Inhibitory sympathetic action on the carotid body responses to sustained hypoxia

The mammalian carotid bodies receive sympathetic innervation from the superior cervical ganglion. The purposes of the present study were: (10 to investigate whether sympathetic innervation influences the carotid body response to hypoxia, and, if so, (2) to determine the involvement fo adrenoceptors in these influence. Chemo-sensory activity was recorded from clearly identifiable action potentials form the carotid sinus nerve in 20 anaesthetized, paralyzed and artificially ventilated cats. Chemoreceptor responses to sustained isocapnic hypoxia (30 min, duration) were compared before and after carotid body sympathectomy (n = 8 cats). In response to low P_CO2, chemoreceptor discharge increased during the first 10 min, and plateaued for the rest of the hypoxic challenge. After sympathectomy, chemoreceptor response in the initial 10 min was the same; whereas, the magnitude of the response in remaining 20 min was significantly greater than controls (P < 0.01). Systemic administration of SKF-86466, an α₂-adrenoceptor antagonist augmented the hypoxic response by 805 n = 6 cats. Ini presence of α₂-antagoonist, sympathectomy had no further effect on the hypoxic response, but the magnitude of potentiation was less than with intact sympathetic innervation (34% vs 80%; P < 0.01; n = 6 cats. From these results,it is concluded that (1) sympathetic innervation exerts an inhibitory influence on chemoreceptor response to sustained hypopxia, and (2) this inhibitory influence is mediated at least in part by α₂-adrenoceptors. The inhibitory effects of sympathetic innervations could be of importance in the efferent regulation of the carotid body activity during sustained hypoxia.     

Secretory responses of intact glomus cells in thin slices of rat carotid body to hypoxia and tetraethylammonium

We have developed a thin-slice preparation of whole rat carotid body that allows us to perform patch-clamp recording of membrane ionic currents and to monitor catecholamine secretion by amperometry in single glomus cells under direct visual control. In normoxic conditions (P(O(2)) approximately 140 mmHg; 1 mmHg = 133 Pa), most glomus cells did not have measurable secretory activity, but exposure to hypoxia (P(O(2)) approximately 20 mmHg) elicited the appearance of a large number of spike-like exocytotic events. This neurosecretory response to hypoxia was fully reversible and required extracellular Ca(2+) influx. The average charge of single quantal events was 46 +/- 25 fC (n = 218), which yields an estimate of approximately 140,000 catecholamine molecules per vesicle. Addition of tetraethylammonium (TEA; 2-5 mM) to the extracellular solution induced in most (>95%) cells tested (n = 32) a secretory response similar to that elicited by low P(O(2)). Cells nonresponsive to hypoxia but activated by exposure to high external K(+) were also stimulated by TEA. A secretory response similar to the responses to hypoxia and TEA was also observed after treatment of the cells with iberiotoxin to block selectively Ca(2+)- and voltage-activated maxi-K(+) channels. Our data further show that membrane ion channels are critically involved in sensory transduction in the carotid body. We also show that in intact glomus cells inhibition of voltage-dependent K(+) channels can contribute to initiation of the secretory response to low P(O(2)).     

Loss of peripheral chemoreflexes to hypoxia after carotid body removal in the rat

Young Sprague-Dawley female rats (16-18 days old) were subjected to bilateral carotid glomectomy or the sham operation under halothane anaesthesia. After recovery, the rats were placed with non-operated peers. One to six months later glomectomised, sham-operated and control animals were anaesthetized with sodium pentobarbital. Respiratory minute volume and arterial pressure were recorded. Respiratory responses to few-breath administration of oxygen or nitrogen, and arterial pressure responses to carotid occlusion or tugging were tested. Oxygen produced transient hypoventilation and nitrogen transient hyperventilation in control, sham and 3 of 31 glomectomised rats. Bilateral vagotomy did not abolish these responses. In only those 3 glomectomised rats was carotid glomus tissue histologically identifiable. Carotid occlusion raised and tugging lowered arterial pressure in all animals. Glomectomy did not affect serum levels of GH, FSH, LH or PRL hormones but produced right ventricular hypertrophy. We conclude that peripheral chemoreception requires the presence of glomus tissue.     

