prevented by prazosin (26%; P < 0.05) in SH rats. Tamsulosin alone reproduced the effects of prazosin in SH rats and prevented the sensitizing effect of the SCG following ES. Within the CB, α1A- and α1B-adrenoreceptors were co-localized on both glomus cells and blood vessels. In conscious SH rats instrumented for recording blood pressure (BP), the CB-evoked pressor response was attenuated after SCGx, and systolic BP fell by 16 ± 4.85 mmHg.ConclusionsThe sympathetic innervation of the CB is tonically activated and sensitizes the CB of SH but not Wistar rats. Furthermore, sensitization of CB-evoked reflex sympathoexcitation appears to be mediated by α1-adrenoceptors located either on the vasculature and/or glomus cells. The SCG is novel target for controlling CB pathophysiology in hypertension.

Carotid body pathophysiology is associated with many cardiovascular-respiratory-metabolic diseases. This pathophysiology reflects both hyper-sensitivity and hyper-tonicity. From both animal models and human patients, evidence indicates that amelioration of this pathophysiological signalling improves disease states such as a lowering of blood pressure in hypertension, a reduction of breathing disturbances with improved cardiac function in heart failure (HF) and a re-balancing of autonomic activity with lowered sympathetic discharge. Given this, we have reviewed the mechanisms of carotid body hyper-sensitivity and hyper-tonicity across disease models asking whether there is uniqueness related to specific disease states. Our analysis indicates some commonalities and some potential differences, although not all mechanisms have been fully explored across all disease models. One potential commonality is that of hypoperfusion of the carotid body across hypertension and HF, where the excessive sympathetic drive may reduce blood flow in both models and, in addition, lowered cardiac output in HF may potentiate the hypoperfusion state of the carotid body. Other mechanisms are explored that focus on neurotransmitter and signalling pathways intrinsic to the carotid body (e.g. ATP, carbon monoxide) as well as extrinsic molecules carried in the blood (e.g. leptin); there are also transcription factors found in the carotid body endothelium that modulate its activity (Kruppel-like factor 2). The evidence to date fully supports that a better understanding of the mechanisms of carotid body pathophysiology is a fruitful strategy for informing potential new treatment strategies for many cardiovascular, respiratory and metabolic diseases, and this is highly relevant clinically.image Abstract figure legend The carotid body has been linked with the development of hypertension and heart failure in animals and humans. This review explores mechanisms of carotid body sensitisation across disease models. We ask whether these mechanisms are common or unique to distinct cardiovascular diseases; such knowledge could inform clinical treatment strategies. We propose that carotid body sensitivity increases progressively from health to hypertension and is maximal in heart failure and that there are both shared and distinct sensitisation mechanisms. This sensitisation is followed by an increase in systemic sympathetic outflow. It remains unknown whether the sensitised carotid body drives sympathetic overactivity or vice versa.image

Twenty-five years ago, a new physiological preparation called the working heart-brainstem preparation (WHBP) was introduced with the claim it would provide a new platform allowing studies not possible before in cardiovascular, neuroendocrine, autonomic and respiratory research. Herein, we review some of the progress made with the WHBP, some advantages and disadvantages along with potential future applications, and provide photographs and technical drawings of all the customised equipment used for the preparation. Using mice or rats, the WHBP is an in situ experimental model that is perfused via an extracorporeal circuit benefitting from unprecedented surgical access, mechanical stability of the brain for whole cell recording and an uncompromised use of pharmacological agents akin to in vitro approaches. The preparation has revealed novel mechanistic insights into, for example, the generation of distinct respiratory rhythms, the neurogenesis of sympathetic activity, coupling between respiration and the heart and circulation, hypothalamic and spinal control mechanisms, and peripheral and central chemoreceptor mechanisms. Insights have been gleaned into diseases such as hypertension, heart failure and sleep apnoea. Findings from the in situ preparation have been ratified in conscious in vivo animals and when tested have translated to humans. We conclude by discussing potential future applications of the WHBP including two-photon imaging of peripheral and central nervous systems and adoption of pharmacogenetic tools that will improve our understanding of physiological mechanisms and reveal novel mechanisms that may guide new treatment strategies for cardiorespiratory diseases.

