Cat Carotid Body Research Articles

Presence of nicotinic acetylcholine receptors in cat carotid body afferent system

With immunocytochemical techniques using a monoclonal antibody for α7 subunits of neuronal nicotinic acetylcholine receptors, we have found these subunits to be exclusively expressed in nerve fibers in the carotid body. Double-immunostaining showed that α7 subunit-positive nerve endings enveloped tyrosine hydroxylase-positive glomus cells. Some carotid sinus nerve fibers and tyrosine hydroxylase-positive petrosal ganglion neurons also expressed α7 subunits. These data support a role for acetylcholine in carotid body neurotransmission.

Dopamine, sensory discharge, and stimulus interaction with CO2 and O2 in cat carotid body.

It is hypothesized that carotid body chemosensory activity is coupled to neurosecretion. The purpose of this study was to examine whether there was a correspondence between carotid body tissue dopamine (DA) levels and neuronal discharge (ND) measured from the carotid sinus nerve of perfused cat carotid bodies and to characterize interaction between CO2 and O2 in these responses. ND and tissue DA were measured after changing from normoxic, normocapnic control bicarbonate buffer (PO2 >120 Torr, PCO2 25-30 Torr, pH approximately 7.4) to normoxic hypercapnia (PCO2 55-57 Torr, pH 7.1-7.2) or to hypoxic solutions (PO2 30-35 Torr) with normocapnia (PCO2 25-30 Torr, pH approximately 7.4) or hypocapnia (PCO2 10-15 Torr, pH 7.6-7.8). Similar temporal changes for ND and tissue DA were found for all of the stimuli, although there was a much different proportional relationship for normoxic hypercapnia. Both ND and DA increased above baseline values during flow interruption and normocapnic hypoxia, and both decreased below baseline values during hypoxic hypocapnia. In contrast, normoxic hypercapnia caused an initial increase in ND, from a baseline of 175 +/- 12 (SE) to a peak of 593 +/- 20 impulses/s within 4.6 +/- 0.9 s, followed by adaptation, whereas ND declined to 423 +/- 20 impulses/s after 1 min. Tissue DA initially increased from a baseline of 17.9 +/- 1.2 microM to a peak of 23.2 +/- 1.2 microM within 3.0 +/- 0.7 s, then declined to 2.6 +/- 1.0 microM. The substantial decrease in tissue DA during normoxic hypercapnia was not consistent with the parallel changes in DA with ND that were observed for hypoxic stimuli.

Expression of dopamine D1-receptor mRNA in the carotid body of adult rabbits, cats and rats

Dopamine is a major neurotransmitter in the carotid body of several animal species and its functional role at the level of peripheral arterial chemoreflex pathway is attributed to the presence of the dopamine D2-receptors. We present evidence that the dopamine D1-receptor mRNA is also expressed in the carotid body of adult rabbits, cats and rats. A DNA fragment of 611 bp of this receptor was first isolated from rabbit. The nucleic acid sequence of this fragment was found to be 84.5% identical to that of rat. This specific 611 bp fragment was used as a probe to detect, either by Northern analysis or by the reverse transcription-polymerase chain reaction, the dopamine D1-receptor mRNA. The results revealed the presence of dopamine D1-receptor transcript in the carotid body as well as in the petrosal ganglion and the superior cervical ganglion from the three animal models studied here. The physiological significance of dopamine D1-receptor expression in the carotid body is discussed.

