Carotid Chemoreceptor Sensitivity Research Articles

AMPA Receptor-Dependent Glutamatergic Signaling is Present in the Carotid Chemoreceptor

Exposure to both sustained and intermittent hypoxia for as little as a day produces sustained augmentation of carotid chemoreceptor sensitivity; however, the molecular basis for this chemoreflex plasticity remains uncertain. We previously reported that NMDA receptor-dependent glutamatergic signaling in rat carotid body played a role in altered hypoxic sensitivity after exposure to cyclic intermittent hypoxia (CIH). Here we found that mRNAs of multiple AMPA and Kainate glutamate receptors were expressed in rat carotid body. The AMPA receptor subunit GluR1 showed intense immunoreactivity in the carotid body, co-localizing with tyrosine hydroxylase in type I cells. Treatment of rat carotid body-derived primary cells with AMPA activated ERK1/2 in a time-dependent manner. Exposing Sprague–Dawley rats to CIH for 8 h/day for 3 weeks significantly enhanced the expression level of GluA1 mRNA as well as GluR1 protein in the carotid body. In addition, our results showed that multiple of vesicular glutamate transporters (VGLUTs) and excitatory amino acid transporters (EAATs) were expressed in the rat carotid body, indicating that glutamate might be as a neurotransmitter stored, released and uptake in the carotid body. Finally, we found that mRNAs of GluA1, GluA2 and GluA3 as well as PSD-95-like membrane-associated granulate kinase family members, PSD-95, PSD-93, and SAP97, were expressed in human carotid body. Our data suggest AMPA receptor-dependent glutamatergic signaling is present in the carotid body and might be involved in the carotid chemoreceptor response to hypoxia.

Interindividual variability in the dose-specific effect of dopamine on carotid chemoreceptor sensitivity to hypoxia.

Human studies use varying levels of low-dose (1-4 μg·kg(-1)·min(-1)) dopamine to examine peripheral chemosensitivity, based on its known ability to blunt carotid body responsiveness to hypoxia. However, the effect of dopamine on the ventilatory responses to hypoxia is highly variable between individuals. Thus we sought to determine 1) the dose response relationship between dopamine and peripheral chemosensitivity as assessed by the ventilatory response to hypoxia in a cohort of healthy adults, and 2) potential confounding cardiovascular responses at variable low doses of dopamine. Young, healthy adults (n = 30, age = 32 ± 1, 24 male/6 female) were given intravenous (iv) saline and a range of iv dopamine doses (1-4 μg·kg(-1)·min(-1)) prior to and throughout five hypoxic ventilatory response (HVR) tests. Subjects initially received iv saline, and after each HVR the dopamine infusion rate was increased by 1 μg·kg(-1)·min(-1). Tidal volume, respiratory rate, heart rate, blood pressure, and oxygen saturation were continuously measured. Dopamine significantly reduced HVR at all doses (P < 0.05). When subjects were divided into high (n = 13) and low (n = 17) baseline chemosensitivity, dopamine infusion (when assessed by dose) reduced HVR in the high group only (P < 0.01), with no effect of dopamine on HVR in the low group (P > 0.05). Dopamine infusion also resulted in a reduction in blood pressure (3 μg·kg(-1)·min(-1)) and total peripheral resistance (1-4 μg·kg(-1)·min(-1)), driven primarily by subjects with low baseline chemosensitivity. In conclusion, we did not find a single dose of dopamine that elicited a nadir HVR in all subjects. Additionally, potential confounding cardiovascular responses occur with dopamine infusion, which may limit its usage.

Should we be ‘Doping’ the peripheral chemoreceptors?

