C. Eyzaguirre

Chemical, electron microscopic and physiological observations on the role of catecholamines in the carotid body

Abstract Chemical determinations revealed large amounts of dopamine, noradrenaline and adrenaline, in the carotid body of the cat. The cathecholamine (CA) content of normally innervated and chronically synmpathectomized carotid bodies was essentially the same. It did not change by vigorous and prolonged carotid body stimulation either in situ or in vitro . High doses of reserpine reduced appreciably the noradrenaline content of the carotid body; the contents of dopamine and adrenaline also were reduced but to a lesser degree. Electron microscopy shoed the presence of numerous dense-cored vesicles in the glomus cells of the carotid body; these vesicles occurred only rarely in the capsular cells and in the carotid nerve terminals. No detectable change in the vesicular content, appearance or distribution was induced by prolonge dhypoxia, reserpinization, adrenalectomy or chronic section of the carotid nerve or sympathetic supply. Acute and chronic reserpinization of cats did not change the sensitivity and reactivity of carotid bodies (either in situ or in vitro to ACh, anoxic or asphyxic stimulation. In vitro , high doses of adrenaline, noradrenaline, dopamine, dl -DOPA and tyramine failed to produced chemoreceptor excitation or marked depression of the chemosensory discharges. The lack of effect of CA on chemoreceptor activity was not changed by reserpinization or inactivation of monaminoxidase with nialamide. Prolonged superfusion with dischloroisoproterenol, a β-adrenergic blocking agent, reduced chemoreceptor discharges by inducing nerve block. It is concluded that CA contained in the carotid body do not play a significant role in the generation of the chemosensory discharges.

The release of acetylcholine from carotid body tissues. Further study on the effects of acetylcholine and cholinergic blocking agents on the chemosensory discharge.

1. Both carotid bodies were removed from cats and placed in a small Perspex channel through which Locke solution was allowed to flow under a layer of paraffin oil. Stimulation of the upstream (‘donor’) organ elicited an increased sensory discharge in the downstream (‘detector’) preparation (Loewi effect).

A comparative physiological and pharmacological study of cat and rabbit carotid body chemoreceptors

Carotid bodies and their nerves were excised from rabbits or cats, cleaned of surrounding connective tissue and placed in a chamber through which mammalian saline equilibrated with different gas mixtures was allowed to flow. Single fibers were isolated and identified as chemosensory by their response to hypoxia, hypercapnia, or NaCN. Mass receptor potentials (recorded at some distance from the sensory nerve endings) were evoked by the same stimuli and registered as close as possible to the carotid body. Both cats and rabbits exhibited receptor depolarization and an increased discharge in response to NaCN, hypoxia, hypercapnia and cyanide. However, the effects of some pharmacological agents were quite different in rabbits and cats. In the rabbit, ACh 10-100 microgram and carbachol 1-10 microgram produced receptor hyperpolarization and discharge depression followed by discharge increase. Nicotine 0.3-20 microgram induced receptor depolarization and increased chemosensory discharge frequency. Nicotinic stimulation was antagonized by D-tubocurarine 10(-6)-10(-4) g/ml. Pilocarpine 2-50 microgram hyperpolarized the receptors and depressed discharge frequency. Pilocarpine-induced depression was reduced by atropine 10(-6) g/ml. Dopamine 5-100 microgram depolarized the receptors and increased the chemosensory discharge frequency. This effect of dopamine was reduced by haloperidol (10(-11)-10(-7) M). In the cat, ACh, carbachol and nicotine (same doses as those used in rabbits) induced receptor depolarization and increased the sensory discharge frequency. Pilocarpine (up to 50 microgram) had little effect on either discharge frequency or the receptor potential. Dopamine 5-100 microgram induced receptor hyperpolarization and depression of discharge frequency, and these effects were reduced by haloperidol.

