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Ventilation is peripherally controlled by afferent activity arising from the peripheral chemoreceptors. In the rat, chemosensory activity is conveyed to the central nervous system through axons of neurons located in the nodose-petrosal-jugular-complex. These neurons have distinct electrophysiological properties, including a persistent Na + current. Acute blockade of this current with phenytoin and other anti-epileptic drugs reduces normoxic chemosensory activity and responses to acute hypoxia. However, because anti-epileptic therapy is prolonged and there is no information on the effects of chronic phenytoin treatment on peripheral chemosensory activity, we studied the effects of long-lasting phenytoin treatment (~25 days) on afferent chemosensory activity, on a wide range of oxygen inspiratory fractions. Osmotic pumps containing dissolved phenytoin (166 mg/mL) or vehicle (daily flow: 60 µL) were implanted subcutaneously in male adult Sprague Dawley rats. At the end of the treatment, the animals were anesthetized and carotid sinus nerve activity was recorded in vivo. Afferent chemosensory activity in normoxia was not significantly different between control (71.2±2.2 Hz) and phenytoin treated (95.4±2.1 Hz) rats. In contrast, carotid body chemosensory responses to acute hypoxic challenges were markedly reduced in phenytoin treated rats, specifically in the lowest part of the hypoxic range (control 133.5±18.0 Hz vs phenytoin treated 50.2±29.4, at 5% F I O 2 ). Chronic phenytoin treatment severely impaired the chemosensory responses to acute hypoxia, suggesting that long-term phenytoin treatment in patients may result in a reduced peripheral respiratory drive together with a reduction in the respiratory responses to hypoxic challenges. [ABSTRACT FROM AUTHOR]

Hypoxia-induced chemosensory activity in the carotid body (CB) may be enhanced by the sympathetic regulation of vascular tone in the CB. In the present study, we recorded cervical sympathetic nerve activity in rats exposed to hypoxia, and examined noradrenaline (NA)- and serotonin (5-HT)-induced intracellular Ca 2+ ([Ca 2+ ] i ) responses in smooth muscle cells and pericytes in isolated blood vessels from the CB. Multifiber electrical activity recorded from the cervical sympathetic trunk was increased during the inhalation of hypoxic gas. NA induced [Ca 2+ ] i increases in smooth muscle cells in arteriole specimens, whereas 5-HT did not cause any [Ca 2+ ] i responses. However, NA did not induce [Ca 2+ ] i increases in pericytes in capillaries, whereas 5-HT did and this response was inhibited by the 5-HT 2 receptor antagonist, ketanserin. In conclusion, cervical sympathetic nerves enhanced by hypoxia may reduce blood flow in the CB in order to increase chemosensitivity. Thus, hypoxic chemosensitivity in the CB may involve a positive feedback mechanism via sympathetic nerves. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: Glomus cells within the carotid body are known to respond to hypoxic stimuli. Recently, these cells have been shown to express the long form of the leptin receptor (Ob-Rb). However, whether these glomus cells expressing the Ob-Rb are activated by hypoxic stimuli is not known. Therefore, in this study we investigated whether intermittent hypoxia (IH) or changes in circulating levels of leptin induced phosphorylated signal transducer and activator of transcription 3 (pSTAT3), the immediate early gene c-fos protein, or fos-related antigen-1 protein (Fra-1) within carotid body glomus cells that expressed the Ob-Rb, and within neurons of the petrosal (PG) and nodose (NG) ganglia. Rats were subjected to IH (120s normoxia, 80s hypoxia for 8h) or normoxia (8h), or intravenous injections of leptin (50 or 200ng/0.1mL) or the vehicle saline. Plasma leptin levels were measured in animals exposed to IH and normoxia. Exposure to 8h of IH increased plasma leptin levels greater than 2-fold compared to normoxic controls. Animals were then perfused with Zamboni''s fixative, and the region of the carotid bifurcation containing the carotid body and PG/NG complex was removed, paraffin embedded and sectioned at 6μm for immunohistochemical processing. Carotid body glomus cells were identified by their expression of tyrosine hydroxylase immunoreactivity. These glomus cells also expressed the OB-Rb and were found to express pSTAT3-, fos-, and Fra-1-like immunoreactivity in response to both IH and systemic leptin injections. IH and leptin injections also increased fos and Fra-1 like expression in the PG, NG and jugular ganglion. Taken together, these data suggest IH alters circulating leptin which in turn activates directly carotid body glomus cells to exert a modulatory effect on the peripheral chemoreceptor reflex. [Copyright &y& Elsevier]