The carotid body chemoreceptors are located bilaterally at the bifurcation of the common carotid artery and are primarily known for their ability to sense and respond to changes in the partial pressure of oxygen in arterial blood. As such, peripheral chemoreceptor hyperactivity has been widely studied in adults with sleep apnoea, who are exposed to multiple, acute hypoxic events during sleep. In response to intermittent falls in the arterial partial pressure of oxygen, patients with sleep apnoea present with increases in heart rate, blood pressure, sympathetic activity and ventilation (e.g. measures of increased carotid chemoreceptor sensitivity). This observed upregulation at the level of the carotid chemoreceptors in response to repeated activation is unlike the typical downregulation/desensitization responses seen in many other physiological systems. In addition to hypoxia, a broad range of factors are now emerging as potent stimulators of carotid body afferent activity (e.g. hypercapnia, hypoglycaemia, hyperinsulinaemia, hypoperfusion). Thus it may not be surprising that carotid chemoreceptor hyperactivity was recently implicated in the pathophysiology of a variety of other cardiovascular and metabolic diseases, such as hypertension, heart failure, insulin resistance and diabetes. In this context, perhaps it is useful to think of these conditions as ‘diseases of sympathoexcitation’ or possibly carotid body-mediated sympathoexcitation. Along these lines, therapies focused on desensitization of the carotid chemoreceptors have shown provocative and successful results in animal models. For example, carotid body denervation appears to prevent the development of hypertension and insulin resistance in a rodent model of overfeeding (Ribeiro et al. 2013) and can eventually improve cardiac function in models of heart failure (Del Rio et al. 2013). Yet, whether denervation is beneficial in long-term treatment of such disorders in humans is still an open question. This is especially true given not all (heart failure) patients respond to chemoreceptor-desensitizing therapies (van de Borne et al. 1996), nor do all patients respond positively to other types of invasive denervation procedures (Joyner, 2012). So how might we identify patients that are likely to respond to carotid body desensitization or even denervation? In this edition of The Journal of Physiology, Niewinski and colleagues used systemic low-dose dopamine in young, healthy humans to lower afferent activity at the level of the peripheral chemoreceptors (Niewinski et al. 2014). Although such doses appear to have minimal impact on basal measures of heart rate, blood pressure, or minute ventilation in healthy adults, low-dose dopamine blunts peripheral chemoreceptor responsiveness to hypoxia as measured by changes in both ventilation and haemodynamic variables. Additionally, when the dopamine infusion was stopped, a transient rise in ventilation was observed – possibly due to release of an accumulating pool of neurotransmitters, given that the post-infusion increase in ventilation correlated with basal levels of carotid chemoreceptor sensitivity. The practicality of low-dopamine as a long-term therapy for ‘diseases of sympathoexcitation’ is questionable. However, techniques used by Niewinski and colleagues in the present investigation could be important in identifying which patients respond to desensitization of the carotid chemoreceptors and are thus potential candidates for carotid body denervation (Niewinski et al. 2013). Since the present investigation is limited to young healthy adults, future studies will be necessary to examine the impact of low-dose dopamine on ventilatory and haemodynamic parameters in disease states known to exhibit increased chemoreceptor activation at baseline. Until then, it is unclear how much patient-to-patient variability exists, what benefit low-dose dopamine has over other clinical measures, and whether a clinical test could be developed using this approach. Furthermore, one must not forget that the carotid chemoreceptors are important in sensing and responding to a number of peripheral stimuli (e.g. hypoglycaemia, hyperinsulinaemia, hyperthermia, hypoperfusion). Thus, removal or desensitization may not only blunt sympathoexcitatory and ventilatory responses to hypoxia (thus impacting natural responses to this environmental stressor), but may impact human physiology in ways that are not fully realized (Johnson & Joyner, 2013). Finally, ‘diseases of sympathoexcitation’ are complex and have effects on multiple organ systems. Because of this, it is hard to imagine that modulation of carotid body afferent activity alone will be sufficient to treat and/or reverse most or all of them.