Carotid body I 1-imidazoline receptors: Binding, visualization and modulatory function

The carotid body is influenced by many neurotransmitter receptors. A novel receptor specific for imidazolines has been implicated in cardiorespiratory regulation in the brain. To test for both I 1-imidazoline and α 2-adrenergic receptors, which also recognize imidazolines, specific [ 125I] p-iodoclonidine binding to carotid body membranes was characterized. The specific α 2-agents epinephrine (100 μM) or SK&F 86466 (10 μM) inhibited only a portion of specific [ 125I] p-iodoclonidine binding in both cat and rabbit carotid bodies, indicating the presence of I 1-imidazoline as well as α 2-adrenergic sites. The distribution of [ 125I] p-iodoclonidine binding sites was visualized autoradiographically. The cat carotid body was intensely labeled by [ 125I] p-iodoclonidine, with both I 1-imidazoline and α 2-adrenergic sites expressed. The relevance of I 1-imidazoline receptors in modulation of chemosensory discharge was determined in seven cats after α 2-adrenergic blockade. Clonidine (100 μg/kg) facilitated chemosensory activity particularly under hypoxia. We conclude that I 1-imidazoline receptors are expressed within the carotid body and may potentiate chemosensory discharge, in contrast to the inhibitory action of α 2-adrenergic receptors.

Anion exchanger and chloride channel in cat carotid body chemotransduction

In order to test the hypothesis that carotid body (CB) chemoreception depends on the functions of anion channels and HCO 3 −/Cl − exchangers, we studied the effects of the anion channel blocker anthracene-9-carboxylic acid (9-ANC), the carbonic anhydrase inhibitor methazolamide, and the HCO 3 −/Cl − exchanger blocker 4,4 diisothiocyanatostilbene-2-2′disulfonic acid (DIDS) on the chemosensory discharges of cat CB, perfused–superfused in vitro at 36.5±0.5°C, with a modified Tyrode solution. The chemosensory responses to hypoxia (PO 2≈50 Torr), hypercapnia (PCO 2≈60 Torr, pH=7.10), nicotine (2–4 nmol) and NaCN (20–40 nmol) were recorded. 9-ANC (2 mM) and DIDS (10 μM) decreased the chemosensory baseline activity, and eliminated the initial peak responses to hypercapnia and hypoxia and increased the time to achieve it. Methazolamide (0.13 mM) did not alter the effect of 9-ANC. The steady state responses to hypoxia and hypercapnia were not diminished after 9-ANC but DIDS lowered the responses. Responses to NaCN effects were all diminished but those to nicotine were not affected. The results suggest that the functions of anion channels and HCO 3 −/Cl − exchangers are important for the resting dischargers and for the fast responses to hypoxia and hypercapnia.

Electrophysiological and immunocytological demonstration of cell-type specific responses to hypoxia in the adult cat carotid body

We have recently shown two types of cat carotid body cells based on the oxygen sensitivity of voltage-gated potassium channels. In the present study, we attempted to determine the correlation between cell types (glomus cells, sheath cells, and subtypes of glomus cells) and oxygen sensitivity of potassium channels. Further, changes in membrane potentials in response to hypoxia were also examined. Carotid body cells harvested from adult cats were cultured, and a whole cell patch clamp method was applied to determine the oxygen sensitivity of outward current. The tested cells were identified by Lucifer Yellow in the patch pipette. Glomus cells and sheath cells were immunocytochemically identified using tyrosine hydroxylase (TH) and glial fibrillary acidic protein (GFAP) as markers. The cells whose outward current was inhibited by hypoxia showed TH-immunoreactivity but not GFAP-immunoreactivity. The cells whose outward current was not sensitive to hypoxia were GFAP-positive or TH-negative. One TH-positive cell had oxygen-insensitive outward current. The resting membrane potentials of the cells having oxygen-sensitive outward current were significantly higher (−55±3 mV) than those of the cells having oxygen-insensitive outward current (−35±2 mV). The former type of cells was depolarized during hypoxia, but not the latter type of cells. These results suggest that most glomus cells of the adult cat carotid body possess oxygen-sensitive potassium channels and are depolarized in response to hypoxia. On the other hand, sheath cells and possibly a small fraction of glomus cells possess oxygen-insensitive potassium channels and their membrane potential is not affected by hypoxia.