Abstract: Hypothyroidism can lead to depressed breathing. We determined if propylthiouracil (PTU)-induced hypothyroidismin hamsters (HH) altered dopamine D1 receptor expression, D1 receptor-modulated ventilation, and ventilatory chemoreflex activation by hypoxia or hypercapnia. Hypothyroidism was induced by administering 0.04% PTU in drinking water for 3months. Ventilation was evaluated following saline or 0.25mg/kg SCH 23390,a D1 receptor antagonist, while awake hamsters breathed normoxic (21% O2 in N2), hypoxic (10% O2in N2) and hypercapnic (5% CO2 in O2)air. Relative to euthyroid hamsters (EH), HH exhibited decreased D1 receptor protein levels in carotid bodies, striatum, and hypothalamic paraventricular nucleus, but not in the nucleus tractus solitarius. Relative to EH, HH exhibited lower ventilation during exposure to normoxia, hypoxia, or hypercapnia, but comparable ventilatory responsiveness to chemoreflex activation. SCH23390 decreased ventilation of EH hamsters exposed to normoxia, hypoxia, and hypercapnia. In HH SCH23390 increased ventilation during baseline normoxia and did not affect ventilation during exposure to hypoxia and hypercapnia, resulting in reduced ventilatory responsivess to chemoreflex activation by hypoxia and hypercapnia. Furthermore, in HH D1 receptor protein levels are decreased in several brain regions and within the carotid bodies. Moreover, D1 receptor-modulation of breathing at rest and during gas exposures were depressed in EH but not HH. [Copyright &y& Elsevier]

Abstract: The enhanced carotid body (CB) chemosensory response to hypoxia induced by chronic intermittent hypoxia (CIH) has been attributed to oxidative stress, which is expected to increase the expression of chemosensory modulators including chemoexcitatory pro-inflammatory cytokines in the CB. Accordingly, we studied the time-course of the changes in the immunohistological expression of TNF-α, IL-1β, IL-6, ET-1, iNOS, eNOS and 3-nitrotyrosine in the CB, along with the progression of enhanced CB chemosensory responses to acute hypoxia in male Sprague-Dawley rats exposed to CIH (5%O2, 12times/h per 8h) for 7, 14 and 21days. Exposure to CIH for 7days resulted in a sustained potentiation of CB chemosensory responses to acute hypoxia, which persisted until 21days of CIH. The chemosensory potentiation was paralleled by an increased 3-nitrotyrosine expression in the CB. On the contrary, CIH produced a transient 2-fold increase of ET-1 immunoreactivity at 7days, a decrease in eNOS immunoreactivity, and a delayed but progressive increase of TNF-α, IL-1β and iNOS immunoreactivity, which was not associated with changes in systemic plasma levels or immune cell invasion within the CB. Thus, present results suggest that the local expression of chemosensory modulators and pro-inflammatory cytokines in the CB may have different temporal contribution to the CB chemosensory potentiation induced by CIH. [Copyright &y& Elsevier]

Abstract: Leptin is known to play a role in the modulation of metabolism and control of breathing acting mainly on central nervous structures, although additional actions on peripheral arterial chemoreceptors have also been suggested in the literature. We therefore examined by means of immunohistochemistry the expression of leptin and leptin receptors in the carotid bodies of rats and humans. Leptin expression and relative expression of leptin receptor isoforms were also studied in rats by real-time PCR. No leptin or leptin receptor immunoreactivities were visible in the type II cells of either series. In rat carotid bodies, diffuse positive stainings for leptin and leptin receptors (both with antibody recognizing all receptor isoforms and antibody specific for Ob-Rb) were observed in type I cells. In human carotid bodies, the mean percentage (±standard error) of leptin immunoreactive type I cells was 39.4%±5.1% and the percentages of leptin receptor immunoreactive type I cells were 57.3%±3.9% with antibody recognizing all receptor isoforms and 33.3%±4.2% with antibody specific for isoform Ob-Rb. Double immunofluorescences with anti-tyrosine hydroxylase (type I cell marker) and anti-glial fibrillary acidic protein (type II cell markers) confirmed the selective location of leptin and Ob-Rb in type I cells. Real-time PCR showed the expression of leptin and Ob-Ra, Ob-Rb, Ob-Rc and Ob-Rf isoform mRNA in the rat carotid body, levels of expression being Ob-Rf>Ob-Rc>>Ob-Ra>>Ob-Rb. Ob-Re mRNA was not detected. The above findings suggest a role of circulating or locally produced leptin in the regulation of chemoreceptor discharge and/or metabolic sensing function, by means of direct action on type I cells. [Copyright &y& Elsevier]