Effect of chronic intermittent hypoxia on leptin and leptin receptor protein expression in the carotid body

This study was done to investigate whether chronic intermittent hypoxia (CIH) induced changes in leptin and leptin receptor protein levels, and known downstream mediators of leptin receptor signaling in the carotid body. Rats were subjected to CIH (120s normoxia, 80s hypoxia) or normoxia for 8h/day to either short term (7 days) or long term CIH exposure (95 days). After both 7 and 95 days of CIH, carotid body leptin protein expression was decreased, while protein levels of the long form leptin receptor (OB-Rb) were elevated. On the other hand, protein expression levels of the short form leptin receptor (OB-R100) were unchanged. Furthermore, phosphorylated signal transducer and activator of transcription 3 (pSTAT3) protein levels were found to be significantly decreased at only the 7 day period. On the other hand, suppressor of cytokine signaling 3 (SOCS3) protein levels were elevated at only the 7 day period, while phosphorylated extracellular-signal-regulated kinase 1/2 (pERK1/2) was elevated only at the 95 day period. In both the normoxia and the CIH groups, carotid body leptin was decreased at the 95 day period compared to 7 days. However, OB-Rb or Ob-R100 protein levels were not changed in the normoxic or CIH group at either time point. Furthermore, pSTAT3 protein levels were found to be significantly higher, while SOCS3 levels were significantly lower in the 95 day CIH group compared to the 7 day CIH group. Taken together, these data indicate that CIH induces changes in leptin and leptin downstream signaling proteins within the carotid bodies which may contribute to alterations in carotid chemoreceptor sensitivity.

Hyperoxia attenuates muscle sympathetic nerve activity following isocapnic hypoxia in humans

Hypoxia may sensitize the carotid chemoreceptors, resulting in a sustained elevation of muscle sympathetic nerve activity (MSNA) that outlasts the hypoxic stimulus. To test this hypothesis, we determined the effect of carotid body inhibition on the sustained elevation of MSNA following isocapnic hypoxia in humans. Seven healthy subjects (5 male, 2 female) breathed 100% O(2) (hyperoxia) for 1 min before (2 interventions) and after (2-3 interventions) 20 min of isocapnic hypoxia (80% arterial oxyhemoglobin saturation). MSNA was continuously recorded from the common peroneal nerve with microneurography. There was no effect of hyperoxia on MSNA before exposure to isocapnic hypoxia. During the isocapnic hypoxia exposure, there was an increase in minute ventilation and heart rate that subsided once hypoxia was terminated. In contrast, there was an increase in MSNA burst frequency that persisted for approximately 25 min after cessation of the stimulus. Hyperoxia resulted in a transient reduction in MSNA burst frequency of 28% (P < 0.05), 15% (P < 0.05), and 9% (P > 0.05) in the three posthypoxia interventions, respectively. Our results suggest that input from the carotid chemoreceptors is obligatory for the sustained elevation of MSNA initiated by chemoreflex stimulation. We attribute the decrease in MSNA to a transient hyperoxia-induced attenuation of carotid chemoreceptor sensitivity.

Hyperoxia attenuates the elevation in muscle sympathetic nerve activity following exposure to hypoxia

Hypoxia may sensitize the carotid chemoreceptors resulting in a sustained elevation of muscle sympathetic nerve activity (MSNA) which outlasts the hypoxic stimulus. To test this hypothesis we determined (n=4) the effect of carotid body deactivation on the sustained elevation in MSNA (common peroneal nerve) following isocapnic hypoxia (arterial O2 saturation 80% for 20 mins) in humans. Ventilation, heart rate and MSNA were elevated in hypoxia; however, only the elevation in MSNA outlasted the hypoxic stimulus, and remained elevated for 20 mins following the hypoxic stimulus. In contrast, carotid body deactivation with hyperoxia (100% O2 for 1 min) transiently reduced MSNA in eight of nine trials (mean decrease 12.6 ± 9.5%, p = 0.005). Our results suggest that input from the carotid chemoreceptors is obligatory for the sustained elevation in MSNA initiated by chemoreflex stimulation. We attribute the decrease in MSNA to a transient hyperoxia‐induced attenuation in carotid chemoreceptor sensitivity.Research support: NSERC, MSFHR