Background: Intraoperative arterial hypotension (IOH) is a common side effect of general anesthesia (GA), associated with poor outcomes in ischemic stroke. While IOH is more prevalent with hypertension, it is unknown whether IOH may differ when GA is induced during ischemic stroke, versus other clinical settings. This is important given that many stroke patients receive GA for endovascular thrombectomy. Methods: We evaluate the cardiovascular responses to volatile GA (isoflurane in 100% o2) before and during middle cerebral artery occlusion stroke in rats instrumented to record blood pressure (BP) and cerebral tissue oxygenation (po2) in the projected penumbra, in clinically relevant cohorts of normotensive (Wistar rat, n = 10), treated hypertensive (spontaneously hypertensive [SH] + enalapril, n = 12), and untreated hypertensive (SH rat, n = 12). Results: During baseline induction of GA, IOH was similar in normotensive, treated hypertensive, and untreated hypertensive rats during the induction phase (first 10 minutes) (–24 ± 15 vs −28 ± 22 vs −48 ± 24 mm Hg; P > .05) and across the procedure (−24 ± 13 vs −30 ± 35 vs −39 ± 27 mm Hg; P > .05). Despite the BP reduction, cerebral po2 increased by ~50% in all groups during the procedure. When inducing GA after 2 hours, all stroke groups showed a greater magnitude IOH compared to baseline GA induction, with larger falls in treated (−79 ± 24 mm Hg; P = .0202) and untreated(−105 ± 43 mm Hg; P < .001) hypertensive rats versus normotensives (−49 ± 21 mm Hg). This was accompanied by smaller increases in cerebral po2 in normotensive rats (19% ± 32%; P = .0144 versus no-stroke); but a decrease in cerebral po2 in treated (−11% ± 19%; P = .0048) and untreated (−12% ± 15%; P = .0003) hypertensive rats. Sham animals (normotensive and hypertensive) showed similar magnitude and pattern of IOH when induced with GA before and after sham procedure. Conclusions: Our findings are the first demonstration that ischemic stroke per se increases the severity of IOH, particularly when combined with a prior history of hypertension; this combination appears to compromise penumbral perfusion.

Background: Aberrant sympathetic nerve activity exacerbates cardiovascular risk in hypertension and diabetes, which are common comorbidities, yet clinically sympathetic nerve activity remains poorly controlled. The hypertensive diabetic state is associated with increased reflex sensitivity and tonic drive from the peripheral chemoreceptors, the cause of which is unknown. We have previously shown hypertension to be critically dependent on the carotid body (CB) input in spontaneously hypertensive rat, a model that also exhibits a number of diabetic traits. CB overstimulation by insulin and leptin has been similarly implicated in the development of increased sympathetic nerve activity in metabolic syndrome and obesity. Thus, we hypothesized that in hypertensive diabetic state (spontaneously hypertensive rat), the CB is sensitized by altered metabolic signaling causing excessive sympathetic activity levels and dysfunctional reflex regulation. Methods: Using a hypothesis-free RNA-seq approach, we investigated potential molecular targets implicated in energy metabolism mediating CB sensitization and its regulation of sympathetic outflow in experimental hypertension. Identified targets were characterized using molecular and functional techniques assessing peripheral chemoreflex sensitivity in situ and in vivo. Results: We discovered GLP1R (glucagon-like peptide-1 receptor) expression in the CBs of rat and human and showed that its decreased expression is linked to sympathetic hyperactivity in rats with cardiometabolic disease. We demonstrate GLP1R to be localized to CB chemosensory cells, while targeted administration of GLP1R agonist to the CB lowered its basal discharge and attenuated chemoreflex-evoked blood pressure and sympathetic responses. Importantly, hyperglycemia-induced peripheral chemoreflex sensitization and associated basal sympathetic overactivity were abolished by GLP1R activation in the CB suggesting a role in a homeostatic response to high blood glucose. Conclusions: We show that GLP1 (glucagon-like peptide-1) modulates the peripheral chemoreflex acting on the CB, supporting this organ as a multimodal receptor. Our findings pinpoint CBs as potential targets for ameliorating excessive sympathetic activity using GLP1R agonists in the hypertensive-diabetic condition.