Abstract: The carotid body is the main peripheral arterial chemoreceptor and it is essential to initiate the cardiovascular and respiratory compensatory reflex responses to a decrease in the arterial oxygen. The carotid body chemoreceptor (type-I) cells respond to hypoxia with membrane depolarization, voltage-gated Ca2+ entry and secretion of transmitters. A key step in this response is the inhibition of a TASK-like background K+ current. It has been reported that TASK-K+ channels can be modulated by G-protein coupled receptors, such as the muscarinic acetylcholine receptor (mAChRs). Since there is a proposed role for ACh as an autocrine/paracrine modulator of the carotid body function, we have investigated the possible regulation of the background K+ current by mAChRs. In identified type-I cells, methacholine (100µM) or muscarine (50µM) increased intracellular Ca2+ levels. In cell-attached patch recordings, TASK-K+ background channel activity was reduced by ∼50% during mAChR activation and by the diacylglycerol analogue oleoylacetylglycerol (OAG, 20µM). The co-application of both metacholine and OAG do not further inhibit K+ channel activity. In addition, two chemically different inhibitors of protein kinase C activity, calphostin C (100nM) and chelerythrine (50µM) are both able to suppress the muscarinic inhibition of the TASK-like K+ channel and to increase channel activity in the absence of mAChR agonists. Our results suggest a muscarinic regulation of the TASK-like K+ current in rat carotid body type-I cells through a PLC/PKC-dependent pathway. Additionally, our findings are consistent with an autocrine/paracrine role for cholinergic autoreceptors present within the carotid body. [Copyright &y& Elsevier]

Abstract: The carotid body (CB) is a polymodal chemosensor of arterial blood located next to the internal carotid artery. The basic chemosensing unit is composed of the neurotransmitter (NT)-containing glomus cells (GCs) and the sensory afferent fibers synapsing onto the GCs. Nicotinic and muscarinic receptors have been found on both the sensory afferent fibers and on the GCs. Neural output from the CB (CBNO) increases when arterial blood perfusing it is hypoxic, hypoglycemic, hypercapnic, or acidic. The increased CBNO due to GC release of excitatory NTs must be preceded by an entrance of calcium into the GCs. With repeated release of ACh from the GCs, cholinergic receptors could become desensitized, particularly nicotinic receptors which function as calcium channels. The purpose of the present study was to see if adenosine (ADO), known to alter receptor sensitivities, could attenuate or eliminate any desensitization of the nicotinic receptors occurring during the repeated application of ACh. Cat CBs were harvested with techniques approved by the University''s Animal Care/Use Committee. The GCs were cultured and prepared for detecting [Ca++]i with standard techniques. Repeated application of ACh produced a progressively decreasing increase in [Ca++]i. With the use of ADO or an A2A ADO receptor agonist the decrease was avoided. Though ADO also increased GC [Ca++]i, the sum of ADO increase and ACh increase, when superfused separately, was less than the increase when they were both included in the same superfusion. This suggested the possible involvement of a new path in the action. Potential mechanisms to explain the phenomena are discussed. [Copyright &y& Elsevier]

Abstract: Neonatal caffeine treatment (adenosine receptor antagonist, 15 mg/kg/day, between postnatal days 3 and 12) affects respiratory patterns in adult male but not female rats as shown by an increase in the respiratory frequency in the early phase of response to hypoxia and an increase in the tidal volume in the late phase of response. Here, we tested the hypothesis that these changes are correlated with modified expression of adenosine receptors in the chemoreflex pathway. Carotid bodies, nucleus tractus solitarii, and superior cervical ganglia were collected from 3-month-old male and female rats that were either naive (not manipulated during the neonatal period) or treated with caffeine (NCT) or water (NWT) between postnatal days 3 and 12 by gavage. Western blot analysis was used to assess the expression of adenosine A1 and A2A receptors and tyrosine hydroxylase, the rate-limiting enzyme for dopamine synthesis. In male rats, there was a 37% increase in the level of A2A receptor and a 17% decrease in tyrosine hydroxylase in the carotid body of NCT (p <0.001) as compared to NWT rats. In the nucleus tractus solitarius, we found a 13% and 19% decrease in A1 receptor expression in NWT and NCT rats (p <0.01), respectively, compared to naive rats. In the superior cervical ganglion, there was no change in A1 receptor, A2A receptor, and tyrosine hydroxylase expression. In female rats, the only changes observed were decreases of 12% and 15% in A1 receptor levels in the nucleus tractus solitarius of NWT and NCT rats (p <0.01), respectively, compared to naive rats. We conclude that NCT induces long-term changes in the adenosine receptor system. These changes may partially explain the modifications of the respiratory pattern induced by NCT in adults. The increased expression of the adenosine A2A receptor (specific to male rats), combined with the decreased tyrosine hydroxylase expression in the carotid body, suggests that NCT affects adenosine–dopamine interactions regulating chemosensory activity. [Copyright &y& Elsevier]