Chronic hypoxia modulates the function and expression of melatonin receptors in the rat carotid body

Melatonin modulates the carotid chemoreceptor response to chemical stimuli, and chronic hypoxia changes circadian activities and carotid body function. The purpose of this study was to test the hypothesis that chronic hypoxia alters the function and expression of melatonin receptors in the rat carotid body. Effects of melatonin on the carotid responses to hypercapnic acidosis and to hypoxia were determined by spectrofluorometric measurement of cytosolic calcium ([Ca(2+)](i)) in fura-2-loaded type-I (glomus) cells dissociated from carotid bodies obtained from normoxic (Nx) or chronically hypoxic (CH) rats breathing 10% oxygen for 4 wk. In the Nx control, melatonin concentration dependently attenuated the peak [Ca(2+)](i) response to hypercapnic acidosis, whereas it augmented the [Ca(2+)](i) response to cyanide or deoxygenated buffer. Yet, melatonin enhanced the peak [Ca(2+)](i) responses to hypercapnic acidosis or hypoxia in the CH glomus cells. An agonist of melatonin receptors, iodomelatonin also elevated the hypercapnic or hypoxic responses in the CH groups. The melatonin-induced changes in the [Ca(2+)](i) responses were abolished by pretreatment with nonselective mt(1)/MT(2) antagonist, luzindole, and by MT(2) antagonists, 4-phenyl-2-propionamidotetraline or DH97. These findings suggest a functional modulation of melatonin receptors in the glomus cells in chronic hypoxia. To evaluate the level of expression of the melatonin receptors, in situ hybridization study with antisense mt(1) and MT(2) receptor mRNA oligonucleotide probes was performed on the Nx and CH carotid bodies. There were significant increases in the expression of mt(1) and MT(2) receptors in the CH comparing with the Nx group. Taken together, our results suggest an upregulation of the carotid expression of melatonin receptors by chronic hypoxia, which modulates the carotid response to melatonin for the circadian influence on breathing.     

Respiratory and cardiovascular responses to hyperoxia, hypoxia and hypercapnia in the renal hypertensive rabbit: role of carotid body chemoreceptors

We tested the hypothesis that in renal hypertension the increased peripheral vascular resistance of neurogenic origin might be due to a reflex through resetting of the carotid body chemoreceptors. The reflex respiratory and cardiovascular functions of the carotid bodies were studied in a one-kidney wrapped hypertension model in conscious rabbits, and compared with a control group of animals, by breathing 100% oxygen, three hypoxic gas mixtures to which were added sufficient CO2 to maintain the PaCO2 constant, and 2 and 4% CO2 in 21% O2 and N2. In the control state (breathing room air) the renal hypertensive animals had a slightly higher respiratory minute volume, a higher level of arterial blood pressure and increased calculated systemic vascular resistance, compared with the normal group, but there was no difference in cardiac output. Hyperoxia had no consistent effect on respiration, heart rate or arterial blood pressure. Increasing degrees of isocapnic hypoxia caused the same degree of hyperventilation and bradycardia in both groups of animals. The arterial blood pressure did not change in either group but there was a transient increase in systemic vascular resistance in the renal hypertensives breathing 9 and 7.5% O2. The respiratory responses to 2 and 4% CO2 were similar in the two groups of animals. In the renal hypertensive animals, serial sections of the carotid bodies showed pathological changes, including subendothelial proliferation in vessels supplying the carotid bodies with narrowing of their lumens, fragmentation of the elastic laminae of the media, hypertrophy of the smooth muscle and extensive fibrosis with occasional haemorrhages. The capillaries, however, were normal. The rostral-caudal lengths of the carotid bodies were similar in the two groups. In view of our findings we conclude that the relatively normal carotid chemoreceptor responses in renal hypertensive rabbits may, in part at least, be the result of the carotid body blood flow through the partially occluded vessels being maintained at near normal levels by the elevated blood pressure.     