Inhibition of dopamine release with simultaneous chemosensory excitation by hypercapnia with and without [Ca 2+] 0 in the cat carotid body

The hypothesis that dopamine (DA) overflow corresponds to carotid sinus nerve (CSN) discharge during hypercapnia and is dependent on [Ca 2+] 0 was tested. We simultaneously measured the time course of DA overflow and CSN discharge of the cat carotid body, perfused/superfused in vitro at 37°C at decreasing [Ca 2+] 0, during transition from normocapnia (P CO 2 ∼30–35 Torr) to hypercapnia (P CO 2 ∼60–65 Torr). In the presence of normal [Ca 2+] 0, hypercapnia instantaneously increased nerve discharge to peak levels followed by a decrease to steady states which were above the basal rate of activity. CSN discharge rate did not differ at decreasing [Ca 2+] 0 between 2.2 and 1.0 mM, and it began to decline at 0.1 mM [Ca 2+] 0, culminating to zero level in most cases, at zero [Ca 2+] 0. DA overflow increased slightly during hypercapnic peak CSN activity. Thereafter it declined to steady state levels below those of normocapnic conditions. Decreases in steady state DA levels were significantly less at 0 mM [Ca 2+] 0 compared to the higher calcium concentrations (0.1, 1.0 and 2.2 mM). Overall, steady state CSN activity and DA overflow were inversely related. Thus, DA release cannot have excitatory implications for carotid chemoreceptors during hypercapnia in the cat.

Suppression of glomus cell K + conductance by 4-aminopyridine is not related to [Ca 2+] i, dopamine release and chemosensory discharge from carotid body

The hypothesis that suppression of O 2-sensitive K + current is the initial event in hypoxic chemotransduction in the carotid body glomus cells was tested by using 4-aminopyridine (4-AP), a known suppressant of K + current, on intracellular [Ca 2+] i , dopamine secretion and chemosensory discharge in cat carotid body (CB). In vitro experiments were performed with superfused–perfused cat CBs, measuring chemosensory discharge, monitoring dopamine release by microsensors without and with 4-AP (0.2, 1.0 and 2.0 mM in CO 2-HCO 3 - buffer) and recording [Ca 2+] i by ratio fluorometry in isolated cat and rat glomus cells. 4-AP decreased the chemosensory activities in normoxia but remained the same in hypoxia and in flow interruption. It decreased the tissue dopamine release in normoxia, and showed an additional inhibition with hypoxia. Also, 4-AP did not evoke any rise in [Ca 2+] i in glomus cells either during normoxia and hypoxia, although hypoxia stimulated it. Thus, the lack of stimulatory effect on chemosensory discharge, inhibition of dopamine release and unaltered [Ca 2+] i by 4-AP are not consistent with the implied meaning of the suppressant effect on K + current of glomus cells.

Changes in Tyrosine Hydroxylase and Substance P Immunoreactivity in the Cat Carotid Body Following Chronic Hypoxia and Denervation

Long-term hypoxia elicits functional changes in the cat carotid body which are manifest as altered chemosensitivity in response to hypoxia. Previous studies have suggested that these functional adjustments may be mediated by changes in neurotransmitter levels in chemosensory type I cells. Neurotransmitter metabolism in the carotid body has also been shown to be regulated by the neural innervation to the organ. The present study using the cat carotid body demonstrates profound changes in the levels of immunoreactivity of the catecholamine-synthesizing enzyme, tyrosine hydroxylase, and the neuropeptide, substance P, in response to a two-week exposure to hypoxia (10% O 2 in 90% N 2). Furthermore, these changes were modulated both by sensory and sympathetic denervation of the organ. For TH, the intensity of immunostaining in type I cells was markedly increased by long-term hypoxia in both normal and chronic carotid sinus nerve-denervated carotid bodies, but this effect was blocked following chronic sympathectomy. Substance P immunoreactivity in type I cells was dramatically attenuated by hypoxia in both intact and chronic carotid sinus nerve-denervated preparations, but this effect was reduced following chronic sympathectomy. Tyrosine hydroxylase- and substance P-positive axon terminals were observed to innervate type I cells. These axons were also present in chronically sympathectomized preparations, but they disappeared following chronic carotid sinus nerve-denervation suggesting that they very likely arise from sensory neurons in the petrosat ganglion. Our data indicate that chronic chemoreceptor stimulation by hypoxia elicits multiple neurochemical adjustments in the cat carotid body. These changes suggest that catecholaminergic enzymes and neuropeptides play a significant role in the adaptive mechanisms of chemoreceptor function which occur in response to chronic physiological stimulation. Furthermore, the data suggest that neurotrophic mechanisms may influence neurotransmitter metabolism in chemosensory type I cells.