Abstract: The carotid body (CB) is a polymodal sensor which increases its neural output to the nucleus tractus solitarii with a subsequent activation of several reflex cardiopulmonary responses. Current reports identify acetylcholine (ACh) and adenosine triphosphate (ATP) as two essential excitatory neurotransmitters in the cat and rat CBs. This study explored the impact of hypoxia, low glucose, and the two together on the release of both ACh and ATP from two incubated cat CBs. The CBs were prepared with standard procedures in accordance with the policies and regulations of the Institutional Animal Care and Use Committee. When normalized to their controls, a significant increase of ACh in the incubation medium was measured in response to hypoxia, low glucose, and the combined stimuli. When normalized to their controls, a significant increase in ATP in the incubation medium was measured in response to hypoxia and to the combined stimuli. Low glucose generated an increase in ATP which was not statistically significant (P >0.05). Second, normalizing the initial 3–4 or 2–3 min Time Segment of the challenge Stage to the final 3–4 or 2–3 min Time Segment of the control Stage for both ACh and ATP generated significant increases in response to hypoxia, low glucose (ACh only), and the combined stimuli. The data suggested the possibility that in the cat the increased CB neural output in response to low glucose might be due primarily to ACh. [Copyright &y& Elsevier]

Abstract: The ventilatory response to hypoxia depends on the carotid body function and sleep–wake states. Therefore, the response must be measured in a consistent sleep–wake state. In mice, EMG with behavioral indices (coordinated movements, CMs; myoclonic twitches, MTs) has been used to assess sleep–wake states. However, in neonatal mice EMG instrumentation could induce stress, altering their behavior and ventilation. Accordingly, we examined: (1) if EMG can be eliminated for assessing sleep–wake states; and (2) behavioral characteristics and carotid body-mediated respiratory control during sleep with EMG (EMG+) or without EMG (EMG−). Seven-day-old DBA/2J and A/J mice were divided into EMG+ and EMG− groups. In both strains, CMs occurred when EMG was high; MTs were present during silent/low EMG activity. The durations of high EMG activity and of CMs were statistically indifferent. Thus, CMs can be used to indicate wake state without EMG. The stress caused by EMG instrumentation may be distinctively manifested based on genetic background. Prolonged agitation was observed in some EMG+ DBA/2J (5 of 13), but not in A/J mice. The sleep time and MT counts were indifferent between the groups in DBA/2J mice. The EMG+ A/J group showed longer sleep time and less MT counts than the EMG− A/J group. Mean respiratory variables (baseline, hyperoxic/hypoxic responses) were not severely influenced by EMG+ in either strain. Individual values were more variable in EMG+ mice. Carotid body-mediated respiratory responses (decreased ventilation upon hyperoxia and increased ventilation upon mild hypoxia) during sleep were clearly observed in these neonatal mice with or without EMG instrumentation. [Copyright &y& Elsevier]

Abstract: Chemoreceptor (glomus) cells of the carotid body are synaptically connected to the sensory nerve endings of petrosal ganglion (PG) neurons. In response to natural stimuli, the glomus cells release transmitters, which acting on the nerve terminals of petrosal neurons increases the chemosensory afferent discharge. Among several transmitter molecules present in glomus cells, acetylcholine (ACh) and adenosine 5′-triphosphate (ATP) are considered to act as excitatory transmitter in this synapse. To test if ACh and ATP play a role as excitatory transmitters in the cat CB, we recorded the electrophysiological responses from PG neurons cultured in vitro. Under voltage clamp, ATP induces a concentration-dependent inward current that partially desensitizes during 20–30 s application pulses. The ATP-induced current has a threshold near 100 nM and saturates between 20–50 μM. ACh induces a fast, inactivating inward current, with a threshold between 10–50 μM, and saturates around 1 mM. A large part of the population of PG neurons (60%) respond to both ATP and ACh. Present results support the hypothesis that ACh and ATP act as excitatory transmitters between cat glomus cells and PG neurons. [Copyright &y& Elsevier]