NO and the hypometabolic and hypothermic responses to hypoxia in the rat

Nitric oxide (NO) has been identified as a possible mediator of the thermoregulatory and respiratory responses to hypoxia. The present study was designed to assess the effects of an NO donor (S-nitroso-N-acetylpenicillamine, SNAP) injected systemically (0.5-2.0 mg/kg) in unanesthetized adult rats studied at ambient temperatures (Ta) of 26, 24, and 15 degrees C. The metabolic rate (VO2), ventilation (V), and colonic temperature (Tc) were recorded while the animals were exposed to normoxia or to hypoxia (FI(O2)=0.11). During normoxia, at all values of Ta, SNAP increased VO2 and V and the decrease in Tc observed following saline injection was prevented or attenuated. However, SNAP did not modify the hypometabolic, hypothermic, and hyperventilatory responses to hypoxia. We concluded that, confirming previous studies, NO may play a role in the control of VO2 and V in normoxia. The failure to observe any effect following the systemic NO donor injection during hypoxia may indicate opposing actions of the drug at different sites resulting in no net changes in ventilatory and metabolic responses to hypoxia.     

Selective inhibition of the carotid body sensory response to hypoxia by the substance P receptor antagonist CP-96,345.

Carotid bodies are sensory organs for monitoring arterial oxygen and CO2. Previous studies have shown that chemoreceptor tissue contains substance P (SP) and exogenously administered SP augments chemosensory discharge. In the present study, we examined the physiological importance of SP in carotid body chemoreception by using a selective nonpeptide SP [neurokinin (NK) 1] receptor antagonist CP-96,345. In experiments performed on anesthetized cats, sensory discharge was recorded from the carotid body in situ. To control for alterations in blood flow, additional studies were conducted on the carotid body in vitro. In in vivo studies, close carotid body (intraarterial) administration of CP-96,345 attenuated the sensory response to hypoxia in a dose-dependent manner with 73% of the response abolished at doses of 0.3-0.6 mg/kg. Comparable doses of the (2R,3R)-enantiomer had no effect on hypoxia-induced excitation, indicating that the effect of CP-96,345 was not due to nonspecific action. In contrast, the carotid body response to high CO2 was not affected by CP-96,345, implying that only the hypoxic response is mediated by NK-1 receptor and confirming that the effect of the SP antagonist was not due to nonspecific actions. Marked attenuation of the sensory response to hypoxia was also obtained in the carotid body in vitro, suggesting that the effects of the NK-1 antagonist were not secondary to cardiovascular changes. These results demonstrate that CP-96,345 attenuates or abolishes the chemosensory response to hypoxia but not to CO2 and suggest that SP mediates the hypoxia-induced sensory excitation in the cat carotid body via NK-1 receptor activation.     

The ventilatory response to hypoxia in the newborn lamb after carotid body denervation

Neonates of various species including lambs respond to hypoxia by a transient hyperventilation followed by a VE depression (diphasic response). To better delineate the role of the carotid chemoreceptors and that of the central depressive/inhibitive effect of hypoxia on minute ventilation, we have studied the VE response of 4-day-old carotid body-deprived lambs (CBD) during successive exposure to moderate and severe (0.12 and 0.07 FIO2) hypoxia. The carotid body denervation was done to abolish most of the chemoreceptor stimulating effect on VE during hypoxia and to allow for central depression/inhibition of VE during hypoxia. In the CBD lambs, baseline VE was 461 +/- 81 (SE) ml X (kg X min)-1. It increased to 532 +/- 79 ml X (kg X min)-1 and to 541 +/- 75 ml X (kg X min)-1, to 0.12 FIO2 and 0.07 FIO2. These VE increases did not reach level of significance (P greater than 0.05). After 2-5 min of both levels of hypoxia VE dropped respectively to 460 +/- 60 ml X (kg X min)-1 and to 459 +/- 38 ml X (kg X min)-1. No marked ventilatory depressions were noted but VE had only returned to baseline. It is concluded that, in the denervated newborn lamb, the centrally mediated depressive effect of hypoxia is small and not sufficient to explain the diphasic VE response of the intact lamb to steady state hypoxia. Analysis of the magnitude of the hyperventilation and the VE damping pre-hypoxic levels occurring with sustained hypoxia in newborns of various species suggests that the immaturity of the O2-sensitive chemoreceptor rather than the central effect of hypoxia is the determinant factor of the diphasic response of newborn mammals to hypoxic hypoxia.     