Effects of CO2-HCO3- on catecholamine efflux from cat carotid body.

Using a chronoamperometric technique with carbon-fiber microelectrodes and neural recordings, we simultaneously measured the effects of the following procedures on catecholamine efflux (delta CA) and frequency of chemosensory discharges (fx) from superfused cat carotid body: 1) the addition of CO2-HCO3- to Tyrode solution previously buffered with N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid, maintaining pH at 7.40; 2) hypercapnia (10% CO2, pH 7.10); 3) hypoxia (PO2 h approximately 40 Torr) with and without CO2-HCO3-; and 4) the impact of several boluses of dopamine (DA; 10-100 micrograms) on hypoxic and hypercapnic challenges. With CO2-HCO3-, hypoxia increased fx which preceded delta CA increases, whereas hypercapnia raised fx but did not consistently increase delta CA. Repeated stimuli induced similar fx increases, but attenuated delta CA. After DA, hypoxia produced larger delta CA, which preceded chemosensory responses. Without CO2-HCO3-, hypoxia produced a similar pattern of delta CA and fx responses. Switching to Tyrode solution with CO2-HCO3- at pH 7.40 raised fx but did not increase delta CA. With CO2-HCO3- and after DA, hypoxic-induced delta CAs were larger than in its absence. Results suggest that DA release is not essential for chemosensory excitation.

Vascular and metabolic effects of nitric oxide synthase inhibition evaluated by tissue PO2 measurements in carotid body.

The suspected role of nitric oxide (NO) in modulating chemosensory activity, and the origin and actual locations of nitric oxide synthase (NOS) in the carotid body (CB) are not clear. Wang et al. (1993) were the first to show that NOS is present in the CB from immunochemistry and NADPH diaphorase staining of rat CBs. Prabhakar et al. (1993) also found NADPH positive nerve fibers around cat CB glomus cells. Wang et al. (1993) attributed the origin of NOS primarily to sensory neurons in the petrosal ganglion. On the other hand, Grimes et al. (1995) presented immunohistochemical evidence that NOS innervation in the cat CB appears to be autonomic, originating from dispersed ganglion cells, and may be parasympathetic in nature. Hohler et al. (1994) also found neuronal NOS primarily associated with autonomic neurons in Wistar rat CBs.KeywordsNitric OxideCarotid BodyNeuronal DischargeGlomus CellDisappearance CurveThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Dopamine increases in cat carotid body during excitation by carbon monoxide: Implications for a chromophore theory of chemoreception

Studies of dopamine (DA) release were conducted with 10 perfused/superfused cat carotid bodies using shallow recessed Nafion polymer-coated microsensors (tips ∼5 μm). Simultaneous measurements of tissue DA and neuronal discharge (ND) from the sinus nerve were made after switching from normoxic, normocapnic control perfusate (20% O 2, 5% CO 2, balance N 2) to a normoxic, normocapnic perfusate equilibrated with a high tension (>550 Torr) of carbon monoxide (CO). When high P CO perfusate was delivered in the dark, ND increased from a baseline of 89±24 (SE) impulses/s, to a peak excitation of 374±44 impulses/s within 15–30 s. Excitation then diminished to a plateau of 281±36 impulses/s within 1–2 min. Both peak and plateau ND were significantly above baseline ( P<0.05). Average tissue DA values increased above basal levels by +7.2±1.0 and +5.6±0.6 μM, respectively, during the peak and plateau ND phases ( P<0.05). Bright light restored the chemosensory activity to baseline, but had no effect on DA. Both chemosensory excitation and tissue DA responses to high CO in the dark were diminished in 3 carotid bodies perfused with Ca 2+-free solutions. Responses were reduced even further with Ca 2+ chelator (EGTA) in the perfusate. The results suggest that the effect of high P CO on DA release and chemosensory excitation are dependent on Ca 2+ in the media, but the two events are not coupled.