Abstract: Exposure to chronic intermittent hypoxia (CIH) for 4 days enhances the cat carotid body (CB) chemosensory responses to acute hypoxia. However, it is not known if CIH enhances the responses of the petrosal ganglion (PG) neurons that innervate the CB chemoreceptor cells. Accordingly, we studied the effects of the CB putative excitatory transmitter acetylcholine (ACh) and adenosine 5´-triphosphate (ATP), and the effects of citotoxic hypoxia (NaCN) applied to the isolated PG from cats exposed to CIH for 4 days. The dose-dependent curve parameters of the frequency of discharges evoked in the carotid sinus nerve by the application of ACh, ATP and NaCN to the isolated PG in control condition were not significantly modified in the CIH-treated cats. Present results suggest that CIH enhances the chemosensory responses to acute hypoxia acting primarily at the chemoreceptor cells, without major changes in the response of PG neurons evoked by the application of putative CB excitatory transmitters to their somata. [Copyright &y& Elsevier]

Abstract: Chronic intermittent hypoxia (CIH) enhances carotid body (CB) chemosensory responses to acute hypoxia. We tested the hypothesis that endothelin-1 (ET-1), an excitatory modulator of CB chemoreception may contribute to the enhanced CB chemosensory responses in cats exposed to cyclic hypoxic episodes repeated during 8 h for 4 days. Accordingly, we measured the ET-1 immunoreactivity (ET-ir) in the CB and plasma. Using a perfused CB preparation, we studied the effects of exogenous ET-1 and bosentan, a non-selective endothelin receptor type A and B antagonist, on the frequency of chemosensory discharges (ƒ x ) during normoxia, mild and severe hypoxia. We found that CIH increased ET-ir in the CB by ∼10-fold leaving ET-1 plasma levels unchanged. Application of ET-1 to control and CIH-treated CBs produced long-lasting dose-dependent increases in ƒ x , although the dose–response curve showed a rightward-shift in the CIH-treated CBs. CIH increased baseline ƒ x and hypoxic chemosensory responses, which were reduced by 50 μM bosentan in CBs from CIH-treated cats. Present results suggest that a local increase of ET-1 in the CB may contribute to the enhanced chemosensory responses induced by CIH predominantly through a vasomotor mechanism. [Copyright &y& Elsevier]

Abstract: Carotid bodies were removed from anesthetized rats kept under normobaric (640 Torr) and hypobaric conditions (380 Torr for 2–3 weeks). Slices (100–150 μm) of the organ were viewed under an inverted microscope for simultaneous stimulation and recording of coupled glomus cells and carotid nerve endings. The latter were identified by their more negative E m, high input resistance (R o) and time-dependent rectification in response to negative current pulses. Also, when nerve endings had an E m more negative than −40 mV showed spontaneous activity in the form of mini-receptor potentials (mrps). Glomus cells had less negative E m and lower R o. Prolonged hypobaric hypoxia did not change the E m of nerve endings and glomus cells. However, in both structures, R o increased. Also, the mrps became smaller and occurred less frequently. Intercellular coupling was recognized when currents applied to one cell spread to adjoining ones. In the case of glomus cells (GC/GC coupling), it was mostly resistive and bidirectional. Coupling between nerve endings and glomus cells was more complex, When a glomus cell was stimulated, current spread to the nerve ending (GC/NE coupling) was similar in magnitude (2–3%) to coupling between GCs. However, when NE was stimulated current spread to GC (NE/GC coupling) was minimal (less than 0.1%) and transient (capacitive). Nerve endings were also bidirectionally and capacitively coupled (NE/NE coupling) with a median of 2,8%. Intracellularly injected Lucifer Yellow or Alexa 488 diffused to neighboring structures. Prolonged hypobaric hypoxia significantly tightened coupling modes GC/NE, NE/GC, and NE/NE but reduced GC/GC coupling. Tighter coupling was accompanied by lower coupling resistance, and the opposite occurred when intercellular coupling decreased. Increased GC/NE and reduced GC/GC coupling during hypobaric hypoxia may be partly responsible for the increased reactivity of these receptors under this condition. [Copyright &y& Elsevier]

Abstract: This study describes the localization and pattern of expression of estradiol and progesterone receptors as well as key enzymes for steroid synthesis (i.e. P450 side-chain-cleavage—P450scc, and P450 aromatase—P450Aro) in the carotid body (CB) and superior cervical ganglion (SCG) of adult, newborn and late fetal male rats, using immunohistochemistry, Western blot and real-time RT-PCR. Our results show a constitutive expression of the β estradiol receptor (Erβ) and the 80 kDa and 60 kDa progesterone receptors (PR-A and PR-C) isoforms in the CB, while in the SCG Erα, Erβ, PR-A and PR-C are expressed. While P450Aro staining was negative, P450scc staining was strong both in the SCG and CB. In late fetal and newborn rats, Erα was not detected in the CB or SCG, but a slight staining appeared for P450 aromatase in the CB, and to a lesser extent in SCG. P450scc was strongly expressed in CB and SCG of late fetal and newborn rats. We conclude that the carotid body shows a constitutive expression of Erβ and PR and may be able to synthesize steroids, including estradiol during late fetal life. [Copyright &y& Elsevier]