Purinergic signaling involving adenosine triphosphate (ATP) acting on P2X2/3 receptors modulates physiological carotid body (CB) afferent discharge and chemoreflex activation however, the upregulation of P2X3 receptors on petrosal sensory neurons is partly responsible for aberrant CB tonicity and hyperreflexia, and the increase in blood pressure in the Spontaneously Hypertensive rat (SHR). The enhanced chemoreflex occurs prior to the onset of hypertension and elevated CB activity is thought to contribute to the underlying pathophysiology of the disease but the exact mechanisms remain unclear. We hypothesize that there is greater release of ATP and/or decreased ATP breakdown in the pre-hypertensive SHR CB compared with that in the Wistar rat, and this evokes an increase in the chemoreflex response and subsequent activation of the sympathetic nervous system. We performed three studies. (i) Using digital droplet PCR (ddPCR), we measured mRNA expression levels of enzymes involved in the breakdown of ATP in CBs extracted from male 4-week-old prehypertensive SHR and Wistar rats; these included: ectonucleotide pyrophosphatase/phosphodiesterase 1-3 (Enpp1-3), ectonucleoside triphosphate diphosphohydrolase 2-3 (Entpd2-3), and ecto-5'-nucleotidase (Nt5e). (ii) We quantified ATP release from CBs in vitro during baseline conditions and during cyanide-evoked ATP release using a colorimetric assay (213-579-1 - Millipore), comparing between male 4-week-old pre-hypertensive SHR and Wistar rats. (iii) In an in situ working heart-brainstem preparation of 4-week-old Wistar rats, simultaneous recordings were made from phrenic (PN), hypoglossal (HN), recurrent laryngeal (RLN), abdominal (ABN), and thoracic sympathetic nerves (tSNA). The peripheral chemoreceptors were stimulated with either potassium cyanide (KCN 20-100 μL, 0.04%) or ATP at varying concentrations (10-100 µL, 50 µM - 2 mM) delivered via the internal carotid artery. In whole CB extracts, ddPCR revealed increased Enpp2-3 and Nt5e expression (p<0.05) in pre-hypertensive SHRs relative to Wistar rats suggesting greater capacity to break down ATP in the pre-hypertensive SHR CB. There were no differences in the expressions of Enpp1 or Entpd2-3 between rat strains. However, the amount of ATP released from the CB in both baseline and during cyanide stimulation was two-fold greater in the pre-hypertensive SHR (P<0.05). Stimulation of the CB with KCN evoked hyperpnoea, bradycardia and sympathoexcitation at all doses. In contrast, CB injection of low dose ATP increased PN rate with little effect on tSNA, whereas higher doses induced apnea accompanied by repetitive burst discharge of post-inspiratory activity in the RLN with tonic elevations in the discharge of both ABN and tSNA. In sum, despite the upregulation of several ATP degrading enzymes in the CBs of the SHR, CB release of ATP is higher in this species. We hypothesize that the increase in ATP release in the SHR CB drives the CB hyperexcitability and that the shift towards an upregulation of ATP degrading enzymes even prior to the development of hypertension is compensatory to oppose the increase in ATP signaling. The dose-dependent effect of exogenous ATP at supraphysiological levels evoking greater CB-mediated expiratory and sympathetic outflow in the Wistar rat mimics aspects of pathophysiological CB chemoreflex signaling in the SHR.