Stimulus-specific mobilization of dopamine and norepinephrine stores in cat carotid body

The catecholamines (CAs), dopamine (DA) and norepinephrine (NE), are synthesized and stored in carotid body chemosensory type I cells. Previous studies in our laboratory demonstrated that low concentrations of nicotine preferentially evoke the release of NE from rabbit type I cells, whereas hypoxia mobilizes DA and NE in proportion to their stores in the tissue. The primary objective of the present study was to examine whether hypoxia, nicotine and elevated concentrations (30 mM) of K + evoke the preferential release of DA vs. NE from cat carotid bodies superfused in vitro. In this species, where tissue stores of DA and NE are nearly equal, hypoxia evoked the preferential release of DA from normal carotid bodies. This pattern of release evoked by low O 2 was also present following chronic removal of the superior cervical ganglion, which eliminated NE contained in the sympathetic innervation to the carotid body. In contrast, nicotine and high-K + preferentially mobilized NE in these sympathectomized animals. Sympathectomy also reduced the percent of DA (but not NE) content released from type I cells in response to any of the three stimuli. Our findings suggest that chemosensory type I cells possess stimulus-specific mechanisms for CA mobilization and that the sympathetic innervation modulates the metabolism and release of CAs in the cat carotid body.

Acid-sensing by carotid body is inhibited by blockers of voltage-sensitive Ca 2+ channels

The membrane potential hypothesis that the responses to hypercapnia of carotid chemosensory activity is mediated by voltage-gated Ca 2+ channels was investigated by measuring directly the chemosensory output from rat and cat carotid bodies, perfused and superfused in vitro. We found that the inorganic and organic blockers of voltage-gated Ca 2+ channels suppressed the hypercapnic responses, thereby supporting the membrane potential hypothesis.

Cat carotid body chemosensory responses to non-hypoxic stimuli are inhibited by sodium nitroprusside in situ and in vitro

We studied the effects of sodium nitroprusside, a nitric oxide donor, on the chemosensory responses to cyanide and nicotine in the cat carotid body. In situ, sodium nitroprusside infusion reduced the cyanide-evoked responses in a dose-dependent manner. In vitro, Tyrode containing nitroprusside reversibly reduced the cyanide- (by 59%) and nicotine-induced (by 45%) chemosensory responses. The present results suggest that chemosensory responses induced by cyanide and nicotine are reduced by increased nitric oxide content, similarly to the hypoxic chemosensory responses.

CO interact with intracellular [H +] with and without CO 2–HCO 3− in the cat carotid chemosensory discharge

To test the hypothesis whether CO 2–HCO 3 − buffer is essential for the expression of chemoreception and to distinguish between pH i and pH o interaction with pCO in the carotid chemosensory response, we superfused-perfused in vitro cat carotid bodies using HEPES-Tyrode's solution with and without CO 2–HCO 3 −, and compared the responses at the same pH o in the absence and presence of light. In the absence of light, pCO (>138 Torr) stimulated the carotid body chemoreceptors in CO 2–HCO 3 − buffer at pH o of 7.40, whereas pCO (69–550 Torr) did not stimulate the neural discharge in HEPES buffer at the pH o of 7.4–7.1 but did so below pH o 7.1. In the presence of light, all the responses were diminished proportionately.