Axon regeneration following a lesion of the carotid nerve: Electrophysiological and ultrastructural observations

Carotid nerves of the cat were crushed and allowed to regenerate in order to study the properties of regerating fibers and the role of carotid body parenchymal cells (glomus or type I, and sustentacular or type II) in the transduction of chemosensory activity. Such activity is reinitiated 6 days after the nerves are crushed close (1-2 mm) to the carotid body. The process of recovery is delayed when a crush is made at successively greater distances (5-6 and 10-12 mm) from the carotid body. Ultrastructural studies show that the reappearance of nerve endings on the glomus-sustentacular cell complex coincides in time with the onset of chemosensory activity. The regenerated nerve endings increase in size and number and appear normal by 48 days. Some barosensory activity can be elicited 6 days after a nerve crush close to the carotid sinus, but rhythmic barosensory discharges only occur after the 21st day when myelinated axons reappear in the carotid sinus adventitia. Results suggest that recovery of chemosensory function depends on the reestablishment of apposition between regenerating carotid nerve fibers and parenchymal cells of the carotid body.

Effects of methionine-enkephalin and substance P on the chemosensory discharge of the cat carotid body

The effects of methionine enkephalin (ME) and substance P (SP) were tested on the chemosensory discharge of the cat carotid body-nerve preparation in vitro. ME superfused in concentrations of 10(-8) to 10(-5) M depressed the sensory discharge, an effect followed by receptor excitation (rebound). Bolus applications of ME (30 ng to 3.0 microgram) induced variable effects (excitation or depression) on the discharge, excitation being more pronounced with the smaller doses. Superfusions with SP (10(-8) to 10(-5) M) either excited or depressed the discharge, excitation being more pronounced with higher SP concentrations (i.e. 10(-6) M). Bolus applications of SP (43 ng to 0.5 micrograms) also excited or depressed the sensory discharge. These variations may be dose-dependent. Superfused ME (10(-6) M) significantly depressed the chemoreceptor response to hypoxia (100% N2) and hypercapnia (6% CO2, pH 7.43). The responses to NaCN and acidity (pH 6.0) were marginally depressed. Superfused SP (10(-6) M) clearly depressed the responses to hypoxia, those to hypercapnia and NaCN were marginally affected but the effects of acidity were not altered. When the peptides were tested against the receptor responses to exogenously applied putative neurotransmitters (ACh, dopamine--DA), it was found that ME tended to depress both the ACh and DA actions whereas SP (10(-6) M) tended to increase their effects. Superfusions with naloxone (10(-6) M) increased the basal chemosensory discharge and this enkephalin blocker partially relieved the depressant effect of ME on the ACh-induced response. It is concluded that carotid body chemoreceptors have excitatory and inhibitory reactive sites to both ME and SP although their precise location is still unknown.

Pharmacology of pH effects on carotid body chemoreceptors in vitro

1. The carotid body and the carotid nerve were removed from anaesthetized cats and placed in a small Perspex channel through which Locke solution (at various pH values and usually equilibrated with 50% O2 in N2) was allowed to flow. The glomus was immersed in the flowing solution while the nerve was lifted into oil covering the saline. Sensory discharges were recorded from the nerve and their frequency was used as an index of receptor activity. At times, a small segment of carotid artery, containing pressoreceptor endings, was removed together with the glomus. In this case, pressoreceptor discharges were recorded from the nerve.

The action of some cholinergic blockers on carotid body chemoreceptors in vivo.

Chemosensory discharges were recordedin vivo from fine filaments of the carotid nerve containing a few or single active units; their frequency was used as an index of receptor activity. The effects of hemicholinium, atropine, mecamylamine and hexamethonium on the chemosensory discharges elicited by ACh and NaCN were studied in 46 adult cats. Hemicholinium in low concentrations (0.5–1 mg/kg, i.v.) did not change the resting discharge rate or the chemoreceptor response to ACh and NaCN. One-2 h after injections of hemicholinium (2–5 mg/kg, i.v.) the responnse to NaCN was markedly depressed, while that to ACh remained unchanged. Larger doses of the drug (10 mg/kg, i.v.) depressed or blocked the responses to both chemicals. These experiments indicate that transmitter substances (probably ACh) stored in the cells are depleted or decreased by the presence hemicholinium, which blocks ACh synthesis. Atropine in low concentrations (0.2–1 mg/kg, i.v.) depressed the receptor response to ACh without affecting the discharges elicited by NaCN. On the other hand, relatively large doses of the drug (2–10 mg, i.v. and i.a.) blocked or depressed both the basal chemoreceptor discharges and the responses to ACh and NaCN. This depressant effect of atropine on the response to both chemicals lasted for 10–20 min. Mecamylamine (2–10 mg, i.a.) blocked the chemoreceptor response to ACh and NaCN. Naturally occuring chemoreceptor discharges were blocked transiently by the drug. Hexamethonium (2–10 mg, i.a.) was ineffective on the chemoreceptor response to ACh and NaCN in 25% of the chemosensory units examined, and in 50% of them the drug depressed or blocked the ACh-induced response without affecting the NaCN-induced effect. In 25% of the units, both the response to ACh and NaCN was blocked or depressed by the drug. Results indicate the approximate population of nicotinic cholinoceptive sites of chemosensory nerve terminals. It is concluded that a cholinergic mechanism is probably responsible for chemoreceptor impulse initiation. Exogenous ACh appears to be acting on extrasynaptic cholinergic receptors, probably located in the stem of the non-myelinated sensory endings, while natural stimuli and NaCN are probably acting on the glomus cells to liberate ACh, which exerts its effects on synaptic cholinergic sites.