Abstract: Petrosal ganglion neurons are depolarized and fire action potentials in response to acetylcholine and nicotine. However, little is known about the subtype(s) of nicotinic acetylcholine receptors involved, although α4 and α7 subunits have been identified in petrosal ganglion neurons. Cytisine, an alkaloid unrelated to nicotine, and its bromo derivatives are agonists exhibiting different affinities, potencies and efficacies at nicotinic acetylcholine receptors containing α4 or α7 subunits. To characterize the receptors involved, we studied the effects of these agonists and the nicotinic acetylcholine receptor antagonists hexamethonium and α-bungarotoxin in isolated petrosal ganglion neurons. Petrosal ganglia were excised from anesthetized cats and cultured for up to 16 days. Using patch-clamp technique, we recorded whole-cell currents evoked by 5–10 s applications of acetylcholine, cytisine or its bromo derivatives. Agonists and antagonists were applied by gravity from a pipette near the neuron surface. Neurons responded to acetylcholine, cytisine, 3-bromocytisine and 5-bromocytisine with fast inward currents that desensitized during application of the stimuli and were reversibly blocked by 1 μM hexamethonium or 10 nM α-bungarotoxin. The order of potency of the agonists was 3-bromocytisine ≫ acetylcholine ≅ cytisine ≫ 5-bromocytisine, suggesting that homomeric α7 neuronal nicotinic receptors predominate in cat petrosal ganglion neurons in culture. [Copyright &y& Elsevier]

Abstract: In response to hypoxia, chemoreceptor cells of the carotid body (CB) release transmitters, which acting on the petrosal ganglion (PG) neuron terminals, increase the chemoafferent discharge. Additionally, vasoactive molecules produced within the CB may modulate hypoxic chemoreception by controlling blood flow and tissue PO2. Endothelin-1 (ET-1) increases basal CB chemosensory discharges in situ, probably due to its vasoconstrictor action. However, the actions of ET-1 on PG neurons or its expression in the PG are not known. Using immunohistochemistry, we found that endothelin-like peptides are expressed in the cat PG and CB under normoxic conditions. Exogenous applications of ET-1 increased the chemosensory activity in the vascularly perfused CB but were ineffective on either the CB or PG superfused preparations, both of which are devoid of vascular control. Thus, our data indicate that the excitatory effect of ET-1 in the carotid chemoreceptor system appears to be mainly due to a vasoconstrictor effect in the CB blood vessels. [Copyright &y& Elsevier]

Recent laboratory and clinical studies demonstrate therapeutic efficacy of intracerebral transplantation of carotid body (CB) in Parkinson''s disease, possibly through secretion of neurotrophic factors. Here, we examined the role of CB in experimental stroke. In the first experiment, we hypothesized that removal of CB would exacerbate cerebral infarction and stroke-related behavioral deficits. Eight-week-old, male Sprague–Dawley rats were randomly divided into two groups: stroke with intact CB and stroke with surgically removed CB. We used the stroke model of temporary middle cerebral artery occlusion. The ipsilateral CB was removed in animals assigned to treatment group exposed to stroke with surgically removed CB. Behavioral tests, using the elevated body swing test, were conducted at days 1–3 after surgery. Cerebral infarction was visualized by TTC staining on day 3 post-surgery. The data revealed no significant differences in behavioral deficits and infarct volumes between the two groups. In the second experiment, CB cell suspension grafts or control adult tissue grafts were intracerebally transplanted into the ischemic penumbra immediately (within 1 h) after stroke surgery. The results revealed significant reduction of behavioral deficits and infarct volumes, accompanied by increased levels of neurotrophic factors, as detected by ELISA, in transplanted ischemic striatum collected from CB-grafted stroke animals. These observations suggest that surgical resection of CB in the periphery did not alter stroke pathology; however, CB when made available in the CNS, via intracerebral transplantation, could protect against stroke possibly through the synergistic release of neurotrophic factors. The present study extends the use of CB as efficacious graft source for transplantation. [Copyright &y& Elsevier]