Novel roles of a local angiotensin‐generating system in the carotid body

The carotid body is a highly vascularized network of chemoreceptor cells, which are located at the bifurcation of the internal and external carotid arteries arising from the common carotid artery. Carotid body chemoreceptors, called glomus (type 1, catechol-secreting) cells release dopamine in response to rapid changes in arterial PO2, PCO2 and pH, thus increasing carotid body sinus nerve activity and provoking centrally mediated cardiopulmonary responses (Gonzalez et al. 1994). The endocrine (circulating) renin–angiotensin system (RAS) plays a role in regulating the blood pressure, electrolyte and fluid homeostasis. The aspartyl protease renin cleaves the precursor of the system, angiotensinogen, to yield angiotensin I, which is subsequently cleaved by angiotensin converting enzyme (ACE) to yield the physiologically active octapeptide angiotensin II (Ang II). Pressor responses to Ang II are largely mediated through activation of the type 1 receptor (AT1R). It is now recognized that the RAS exists in diverse mammalian cells and organs, where it plays diverse roles in their respective tissues and organs. Apart from the haemodynamic function, they include, but are not limited to, cell proliferation, apoptosis, oxidative stress and inflammatory as well as fibrotic actions. This so-called local RAS which adds to and/or differs from the circulating RAS, frequently operated in a paracrine and/or autocrine manner (Lavoie & Sigmund, 2003). Newly discovered functions of locally generated Ang II in tissues and organs are of particular interest in the physiological and pathophysiological aspects. Such a system has recently been proposed to exist in the carotid body: here, key RAS components (including angiotensinogen, ACE, AT1R and AT2R) are expressed in the absence of renin, suggesting the existence of a renin-independent biosynthetic pathway for Ang II or an intrinsic angiotensin-generating system in the carotid body (Lam & Leung, 2002). This system may play a key role in modulating the carotid body response to hypoxia. Ang II itself increases carotid body efferent activity, presumably via AT1R activation (Allen, 1998). Meanwhile, chronic hypoxia up-regulates several key RAS components in the carotid body, including a time-course-dependent activity of ACE (Lam & Leung, 2003; Lam et al. 2004). Specifically, expression of the AT1R on glomus cells is enhanced, consistent with increased expression of the AT1R at gene and protein level (Leung et al. 2000). Chronic hypoxia thus enhances AT1R-mediated efferent activity (Leung et al. 2000). In addition, AT1R activation increases intracellular calcium levels in dissociated glomus cells, an effect which is enhanced three-fold by chronic hypoxaemia (Fung et al. 2002). Taken together, all data suggest that chronic hypoxia is a major factor that increases AT1R expression and function in the carotid chemoreceptor, with likely changes in cardiopulmonary function. Thus the excitatory action of Ang II on the glomus cells may increase the chemosensitivity of the carotid body and may counteract the blunting effect of chronic hypoxia. Notwithstanding this involvement of a local angiotensin-generating system in the carotid body, details of the molecular and cellular mechanisms underlying the modulation require extensive study. A paper by Li & Schultz (2006) in this issue of The Journal of Physiology adds valuable data to this story. Using a rabbit model of congestive heart failure (CHF), the authors demonstrate a role for local glomus Ang II/AT1R signalling in increasing the sensitivity of Kv channels to hypoxia. High concentrations of Ang II (> 1 nm) directly inhibit Kv currents (Ik) while changes in Kv channel protein expression may contribute to the suppression of Ik and enhanced sensitivity of Ik to hypoxia in the CHF state. This study has two significant implications: Ang II can enhance the oxygen sensitivity of Kv channels via the mediation of AT1R and the cellular Ang II–AT1R pathway is functional in carotid glomus cells of CHF but not normal animals. In spite of this solid evidence, the precise mechanism by which CHF up-regulates the Ang II–AT1R and its function in the carotid body have yet to be resolved. In this regard, chronically impaired cardiac output resulting from CHF might be sufficient to render a prolonged deprivation of oxygen delivery to carotid glomus cells, a state akin to chronic hypoxia, thus leading to activation of its cellular RAS pathway. These data indicate a paracrine/autocrine role of Ang II in the modulation of enhanced carotid chemoreceptor sensitivity to chronic hypoxia characterized in chronic heart failure. In summary, the carotid body is the major organ central to the physiological responses to various forms of hypoxic stress: acute, chronic and intermittent hypoxia. Thus, activation of the RAS by hypoxia might be commonly associated with chronic cardiopulmonary diseases and sleep apnoea. Of great interest in this context is the emerging data of evidence for the existence of a local carotid angiotensin-generating system, which is up-regulated by chronic hypoxia to alter carotid chemoreceptor sensitivity. The data of Li and Schultz thus support this concept.