Amiloride and carotid body chemoreception of hypercapnia and hypoxia

The sodium-proton (Na(+)-H+) antiporter has been found in virtually every tissue where its presence has been investigated. Its principal physiological role is to regulate intracellular pH (pHi). Amiloride (10(-3)-10(-4) M) is a known blocker of the antiporter when Na is present in normal physiological concentrations (130-140 x 10(-3) M). In order to determine if the Na(+)-H+ antiporter participated in the chemoreception of hypercapnia or hypoxia anesthetized, paralyzed, artificially ventilated cats were fitted with a loop in the right common carotid artery for the selective perfusion of the carotid body. Neural activity (imp/10 sec) was recorded from single or few fiber preparations during hypercapnia (PaCO2 = 48-64 Torr) while the carotid body was perfused with Krebs-Ringer bicarbonate solution for 2.5 min, then with its own hypercapnic arterial blood (4 min), then with Krebs-Ringer bicarbonate solution containing 0.6-0.8 x 10(-3) M amiloride (2.5 min), then with its own hypercapnic blood (4 min). After 20 min of rest the protocol was repeated during hypoxia (PaO2 = 35-45 Torr). The carotid body response to hypercapnic blood was unaffected by a preceding perfusion of the amiloride-containing solution but the response to hypoxic blood was decreased by 25% by the amiloride-containing solution. The data suggest the possibility of different mechanisms being involved in the chemoreception of hypercapnia and hypoxia.     

Ventilatory responses to hypercapnia and hypoxia following chronic hypercapnia in the rat

This study investigated the effects of an 18 week exposure to 10% CO(2) in air on minute ventilation (V(E)), breathing pattern and the chemoresponiveness of rats to hypoxic and hyperoxic stimuli. We found that V(E) remained elevated over the 18 weeks. Nonetheless, the breathing pattern changed significantly. Tidal volume increased and the durations of inspiration and the total cycle decreased. After the sustained hypercapnia the mean Pa(CO(2)) was 72.0+/-5.1 (S. D.) mmHg. Every 6 weeks the chemoresponiveness of the CO(2)-exposed rats was tested by an acute exposure sequentially to room air, then a 6% O(2), 10% CO(2) and 84% N(2) gas mixture, and finally a 90% O(2) in 10% CO(2) mixture. On either room air or the hyperoxic-hypercapnic mixture V(E) fell to its pre-hypercapnic level. On the hypoxic-hypercapnic mixture V(E) increased significantly. These results demonstrate that the initial stimulating effect of 10% CO(2) on V(E) persisted for the entire 18 weeks without altering hypoxic or hyperoxic ventilatory responses.     

Carotid body chemoreceptor and ventilatory responses to sustained hypoxia and hypercapnia in the cat

To understand the role of carotid chemoreceptor activity in the ventilatory responses to sustained hypoxia (30 min) the following measurements were made in cats anesthetized with alpha-chloralose: (1) carotid chemoreceptor and ventilatory responses to isocapnic hypoxia and to hypercapnia during hyperoxia; (2) carotid chemoreceptor responses to isocapnic hypoxia after dopamine receptor blockade; and (3) ventilatory responses to hypoxia after bilateral section of carotid sinus nerves (CSN). Transition to hypoxia (PaO2 approximately equal to 52 Torr) from hyperoxia gradually increased carotid chemoreceptor activity by ten fold and ventilation by two fold without any detectable overshoot. Termination of isocapnic hypoxia with hyperoxia (PaO2 greater than 300 Torr) at 30 min promptly restored the carotid chemoreceptor activity to prehypoxic level. Ventilation also decreased promptly, but remained above the control value. Induction of hypercapnia (from 31.8 Torr to 43.9 Torr) during hyperoxia was followed by a prompt increase in the chemoreceptor activity by four fold which subsequently diminished, and by a gradual four fold increase in ventilation. Termination of hypercapnia after 30 min was followed by a prompt return of chemoreceptor activity and by a slow return of ventilation to near control levels. Dopamine receptor blockade increased carotid chemoreceptor responsiveness to acute hypoxia but did not alter the response pattern during sustained hypoxia. After bilateral CSN section, ventilation decreased during maintained hypoxia. Thus, a stimulatory peripheral and inhibitory central effects of hypoxia could produce a biphasic ventilatory response to short-term hypoxia in the anesthetized cat with intact CSN but did not manifest it. The results suggest that the chemosensory input not only promptly stimulates ventilation but also prevents the subsequent depressant effect of hypoxia on the brain-stem respiratory mechanisms and hence presumably a biphasic ventilatory response in the anesthetized cat.     