Dissociated rat glomus cells were loaded with Fura-2 AM to study the effects of hypoxia, and carotid body transmitters on intracellular calcium, [Ca2+]i. The mean control [Ca2+]i was 55 nM in isolated cells and 67 nM in clusters. The following procedures changed [Ca2+]i:(1)0[Ca2+]o+EGTA reduced [Ca2+]i by about 50%, suggesting that the remaining calcium originated from intracellular organelles. [Ca2+]i increased when [Ca2+]o was doubled.(2)Hypoxia by sodium dithionite (Na2S2O4) induced large [Ca2+]i increases in clustered and isolated cells. Smaller rises occurred with 100% N2 hypoxia. The augmented [Ca2+]i, induced by Na2S2O4, was reduced (not eliminated) in 0[Ca2+]o+EGTA, suggesting that some calcium was intracellularly released. Nifedipine depressed (did not block) the Na2S2O4-induced calcium increase, implying some inflow via other (N, T or P/Q) voltage-dependent or voltage-independent calcium channels.(3)Cholinergic agents (ACh, nicotine, muscarine, bethanechol and pilocarpine) increased [Ca2+]i. The ACh effect was produced exclusively by calcium inflow since it was eliminated in 0[Ca2+]o+EGTA. Cholinergic effects were depressed (not obliterated) by d-tubocurarine (d-TC), hexamethonium (C6) and atropine.(4)ACh, nicotine and pilocarpine potentiated the excitatory effect of Na2S2O4 on [Ca2+]i. Bethanechol depressed this excitation whereas muscarine had inconsistent effects.(5)Atropine and C6 depressed [Ca2+]i increases elicited by Na2S2O4 but the effects of d-TC were variable.(6)Dopamine (DA) had variable effects. It increased [Ca2+]i in 75% of cases, and reduced the Na2S2O4 -induced calcium increase.Thus, calcium increases during Na2S2O4 occur by direct effects on the glomus cells and feedback action through released ACh and DA. [Copyright &y& Elsevier]

It is well established that the carotid body receptors (CBR), at the bifurcation of the carotid artery, inform the brain of changes in the concentration of CO2 and O2 in arterial blood. More recent work suggests that these receptors are also extremely sensitive to blood glucose levels suggesting that they may play an important role as sensors of blood components important for brain energy metabolism. Much less is known about changes in brain glucose metabolism in response to CBR activation. Here we show that 2–8 min after local injection of sodium cyanide (NaCN) into the CBR or after electrical stimulation of the carotid sinus nerve in dogs and rats, brain glucose uptake increased fourfold. Cerebrospinal fluids (CSF) transferred from dogs, 2–8 min after CBR stimulation, into the cisterna magna of non-stimulated dogs or rats induced a similar increase in brain glucose uptake. CSF from stimulated dogs was also active when injected intravenously in anesthetized or awake rats. The activity was destroyed when the stimulated CSF was heated to 100 °C or treated with trypsin. We conclude that a peptide important for brain glucose regulation appears in the CSF shortly after CBR stimulation. [Copyright &y& Elsevier]

Several molecules have been proposed as excitatory transmitters between glomus (type 1) cells and nerve terminals of petrosal ganglion (PG) neurons in the carotid body (CB). We tested whether ACh and ATP have a role to play as excitatory transmitters in the cat CB by recording intracellularly from identified PG neurons functionally connected to the CB in vitro. PG neurons projecting to the CB were classified according to their intracellular responses as: (a) neurons with humped action potentials (hAP neurons) responding phasically to long-lasting depolarizing pulses (53/67), and (b) neurons with smooth action potentials (non-hAP neurons) that fire tonically during long-lasting depolarizations (14/67). CB stimulation by stop flow and/or acidosis induced activity in 28 of 39 hAP-type neurons, being classified as chemosensory, but in none of the non-hAP neurons. Hexamethonium (10 μM) and suramin (100 μM) reversibly abolished the increased discharges evoked in chemosensory neurons (8/9) by stop flow or acidosis. Moreover, 24 of 27 chemosensory neurons responded to ganglionar application of ACh and ATP, while two neurons responded only to ACh and one to ATP. Mechanical deformation of the carotid sinus induced firing activity in 10 of 13 non-hAP neurons, but in none of the hAP neurons tested. Interestingly, 4/10 non-hAP neurons, which responded to carotid sinus mechanical stimulation also responded to ganglionar application of ATP, but were insensitive to ACh. Present results favor the hypothesis that ACh and ATP are excitatory transmitters in the cat CB, acting—at least—on the PG neuron terminals in the CB. [Copyright &y& Elsevier]