Different effects of hypoxia on the membrane potential and input resistance of isolated and clustered carotid body glomus cells

Intracellular recordings were made from cultured glomus cells of rat carotid bodies. Hypoxia (PO2 1-61 Torr), induced by Na-dithionite (Na2S2O4), differentially affected cells that were clustered and those that were isolated. More than 80% of clustered cells depolarized and their input resistance decreased, whereas about 60% of isolated cells hyperpolarized and their input resistance increased. In both groups, the mechanisms for cell depolarization or hyperpolarization appeared similar. Differences between clustered and isolated cells seemed to be conditioned by the sustentacular cells. Their processes surrounded clustered cells but were absent around isolated ones. However, we may have also functionally different glomus cells which may or may not correspond to the different types described by anatomists.

Changes in glomus cell membrane properties in response to stimulants and depressants of carotid nerve discharge

Intracellular recordings were made from glomus cells in the excised, intact or sliced (150-200 microns) carotid body. Carotid nerve discharge was also recorded from intact preparations. Slices were prepared for visual (Nomarski) control of microelectrode impalement. Resting potential (Em), input resistance (Ro) and voltage noise (Erms) were measured in control conditions and in response to several stimulants (interruption of flow, hypoxic and histotoxic [NaCN]anoxia, hypercapnia, asphyxia and acidity) and depressants (alkalinity, cooling) of the carotid nerve sensory discharge. Different glomus cells responded differently to the same stimulus but significant trends were found. The more common responses to zero flow and anoxia (hypoxic and histotoxic) were depolarization (64%) and decreases in Erms (63%) and Ro (71%). When extracellular pH was varied from 8.5 to 5.0, the preponderant responses were cell depolarization, and increases in noise and input resistance as pH decreased. Consequently, cell depolarization induced by zero flow and anoxia tended to be accompanied by reduced Ro, whereas that induced by acidity generally showed increased Ro. Changes in voltage noise usually followed variations in Ro. When nerve discharge frequency was plotted against delta Em or delta Erms there were positive correlations during acid stimulation. However, these correlations were complex (parabolic) during flow interruption and anoxia: an increase in discharge occurred in response to cell depolarization and to hyperpolarization. These results suggest that hypoxia and hypercapnic or acidic stimuli act on glomus cells by different mechanisms. This finding is consistent with evidence obtained by recording carotid nerve discharges in intact animals.

Electrical communication between glomus cells of the rat carotid body

Glomus cells of rat carotid bodies can be electrotonically coupled. This was determined by simultaneous intracellular recording and stimulation of two neighboring cells. Voltage applied into one cell (V1), was detected in the other cell as E2. The ratio E2/V1 or coupling coefficient (KC), varied from 0.003 to 1. R0 or input resistance (24.1-3,500 M omega), was calculated from the voltage elicited in the injected cell by current injection (V1/I1). The coupling resistance (RC) was estimated by using Bennetts model and was inversely related to KC. It ranged from 8.5 to 46,112 M omega. Values for KC are provisional since we may not have always recorded from immediately adjacent cells. Similarly, calculations of R0 and RC may not be accurate since, in all probability, there is a multicellular network. Stimulation by hypoxia (100% N2 or Na2S2O4), acidity (lactic acid or 100% CO2), dopamine, ACh, nicotine and bethanechol depolarized the majority of glomus cells, their input resistance decreased and cells became uncoupled. Fewer cells were either unaffected or coupling increased. There was a significant and negative correlation between changes in coupling coefficient and in coupling resistance.