Induced recovery of hypoxic phrenic responses in adult rats exposed to hyperoxia for the first month of life.

1. Adult rats exposed to hyperoxia for the first month of life have permanently attenuated ventilatory and phrenic nerve responses to hypoxia. We tested the hypothesis that the blunted hypoxic phrenic response in hyperoxia-treated rats (inspired O(2) fraction, F(I,O2) = 0.6 for 28 post-natal days) could be actively restored to normal by intermittent (alternating 12 % O(2)/air at 5 min intervals; 12 h per night for 1 week) or sustained (12 % O(2) for 1 week) hypoxia. 2. Phrenic responses to isocapnic hypoxia (P(a,O2) = 60, 50 and 40 +/- 2 mmHg) were assessed in the following groups of anaesthetized, vagotomized adult Sprague-Dawley rats (age 4 months), treated with a neuromuscular blocking agent and ventilated: control, hyperoxia-treated and hyperoxia-treated exposed to either intermittent or sustained hypoxia as adults. Experiments on intermittent and sustained hypoxia-treated rats were performed on the morning following hypoxic exposures. 3. Both intermittent and sustained hypoxia enhanced hypoxic phrenic responses in hyperoxia-treated rats when expressed as minute phrenic activity (P < 0.05). Increases in phrenic burst amplitude during hypoxia were greater in hyperoxia-treated rats after intermittent hypoxia (P < 0.05), and a similar but non-significant trend was observed after sustained hypoxia. Hypoxia-induced changes in phrenic burst frequency were not significantly different among groups. 4. The estimated carotid body volume in control rats (11.5 (+/- 0.7) x 10(6) microm(3)) was greater than in the other treatment groups (P < 0.05). However, carotid body volume was significantly greater in hyperoxia-treated rats exposed to sustained hypoxia (6.3 (+/- 0.3) x 10(6) microm(3); P < 0.05) compared to hyperoxia-treated rats (3.3 (+/- 0.2) x 10(6) microm(3)) or hyperoxia-treated rats exposed to intermittent hypoxia (3.8 (+/- 0.3) x 10(6) microm(3)). 5. Hypoxic phrenic responses in hyperoxia-treated rats 1 week after intermittent hypoxia were similar to responses measured immediately after intermittent hypoxia, indicating persistent functional recovery. 6. The results indicate that diminished hypoxic phrenic responses in adult rats due to hyperoxia exposure for the first 28 post-natal days can be reversed by intermittent or sustained activation of the hypoxic ventilatory control system. Although the detailed mechanisms of functional recovery are unknown, we suggest that sustained hypoxia restores carotid chemoreceptor sensitivity, whereas intermittent hypoxia primarily augments central integration of synaptic inputs from chemoafferent neurons.

Resetting and postnatal maturation of oxygen chemosensitivity in rat carotid chemoreceptor cells.