Neurotransmitters appear to be involved in chemotransmission of the carotid body, a major arterial chemoreceptor. Substantial data indicate that acetylcholine (ACh) is an excitatory neurotransmitter in the carotid body, regulating the excitability of afferent nerve endings and glomus cells (putative chemoreceptor cells). In this study we characterized properties of nicotinic ACh receptors (nAChRs) in cultured cat glomus cells using immunocytochemistry and whole cell patch clamp techniques. Cultured glomus cells expressed immunoreactivity for α3, α4, and β2 subunits of nAChRs. An application of ACh elicited inward current. Nicotinic AChRs of glomus cells showed high affinity for ACh. The current–voltage relationship showed strong inward rectification at positive membrane potential. α-Conotoxin MII (20 nM), dihydro-β-erythroidine (DHβE; 1 nM), and hexamethonium (300 μM) significantly inhibited ACh-induced current. These results indicate that cultured cat glomus cells possess functional nAChRs, and that their characteristics are consistent with those of α3, α4 and β2 containing nAChRs. [Copyright &y& Elsevier]

In the present study, we investigated the immunohistochemical localization of two-pore K+-channels TASK-1, TASK-2, TASK-3 and TRAAK in the rat carotid body. Type I cells were positive for TASK-1, TASK-2, TASK-3 and TRAAK. Intrinsic nerve cell bodies were also strongly positive for TASK-1, TASK-2 and TRAAK, but negative for TASK-3. In addition, some type II cells, Schwann cells in the nerve bundles and fibroblast between type I cell clusters were also immunostained for TASK-1. Smooth muscle cells of the carotid body artery were intensely positive for TASK-3. Our results indicate that TASK-1 immunoreactivity was ubiquitously distributed in many cell types and immunoreactivities for TASK-2, TASK-3 and TRAAK were cell type specific distribution patterns in the rat carotid body. [Copyright &y& Elsevier]

Chemotransduction of arterial hypoxemia by the cat carotid body is generally thought to begin with a hypoxia-induced depolarization of the glomus cells (GCs) of the carotid body (CB). This depolarization activates voltage-gated calcium channels with the subsequent entry of calcium, movement of transmitter-containing vesicles to the synaptic-like juncture between the GC and apposed sensory afferent neuron. The vesicles exocytotically release their transmitters which then proceed to the receptors on both the postsynaptic neuron and on the GCs themselves (autoreceptors). Action potentials and their modulation in the sensory fibers are the result, along with the modulation of further neurotransmitter release from the GCs. The purpose of the present study was to: (1) determine the parameters of an incubated cat CB preparation capable of releasing measurable amounts of catecholamines (CAs) in response to hypoxia; (2) determine the impact of muscarinic activities on CA release during the hypoxic challenge; (3) determine if the muscarinic activity preferentially modified the release of one CA more than another; (4) determine if there were any differences in the pattern of hypoxia-induced release of dopamine (DA) vs. norepinephrine (NE). CBs were harvested from deeply anesthetized cats. Cleaned of fat and connective tissue, they were incubated in Krebs Ringer bicarbonate solution at 37 °C, and bubbled with a hyperoxic mixture of gases (95% O2–5% CO2) for 30 min. The first series of experiments to address the CB’s hypoxia-induced release of CAs explored the effects of incubating CBs for 2 h with hyperoxia vs. normoxia (21% O2–6% CO2) followed by a 30 min hypoxic challenge, with or without l-dihydroxyphenylalanine (l-DOPA). In the second series of experiments the CBs, after the first 30 min of hyperoxia, were next challenged with hypoxia (4% O2–5% CO2) for intervals of 3–20 min with intervening recovery periods of hyperoxia to determine the effect of the duration of the hypoxic exposure on CA release. In the third series of experiments the CBs, after the first 30 min of hyperoxia, were challenged with hypoxia for intervals of 10–40 min in the presence or absence of an M1 or M2 muscarinic receptor antagonist. CAs released into the incubation medium were analyzed by means of high performance liquid chromatography–electrochemical detection using standard procedures. Incubated cat CBs challenged for 2 h with hyperoxia followed by 30 min of hypoxia, released much more measurable amounts of CAs in the presence of 40 μM l-DOPA than without it. Moving from hyperoxia to hypoxia produced a better yield than moving from normoxia to hypoxia, and at least 10–20 min exposures were needed for measurable amounts of CAs. The M1 muscarinic receptor antagonist, pirenzepine, reduced the hypoxia-induced release of CAs during each exposure. Further, the reduction appeared to be dose-related. The M2 muscarinic receptor antagonist, methoctramine, enhanced the hypoxia-induced release of CAs during each exposure. These data support a role for acetylcholine (ACh) in the hypoxia-induced release of CAs, and suggest a significant, if modest, muscarinic dimension to it. And although hypoxia induced a greater release of DA than of NE, the muscarinic modulation of the release (both decreasing it and increasing it) may have had a greater impact on NE release than on DA release. Finally, the patterns of hypoxia-induced release of DA and NE from incubated cat carotid bodies are significantly different. [Copyright &y& Elsevier]