While a considerable body of literature has characterized the clinical features induced by organophosphate pesticides, the field lacks scrutiny into cardio-respiratory changes in different phases of poisoning. Herein, we evaluated the impact of chlorpyrifos (CPF) and its active metabolite chlorpyrifos-oxon (CPO) on the cardiorespiratory system during acute and subacute phases of poisoning using an in situ experimental rodent model. CPF (30 mg/kg) was injected intraperitoneally to rats beforehand (24 h) whereas CPO (15 mg/kg) was added into the perfusate reservoir to evaluate the effects on the motor outputs throughout the three phases of the respiratory cycle: inspiration, post-inspiration and late expiration. Phrenic, recurrent laryngeal (RLN) and thoracic sympathetic nerve activity (tSNA) were recorded. Heart rate was derived from the electrocardiogram (ECG) and the baro- and chemo-reflexes tested. CPF and CPO led to a time-dependent change in cardiorespiratory motor outputs. In the acute phase, the CPO induced bradypnea, transiently reduced the inspiratory time (TI), and increased the amplitude of phrenic. Post-inspiratory (PI) discharge recorded from the RLN was progressively reduced while tSNA was increased. CPO significantly depressed the chemoreflex but had no effect on baroreflex. During subacute phase, CPF prolongated TI with no effect on respiratory rate. Both the RLN PI discharge, the chemoreflex and the baroreflex sympathetic gain were reduced. In addition, both CPF and CPO shifted the cardiac sympatho-vagal balance towards sympathetic dominance. Our data show that different phases of poisoning are associated with specific changes in the cardio-respiratory system and might therefore demand distinct approaches by health care providers.

The motivation for this review comes from the emerging complexity of the autonomic innervation of the carotid body (CB) and its putative role in regulating chemoreceptor sensitivity. With the carotid bodies as a potential therapeutic target for numerous cardiorespiratory and metabolic diseases, an understanding of the neural control of its circulation is most relevant. Since nerve fibres track blood vessels and receive autonomic innervation, we initiate our review by describing the origins of arterial feed to the CB and its unique vascular architecture and blood flow. Arterial feed(s) vary amongst species and, unequivocally, the arterial blood supply is relatively high to this organ. The vasculature appears to form separate circuits inside the CB with one having arterial venous anastomoses. Both sympathetic and parasympathetic nerves are present with postganglionic neurons located within the CB or close to it in the form of paraganglia. Their role in arterial vascular resistance control is described as is how CB blood flow relates to carotid sinus afferent activity. We discuss non-vascular targets of autonomic nerves, their possible role in controlling glomus cell activity, and how certain transmitters may relate to function. We propose that the autonomic nerves sub-serving the CB provide a rapid mechanism to tune the gain of peripheral chemoreflex sensitivity based on alterations in blood flow and oxygen delivery, and might provide future therapeutic targets. However, there remain a number of unknowns regarding these mechanisms that require further research that is discussed.