Effects of carotid body sympathetic denervation on ventilatory acclimatization to hypoxia in the goat

Our objective was to test the hypothesis that diminishing sympathetic input to the carotid body (CB) during prolonged exposure to hypoxia results in increased CB afferent activity and increased ventilatory drive. Six awake goats were studied prior to and following sectioning of the efferent sympathetic input to the CB from the superior cervical ganglion. Ventilatory responses to acute and prolonged isocapnic hypoxia (Pa_O2 40 Torr) and drugs (norepinephrine and dopamine, 0.5, 1.0 and 5.0 μg·kg⁻¹·min⁻¹) were collected prior to the denervation. One week and 3–4 weeks following the sympathetic denervation, the animals were restudied following the above protocol. Ventilation was significantly lower following sympathetic denervation in normoxia and during the hypoxic exposure. However, the response to acute hypoxia and the time-course of ventilatory acclimatization to hypoxia was not altered by sympathetic denervation. All doses of norepinephrine and dopamine significantly inhibited V̇e in a dose-dependent manner. Sympathetic denervation did not significantly alter the response to the drug infusions. The sympathetic innervation to the CB does not appear to play a role in either the acute or prolonged ventilatory responses to hypoxia in the awake goat, but may affect overall ventilation.     

Adrenomedullin expression and function in the rat carotid body

Adrenomedullin (AM) immunoreactivity has been found in granules of the glomus (type I) cells of the carotid bodies in rats. The identity of these cells was ascertained by colocalization of immunoreactivities for AM and tyrosine hydroxylase in their cytoplasm. Exposure of freshly isolated carotid bodies to synthetic AM resulted in a concentration- and time-dependent degranulation of glomus cells as measured by dopamine (DA) release. DA release reached a zenith 30 min after exposure to AM (94.2% over untreated controls). At this time-point, the response to AM was similar to the one elicited by 5 min of exposure to 100 mM K+. Nevertheless, injection of 1 micro l 60 nM AM/g body weight into the tail vein of the rats did not induce statistical differences in DA release from the carotid bodies. Exposure of the oxygen-sensitive cell line PC-12 to hypoxia elicited an increase in AM mRNA expression and peptide secretion into serum-free conditioned medium. Previous data have shown that elevation of AM expression under hypoxia is mediated through hypoxia-inducible factor-1, and that exposure of chromaffin cells to AM results in degranulation. All these data suggest that AM is an important autocrine regulator of carotid body function.     

Role of alpha 2-adrenergic receptors in the carotid body response to isocapnic hypoxia.

Alpha-2 adrenergic receptors have been identified in numerous tissues containing norepinephrine. Carotid bodies are sensory organs that detect changes in partial pressure of arterial oxygen and contain substantial amounts of norepinephrine. However, neither the presence nor the functional significance of alpha 2-receptors in the carotid body is known. The purpose of the present study was two-fold: (1) to determine the alpha 2-receptor density in the carotid body and (2) to assess their participation in the chemoreceptor responses to hypoxia. Experiments were performed on 27 anesthetized, paralyzed and artificially ventilated cats. Alpha-2 receptor binding determined by [125I]p-iodoclonidine averaged 10 +/- 2 fmol/mg of protein (n = 18 carotid bodies). Intracarotid infusion of an alpha 2-agonist (guanabenz; 0.5, 1, and 5 micrograms/min for 5 min) caused a dose-dependent depression of the baseline activity and 5 micrograms/min reduced the magnitude of the chemoreceptor response to isocapnic hypoxia by 61% (n = 8). Systemic administration of an alpha 2-antagonist (SKF-86466, 0.5-2 mg/kg) prevented the effects of guanabenz on the chemoreceptor activity. Furthermore, alpha 2-antagonist (0.5-2 mg/kg, i.v.) alone significantly increased baseline discharge by 68% and potentiated the chemoreceptor response to isocapnic hypoxia by 46% (n = 10). These results demonstrate that (1) alpha 2-adrenergic receptors are present in the carotid body and (2) they exert an inhibitory influence on chemoreceptor response to hypoxia. It is suggested that norepinephrine may tune the carotid body responses to hypoxia in part by its action on alpha 2-receptors.     