1. Carotid chemoreceptor sensitivity is minimal immediately after birth and increases with postnatal age. In the present study we have investigated the peri- and postnatal developmental time course of [Ca2+]i responses to hypoxia in clusters of type I cells isolated from near-term fetal rats and rats that were 1, 3, 7, 11, 14 and 21 days old, using the Ca2+-sensitive fluoroprobe fura-2. 2. In type I cells from all age groups a graded increase in [Ca2+]i occurred in response to lowering the PO2 from 150 mmHg to 70, 35, 14, 7, 2 and 0 mmHg. The graded [Ca2+]i response to hypoxia was hyperbolic at all ages. 3. Type I cells from rats near-term fetal to 1 day old exhibited small [Ca2+]i responses, mainly to the most severe levels of hypoxia. After day 1, an increase in the [Ca2+]i responses to submaximal hypoxia stimulation resulted in a rightward shift in the O2 response curve. Using the Delta[Ca2+]i between 35 and 2 mmHg PO2 as an index of O2 sensitivity, type I cell O2 sensitivity increased approximately 4- to 5-fold between near-term fetal to 1 day old and 11 to 14 days of age. 4. Exposure to elevated extracellular potassium (10, 20 and 40 mM K+) caused a dose-dependent [Ca2+]i rise in type I cells from all age groups. There were no age-related changes in [Ca2+]i responses to any level of K+ between near-term fetal and 21 days. 5. We conclude that the maximal type I cell [Ca2+]i response to anoxia, as well as the sensitivity to submaximal hypoxic stimulation, of rats aged from near-term fetal to 21 days depends on the level of postnatal maturity. The lack of an age-related increase in the [Ca2+]i response to elevated K+ during the timeframe of maximal development of O2 sensitivity suggests that resetting involves maturation of O2 sensing, rather than non-specific developmental changes in the [Ca2+]i rise resulting from depolarization.

Maturation of carotid chemoreceptor sensitivity to hypoxia: in vitro studies in the newborn rat.

1. A preparation was developed to record single-fibre chemoreceptor afferent activity from carotid bodies of newborn and adult rats in vitro. The response to severe hypoxia was studied as a function of developmental age in four age groups: 1-2, 4-7, 10-15 days and adult (25-30 days). 2. During superfusion with HEPES-saline at room PO2 and at 26 or 35 degrees C, afferent chemoreceptor activity could be recorded in all age groups. No significant difference was found among groups in baseline discharge frequency at 26 or 35 degrees C. 3. All chemoreceptors responded to anoxia (PO2 congruent to 0 Torr) with a rapid increase in discharge frequency. At 35 degrees C, peak discharge frequency of single chemoreceptor afferents was significantly greater in the adult (15.7 +/- 1.6 Hz, mean +/- S.E.M., n = 18) and 10-15 days (11.2 +/- 4.2, n = 8) compared to rats of 1-2 days (4.3 +/- 0.7, n = 14) and 4-7 days of age (3.9 +/- 1.0, n = 7). 4. At 2 min into the anoxia period, all chemoreceptor activities were reduced from their peak discharge levels. At 35 degrees C, this decrease was significantly greater in the adult compared to the newborn. 5. During the period of decreased activity during anoxia, the chemoreceptor discharge could not be increased by inter-stream injection of acetylcholine (100 micrograms) or dopamine (100 micrograms), although these drugs were effective in increasing discharge rate prior to hypoxia. 6. We conclude that: (1) postnatal maturation of chemoreceptor sensitivity to hypoxia is present in vitro, (2) maturation occurs between the first and second week after birth in the rat, and (3) the decrease in activity during prolonged anoxia is not greater in the newborn compared to the adult. Thus, maturational changes occur in the sensitivity of the glomus cell-nerve ending complex to hypoxia.

Role of the carotid chemoreceptors in the hyperpnea of exercise in the cat

The role of the carotid chemoreceptors in the hyperpnea of exercise was investigated. The activity of the sinus nerve of the cat was monitored while the blood supply to the carotid body was controlled independently of the systemic circulation. By this technique, fluctuations in the arterial blood gases during a short interval of exercise induced by electrical stimulation of hindlimb muscles were unable to affect the chemoreceptor activity. While minute ventilation increased by an average of 51%, chemoreccptor discharge was found to be unchanged in 12 experiments, 6 while perfusing with normoxic blood and 6 while perfusing with hypoxic blood. Thus, it must be concluded that alteration of carotid chemoreceptor sensitivity does not occur during artificially induced exercise in anesthetized cats. However, the difference in the time course of ventilation following the initiation of artificially induced exercise between cats and other species does not allow it to be ruled out in other species, including man. Indirect evidence is against such a role.