Previous studies have demonstrated that the integrity of the carotid bodies (CBs) is essential for the development and maintenance of hypertension via exacerbated sympathetic drive. CBs of spontaneously hypertensive rats (SHR) exhibit both increased tonicity and hyperreflexia. However, what drives this CB hyper-excitability is not fully understood. We hypothesised that CB hyperreflexia is mediated by its sympathetic innervation, which originates from the superior cervical ganglion (SCG). Experiments were carried out in Wistar and SH rats (60-80g) using the in situ working heart-brainstem preparation (WHBP). Phrenic and thoracic sympathetic nerves, heart rate and perfusion pressure were recorded and the chemoreflex activated using NaCN (50-100µL, 0.04%). Drug infusions into the CB were made via a cannula placed in the internal carotid artery with its tip juxta-positioned to the carotid body artery. To assess if the SCG modulates the chemoreflex, we stimulated the SCG electrically. This enhanced the chemoreflex evoked sympathoexcitation in both rat strains by 40-50% (P<0.05); this hyperreflexia was not different between rat strains and restricted to the chemoreflex sympathetic response with no obvious change in heart rate, perfusion pressure or phrenic nerve responses. The SCG evoked hyperreflexia of the sympathetic chemoreflex was prevented by Tamsulosin, an alpha1-adrenoceptor antagonist, applied to the CB. To mimic the effect of SCG stimulation, we applied phenylephrine (an alpha1 adrenoceptor agonist) to the CB, whichenhanced the chemoreflex sympathoexcitation by 33% (P<0.05). Next, we ascertained whether there was any endogenous modulation of the chemoreflex by the SCG in SH rats. Both SCG ganglionectomy and alpha1 adrenoceptor antagonism reduced the chemoreflex evoked sympathoexcitation (P<0.01). To equivalent levels observed in Wistar rats. Notably, respiratory-sympathetic coupling was also reduced by alpha1 adrenoceptor antagonism of the CB in SH rats. Immunohistochemistry showed positive co-localisation of alpha1A- and alpha1B- adrenoreceptors on both glomus cells and blood vessels within the CB. We conclude, sympathetic activity modulates CB reflex sensitivity via alpha1-adrenoreceptors and this appears to be a mechanism underpinning the pathological hyperreflexia of the CB in SH rats. We propose that a positive feedback loop whereby CB activity drives the sympathetic nervous system and increased sympathetic innervation of the CB causes its sensitisation is a pivotal mechanism for the development of hypertension. Our data support the SCG as a novel target for controlling blood pressure in hypertension.

Previous studies have demonstrated that the integrity of the carotid bodies is essential for the development and maintenance of hypertension. In this condition, both increased tonicity and hyperreflexia are displayed by chemosensitive neurons in the petrosal ganglia of spontaneous hypertensive rats (SHR). However, what drives carotid body hyper‐excitability is not known. We hypothesised this was triggered by the sympathetic nerves innervating the carotid body, which originate from the superior cervical ganglion (SCG). The aim of our study was test if activation of the SCG could modulate carotid body activity. Experiments were carried out in 6 juvenile Wistar rats (60–80g) using the decerebrated in situ working heart‐brainstem preparation (WHBP). Simultaneous recordings of phrenic (PN) and thoracic sympathetic nerves (tSNA) were obtained using glass suction electrodes. Heart rate (HR) was derived from the electrocardiogram (ECG). Chemoreflex was activated using NaCN (50μL, 0.04%, i.a.) and the SCG was stimulated electrically (ES; 30Hz, 2 ms, 10V) using a Grass SD9 stimulator via a bipolar microelectrode. The chemoreflex was evoked before and immediately after ES of the SCG. Changes in sympathetic activity, perfusion pressure, phrenic and heart rate were analysed quantitatively. Data are displayed as mean ± SEM with a level of significance set at P<0.05; differences were tested using paired student‐t test.The only component of the chemoreflex augmented by SCG stimulation was the evoked increase in sympathetic activity. In Wistars, the control response of 29.3 ± 3.2% from baseline was augmented to 46.04 ± 3.58% after ES (P=0.006). Within 10 min of the stimulation, the sympatho hyperreflexia had returned to control (20.24 ± 5.96%). SHR also presented sympatho hyperreflexia (43.0 ± 6.3 vs 62.9 ± 9.2 %, P= 0.015) but the change of sensitization was of similar magnitude between rat strains. The SCG induced carotid body hyperreflexia was prevented by application of lignocaine into the SCG. We conclude that stimulation of the efferent sympathetic nerves innervating the carotid body can increase the gain of the chemoreflex symathoexcitatory response. We are currently assessing whether chemoreflex hyperreflexia observed in neurogenic hypertension is mediated by sympathetic nerves innervating the carotid body.Support or Funding InformationHealth Research Council of New Zealand

