L. Monti-Bloch

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.

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.

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.

Effects of Putative Neurotransmitters of the Carotid Body on its Own Glomus Cells.

Carotid body glomus cells produce and release acetylcholine (ACh), catecholamines, and neuropeptides, and there is biochemical evidence that these cells possess receptors for these substances. Thus, we studied the effects of cholinergics [ACh, nicotine (Nic), bethanechol (BN)] and peptides [met‐enkephalin (ME), substance P (SP)] on the membrane potential (Em), voltage noise (Erms), and input resistance (R0) of glomus cells. Sliced carotid bodies (for cell visualization) of cats, rabbits, and mice were used.

Nicotinic and muscarinic reactive sites in mammalian glomus cells

Nicotinic and muscarinic sites on glomus cell membranes of cats, rabbits and mice were determined in carotid body slices. Cells were impaled under Nomarski optics. Resting potentials were 11.1-73.1 mV and input resistances were 11-250 M omega. Nicotine, pilocarpine or bethanechol depolarized glomus cells and their input resistance decreased. Curare or alpha-bungarotoxin reduced nicotine effects. Atropine had similar effects on the responses to pilocarpine or bethanechol.

Carotid body grafts induced chemosensitivity in muscle nerve fibers of the cat

The ability of carotid body parenchymal cells to change the physiological properties of nerve fibers was tested in 8 cats by transplanting the carotid body to the tenuissimus muscle and reinnervating the organ with the muscle nerve. Ninety-five to 174 days after the transplant, electrophysiological recordings were obtained from the reinnervating tenuissimus nerves. Forty percent of the regenerating nerve fibers having spontaneous activity responded to muscle stretch, hypoxia, NaCN, ACh, nicotine and dopamine, apparently because of dual innervation of glomus cells and muscle spindles. Five of 6 transplants, examined electron microscopically, contained viable glomus cells innervated by axon terminals. Neither the normal tenuissimus nerve nor the same nerve regenerating into adipose tissue grafts or to one non-viable carotid body transplant showed chemosensory properties although these grafts contained numerous, small myelinated axons indicative of extensive nerve regeneration. It is concluded that carotid body cells are capable of inducing chemosensory properties in axons that, normally, subserve a mechanosensory function.

Effects of temperature on denervated carotid body (glomus) cells

Cat carotid bodies were denervated for 3-31 days by sectioning the carotid nerve. The normal response of glomus cells to cooling by about 6 degrees C, namely, membrane depolarization and reduced input resistance (Ro) was altered. At 3 days, cooling induced normal depolarization although Ro decreased more markedly. At 7 days, cooling depolarized 53% of the cells which was accompanied by variable Ro changes. Forty-seven percent of the cells were hyperpolarized and their Ro increased. At 16 days, a fall in temperature depolarized 25% of the cells which showed variable changes in Ro, whereas the rest showed hyperpolarization and increased Ro. At 31 days, cooling weakly depolarized 64% of the cells together with a small reduction in Ro whereas 28% showed some hyperpolarization and larger Ro. It is concluded that sensory denervation alters the membrane of glomus cells, the preneuronal component of the receptor complex.

Effects of denervation on the glomus cell membrane

Trophic influences of the carotid nerve (CN) on carotid body (CB) glomus cells were studied by comparing the membrane potential (Em), input resistance (Ro) and voltage noise (Erms) of normal and 3-31-day denervated cells had more negative Ems. Higher Ros were recorded at 3 and 6 days. Erms sharply increased at 3 days, returned to normal at 6-15 days and was below normal at 31 days. A transmitter (ACh) and NaCN, producing histotoxic anoxia, were used for stimulation. These substances either depolarized or hyperpolarized innervated cells and increased or decreased voltage noise. Denervation selectively changed these patterns but only for a short time. ACh preferentially depolarized the cells, only at 3 days, whereas its effects on noise did not change. The Em responses to NaCN remained unaltered although at 3-6 days noise increases were smaller and depressions exaggerated. Possible reasons for these effects are discussed.

EFFECTS OF DIFFERENT STIMULI ON THE GLOMUS CELL MEMBRANE

Publisher Summary This chapter explores the effects of different stimuli on the glomus cell membrane. The study described in the chapter involved removing the carotid body and sinus nerve from anesthetized cats and placing the preparation in a chamber through which flowed physiological saline equilibrated with 50% O2. The nerve discharges were registered with wire electrodes connected to an a.c. recording system. The carotid body was impaled with microelectrodes filled with 3M KCl, and intracellular potentials were recorded with a high impedance electrometer connected to the d.c. amplifier of an oscilloscope. The intracellularly recorded elements were often stained by using microelectrodes filled with a procion dye, which was ejected from the micropipette after recordings were made. The tissues were then prepared for histology and observation with interference contrast optics. The other method consisted of cutting the carotid body into 100 to 200 μm slices, which were placed in a chamber mounted on the stage of an inverted microscope equipped with Nomarski optics. Impalements of carotid body cells yielded membrane potentials (MPs) of 10–60 mV and mean about 20 mV. These values are low, probably because of partial membrane injury produced by insertion of the electrode into these small cells.