Differences in time-related cardiopulmonary responses to hypoxia in three rat strains

The cardiopulmonary profile of three rat strains (Sprague-Dawley, Wistar and High altitude-sensitive) was compared upon exposure to hypoxia (9% O2) for 0, 7 or 14 days. No differences were observed among the in vitro contractile (ET-1) and relaxant (carbachol) responses of pulmonary artery isolated from the three strains during normoxia. Chronic hypoxia decreased ET-1 contractile responses and diminished relaxant responses to carbachol similarly in all strains. In Sprague-Dawley, Wistar and High altitude-sensitive rats, pulmonary arterial pressure rose time-dependently and was elevated by 108%, 116% and 167%, respectively, after 14 days of hypoxia compared to normoxic controls. Right ventricular hypertrophy was increased by 51%, 93% and 55%, respectively, at 14 days. Hypoxia-induced hypertrophy and medial thickening in the pulmonary vasculature were more pronounced in High altitude-sensitive rats. Sprague-Dawley exhibited hypoxia-induced airway hyperresponsiveness to intravenous methacholine, but there were no hypoxia- or strain-related differences in in vitro tracheal contractility. Although each strain exhibited greater sensitivity for a particular hypoxia-induced parameter, pulmonary vascular functional and structural changes suggest that High altitude-sensitive rats represent a choice model of hypoxia-induced pulmonary hypertension.     

Ventilatory responses to hypocapnic vertebral artery perfusion in intact and carotid body denervated dogs

The ventilatory responses to step changes in vertebral artery PCO2 were investigated in intact and carotid body denervated dogs. The steady-state ventilatory responses of the denervated dogs were less than those of intact dogs. However, when expressed as a ratio to the control ventilation there was no difference between the two groups. While the arterial PCO2 was held at 56 mm Hg by adding CO2 to the inspired air the perfusion of the vertebral arteries was switched from the dog's own arterial supply to hypocapnic blood. The ventilation of the denervated dogs decreased at a faster rate (half time = 130 +/- 9 sec) to a level less than the room air control ventilation. The ventilation in the intact dogs decreased at a slower rate (half time = 184 +/- 23 sec) and was maintained above the room air control level after ten minutes of hypocapnic perfusion. Increasing the medullary blood flow, as measured with radiolabeled microspheres, augmented the rate of decline of ventilation in intact dogs. We conclude, (1) the influence of the peripheral chemoreceptors appears to increase as central drive is decreasing, and (2) the remaining time course of the decrease in ventilation is related to the rate of brain stem perfusion.     

The effect of hypoxia on the response of the carotid body chemoreceptor to potassium in the anaesthetized cat

The effect of hypoxia on the response of the carotid chemoreceptor to potassium has been investigated in anaesthetized cats. After an initial period of ventilation on air, FIO2 was reduced to 0.1-0.13 to give a mean PaO2 of 44 mm Hg. KCl was infused intravenously to raise arterial K+ to approximately 6 mM and hold it at that level. For 8 experiments in 7 cats, mean chemoreceptor discharge increased from 1.9 impulses.sec-1 on air, to 7.3 impulses.sec-1 during hypoxia to a peak of 12.2 impulses.sec-1 after the first 0.25 min of the potassium infusion. The initial speed of response and pattern of adaptation were similar to those seen in normoxic cats, but the combined effects of hypoxia and hyperkalaemia on carotid chemoreceptor discharge were greater than the sum of the individual effects.