In a previous work we showed that the organophosphate pesticide (OP) chlorpyrifos (CPF) reduces the protective chemoreflex and baroreflex responses in rats. However, whether the antidotes atropine (ATR) and pralidoxime (2-PAM) are capable of restoring these reflex functions remains unexplored. Rats were poisoned with CPF (30 mg.kg(-1), i.p.) and one hour after the intoxication, ATR (10 mg.kg(-1), i.p.) and 2-PAM (40 mg.kg(-1), i.p.) were administrated separately or in combination. Cardiorespiratory parameters were recorded in awake rats 24 h after CPF. Systolic blood pressure (SBP) and heart rate (HR) variability and spontaneous baroreflex sensitivity (sBRS) were derived from undisturbed recordings (30 min), while chemoreflex was assessed through potassium cyanide (KCN) i.v. injections (10, 20, 40, 80 mu g/rat). CPF poisoning increased SBP variability and low frequency/high frequency (LF/HF) ratio of the HR variability spectrum, indicating autonomic imbalance with increased cardiac sympathetic tone. sBRS was not changed. Treatment with 2-PAM restored SBP variability, whilst both antidotes increased LF/HF ratio. CPF poisoning reduced the hypertensive, bradycardic and tachypneic chemoreflex responses. Chemoreflex-induced hypertensive response was restored by 2-PAM treatment, while ATR recovered the bradycardic response. Both antidotes restored the chemoreflex tachypneic response. Our data show distinct effects of ATR and 2-PAM on cardiorespiratory parameters affected by OP poisoning. While 2-PAM rescued the chemoreflex hypertensive response, ATR reversed chemoreflex bradycardic dysfunction. Although 2-PAM clinical use is questioned in some countries, our data indicate that summation of effects of both antidotes appears beneficial on the cardiorespiratory system and peripheral chemoreflex function.

Previous studies showed that chlorpyrifos (CPF) acute exposure impaired cardiorespiratory reflexes. Evidence also indicates that continuous exposure to organophosphorus compounds impairs cardiovascular function. However, the effect of intermittent exposure to CPF, as may be experienced in the real world, on tonic and reflex cardiorespiratory function remains unexplored. Wistar rats were injected with saline or CPF for 4 weeks (3 times/week) or 12 weeks (once/week) at the doses of 7 mg/kg and 10 mg/kg. After exposure, blood pressure (BP), heart rate (HR), respiratory rate (fR), tidal volume (VT), and minute volume (VE) were recorded. Systolic BP and pulse interval (PI) variability, HR spectrum, spontaneous baroreflex and chemoreflex function were also evaluated. Plasma butyrylcholinesterase and brainstem acetylcholinesterase activities were quantified. Enzymatic activity of the CPF animals was reduced after both treatment periods. Baseline BP, HR, and fR, as well as systolic BP and PI variability indices, did not change, after CPF treatment. VT and VE were elevated in CPF animals. CPF exposure increased the very low-frequency component of the HR spectrum. Baroreflex gain was reduced after CPF 4-week exposure. Chemoreflex bradycardia was reduced in the CPF-treated rats. These data show that intermittent exposure to CPF impairs cardiorespiratory function in rats. These results may have important clinical implications for workers seasonally exposed to these compounds.

Acute organophosphate (OP) poisoning induces well-known signs of toxicosis related to acetylcholinesterase (AChE) inhibition. However, the relationship between acute OP poisoning and the onset of psychiatric disorders remains unclear. Thus, we investigated behavioural and biochemical consequences of acute exposure to the OP chlorpyrifos in male rats and also the effectiveness of the antidotes atropine and pralidoxime on reversing these changes. A sub-lethal dose of commercial chlorpyrifos (20 mg/kg, i.p.) elicited signs of acute toxicosis during the first hours after its injection in rats. Twenty-four hours after treatment, this single dose of chlorpyrifos induced a depressive-like behaviour in the rat forced swimming test without impairing locomotor activity. At this time (24 h), chlorpyrifos decreased plasma butyrylcholinesterase (BChE) activity and hippocampal, striatal and prefrontal cortical AChE activity in rats. The behavioural and biochemical consequences of acute chlorpyrifos poisoning do not seem to be long lasting, since 30 days later they were absent. We evaluated whether these behavioural and biochemical consequences of acute chlorpyrifos treatment would be reversed by the antidotes atropine (10 mg/kg i.p.) and/or pralidoxime (40 mg/kg; i.p.) given 1 h after poisoning. Pralidoxime partially reactivated the AChE activity in the prefrontal cortex, but not in the hippocampus and striatum. Atropine attenuated the depressive-like behaviour induced by chlorpyrifos in rats. Our results suggest that acute chlorpyrifos poisoning induces a transient depressive-like behaviour possible related to hippocampal AChE inhibition. They suggest that treatment with atropine and pralidoxime seems to be insufficient to counteract all the effects of OP acute poisoning, at least in rats.