Expression of dopamine D2 receptor mRNA isoforms in the carotid body of rat, cat and rabbit

Using the Reverse transcription-Polymerase chain reaction, we detected dopamine D2 receptor mRNA short and long isoforms in the adult carotid body of rats, cats, and rabbits. For these animals, the relative short/long ratios were 0.60, 0.65 and 0.57, respectively. Our results suggest that the variety of dopamine effects on carotid chemoreceptor activity, that has been related to species differences, may not be dependent on the expression levels of the dopamine D2 receptor mRNA isoforms in the studied species.

Evidence for two types of nicotinic receptors in the cat carotid body chemoreceptor cells

Current concepts on the location and functional significance of nicotinic receptors in the carotid body rest on α-bungarotoxin binding and autoradiographic studies. Using an in vitro preparation of the cat carotid body whose catecholamine deposits have been labeled by prior incubation with the tritiated natural precursor [ 3H]tyrosine, we have found that nicotine induces release of [ 3H]catecholamines in a dose-dependent manner (IC 50=9.81 μM). We also found that mecamylamine (50 μM) completely abolished the nicotine-induced release, while α-bungarotoxin (100 nM; ≈20 times its binding K d) only reduced the release by 56%. These findings indicate that chemoreceptor cells, and perhaps other carotid body structures, contain nicotinic receptors that are not sensitive to α-bungarotoxin and force a revision of the current concepts on cholinergic mechanisms in the carotid body chemoreception.

Potential role of H 2O 2 in chemoreception in the cat carotid body

The hypothesis that H 2O 2 plays a critical role in hypoxic chemoreception in the cat carotid body (CB) was tested using a perfused–superfused preparation in vitro, measuring chemosensory discharge and CB tissue P O 2 ( P tiO 2 ). According to the hypothesis NADPH mediated, P O 2 dependant increase in H 2O 2 production would hyperpolarize the glomus cell, decreasing the chemosensory discharge. Thus, lactate and aminotriazole which would increase H 2O 2 concentration, would decrease the chemosensory discharge during hypoxia. However, 2.5–5.0 mM lactate and 25 mM aminotriazole did not diminish the hypoxic response. But, 2.5 mM lactate decreased the chemosensory discharge during normoxia which can be explained by an increase of CB P tiO 2 . Diethyldithiocarbamic acid (5 mM), which blocks the conversion of superoxide to H 2O 2, also diminished the chemosensory discharge, presumably due to an increased CB P tiO 2 . Menadione (increasing H 2O 2) and t-butyl hydroperoxide irreversibly decreased the chemosensory discharge, and the data are not useful. H 2O 2 increased the P O 2 of the perfusate, and therefore could not be tested against P O 2 . Thus, perturbation of endogenous or exogenous H 2O 2 did not provide any evidence for its critical role in O 2 chemoreception.

Further cholinergic aspects of carotid body chemotransduction of hypoxia in cats.

From the 1930s into the 1970s, the role of acetylcholine (ACh) in the carotid body's chemotransduction of hypoxia was debated. Since the late 1970s, the issue has been pursued only intermittently or not at all. The purpose of this study was to test again with a new preparation the hypothesis that ACh is an excitatory neurotransmitter in the cat carotid body's chemotransduction of hypoxia. We tested the effect of the specific nicotinic blocker mecamylamine and the muscarinic blocker of all five muscarinic receptors, atropine. We further tested the effects of M1 and M2 muscarinic-receptor blockers. The carotid body region was selectively perfused with hypoxic Krebs-Ringer bicarbonate (KRB) solutions that were blocker free or contained varying doses of the blockers. Both mecamylamine and atropine reduced the response to hypoxic KRB in a dose-related manner. The M2 muscarinic-receptor blockers gallamine and AFDX 116 increased the response to hypoxic KRB, whereas the M1 muscarinic-receptor blocker pirenzepine reduced the response to hypoxic KRB. These data are consistent with an excitatory role for ACh in the carotid body chemotransduction of hypoxia in the cat.