Although it is well-established that severe poisoning by organophosphorus (OP) compounds strongly affects the cardiorespiratory system, the effects of sub-lethal exposure to these compounds on the neural control of cardiovascular function are poorly explored. The aim of this study was to evaluate the effects of acute sub-lethal exposure to chlorpyrifos (CPF), a commonly used OP insecticide, on three basic reflex mechanisms involved in blood pressure regulation, the peripheral chemoreflex, the baroreflex and the Bezold-Jarisch reflex. Adult male Wistar rats were injected intraperitoneally with a single dose of CPF (30 mg/kg) or saline (0.9%). 24 h after injections, cardiovascular reflexes were tested in awake rats. Potassium cyanide (KCN) and phenylbiguanide (PBG) were injected intravenously to activate the chemoreflex and the Bezold-Jarisch reflex, respectively. The baroreflex was activated by phenylephrine and sodium nitroprusside infusions. Blood samples were taken for measurements of butyrylcholinesterase (BChE) activity while acetylcholinesterase (AChE) activity was measured in brainstem samples. Animals treated with CPF presented signs of intoxication such as ataxia, tremor, lacrimation, salivation, tetany, urination and defecation. The hypertensive and the bradycardic responses of the chemoreflex as well as the hypotensive and bradycardic responses of the Bezold-Jarisch reflex were attenuated in CPF treated animals (P < 0.05). Concerning the baroreflex responses, CPF treatment reduced the bradycardia plateau, the range and the gain of the reflex (P < 0.05). Plasma BChE and brainstem AChE were both reduced significantly after CPF treatment (P < 0.05). Our results showed that acute sub-lethal exposure to CPF impairs the cardiovascular responses of homeostatic and defensive cardiovascular reflexes. These effects are associated with a marked inhibition of plasma BChE and brainstem AChE.

Organophosphate (OP) poisoning induced disruption of glucose homeostasis is well established. OP poisoning leads to accumulation of acetylcholine (ACh) due to the inhibition of acetylcholinesterases (AChE). On the other hand the incidence of type 2 diabetes mellitus (T2DM) is shown to rise along with the use of pesticides in Southeast Asia. Attenuation of the ‘incretin effect’ is seen in T2DM. This effect is regulated by a complex loop of mechanism involving ACh driven muscarinic receptors. We hypothesize that OP poisoning leads to disruption of glucose homeostasis by attenuation of the incretin effect. Inhibition of the Glucagon Like Peptide-1 (GLP-1) secretion is our main focus of interest. Positive finding of the hypothesis will open possibility of using incretin based treatment modalities to treat or prevent acute OP induced disruption of glucose homeostasis.

In this study, we report the preparation of a new tetra-substituted epoxide aldehyde cyclopentane, which acts as a starting material for the synthesis of plinol, from (R)-(+)-epoxy-limonene. The synthesis was performed in three steps and resulted in a good yield. The structural determination was performed by H-1 and C-13 NMR, and the relative stereochemistry was defined by nuclear Overhauser effect (NOE) experiments with computer calculations of molecular modeling, particularly with respect to indirect coupling constant calculations.