Acidic regulation of junction channels between glomus cells in the rat carotid body. Possible role of [Ca]i

The purpose of this work was to characterize the gap junctions between cultured glomus cells of the rat carotid body and to assess the effects of acidity and accompanying changes in [Ca²⁺]_i on electric coupling. Dual voltage clamping of coupled glomus cells showed a mean macrojunctional conductance (G_j) of 1.16 nS±0.6 (S.E.), range 0.15–4.86 nS. At normal pH_o (7.43), a steady transjunctional voltage (ΔV_j=100.1±10.9 mV) showed multiple junction channel activity with a mean microconductance (g_j) of 93.98±0.6 pS, range 0.3–324.5 pS. Single-channel conductances, calculated as variance/mean g_j, gave a mean value of 16.7±0.2 pS, range 5.13–39.38 pS. Manual measurements of single-channel activity showed a mean g_j of 22.03±0.2 pS, range 1.3–160 pS. Computer analysis of the noise spectral density distribution gave a channel mean open time of 12.7±1.5 ms, range 6.37–23.42 ms. The number of junction channels, estimated in each experiment from G_j/single-channel g_j, showed a range of 7 to 258 channels (mean, 107.2). Optical measurements of [Ca²⁺]_i gave a mean value of 80.2±4.27 nM at pH_o of 7.43. Acidification of the medium with lactic acid (1 mM, pH 6.3) induced: 1) Variable changes in G_j (decreases and increases); 2) A significant decrease in mean g_j (to 80.36±0.34 pS) and in single-channel conductance (g_j=12.8±0.2 pS in computer analyses and 17.23±0.2 pS when measured by hand); 3) Variable changes in open times, resulting in a similar mean (12.8±1.5 ms) and 4) No change in the number of junction channels. When pH_o was lowered to 6.3 [Ca²⁺]_i did not change significantly (there were increases and decreases). However, when pH_o was lowered to 4.4, [Ca²⁺]_i increased significantly to 157.1±8.1 nM. It is concluded that saline acidification to pH 6.3 depresses the conductance of junction channels and this effect may be either a direct effect on channel proteins or synergistically enhanced by increases in [Ca²⁺]_i. However, there are no studies correlating changes of [Ca²⁺]_i and intercellular coupling in glomus cells. Stronger acidification (pH_o 4.4), producing much larger changes in [Ca²⁺]_i, may enhance this synergism. But, again, there are no studies correlating these effects.     

Electrical coupling between cultured glomus cells of the rat carotid body: observations with current and voltage clamping

Electrically coupled pairs of cultured rat glomus cells were used. In one group of experiments, both cells were current-clamped. Delivery of positive or negative pulses to Cell 1 elicited appreciable voltage noise in this cell and large action potentials (probably Ca²⁺ spikes) in about 10% of them. Both passive and active electrical events spread to Cell 2, presumably through the gap junctions between them. The coupling coefficient (K_c) was larger for the spikes than for non-regenerative voltage noise. In another group of experiments, Cell 1 was current-clamped and Cell 2 was voltage-clamped at Cell 1 E_M. Pulses of either polarity, delivered to Cell 1, produced current flow through the intercellular junction and allowed direct measurement of junctional currents (I_j) and total conductances (G_j). I_j had a mean value of about 12.5 pA and G_j of 391 pS. Unitary (presumably single channel) conductance (g_j) was about 78 pS.     

A new mutation in GJC2 associated with subclinical leukodystrophy

Recessive mutations in GJC2, the gene-encoding connexin 47 (Cx47), cause Pelizaeus–Merzbacher-like disease type 1, a severe dysmyelinating disorder. One recessive mutation (p.Ile33Met) has been associated with a much milder phenotype––hereditary spastic paraplegia type 44. Here, we present evidence that a novel Arg98Leu mutation causes an even milder phenotype––a subclinical leukodystrophy. The Arg98Leu mutant forms gap junction plaques in HeLa cells comparable to wild-type Cx47, but electrical coupling was 20-fold lower in cell pairs expressing Arg98Leu than for cell pairs expressing wild-type Cx47. On the other hand, coupling between Cx47Arg98Leu and Cx43WT expressing cells did not show such reductions. Single channel conductance and normalized steady-state junctional conductance–junctional voltage (G _j–V _j) relations differed only slightly from those for wild-type Cx47. Our data suggest that the minimal phenotype in this patient results from a reduced efficiency of opening of Cx47 channels between oligodendrocyte and oligodendrocyte with preserved coupling between oligodendrocyte and astrocyte, and support a partial loss of function model for the mild Cx47 associated disease phenotypes.     

Effects of hypoxia induced by Na2S2O4 on intracellular calcium and resting potential of mouse glomus cells

Isolated and cultured glomus cells, obtained from mouse carotid bodies, were superfused with Ham's F-12 equilibrated with air (mean PO₂, 119 Torr; altitude 1350 m). [Ca²⁺]_o was 3.0 mM. In one experimental series, dual cell penetrations with microelectrodes measured intracellular calcium ([Ca²⁺]_i) and the resting potential (E_m). In another series, [Ca²⁺]_i was measured with Indo-1/AM, dissolved in DMSO. Normoxic cells had a mean E_m of −42.4 mV and [Ca²⁺]_i was about 80 nM (measured with both methods). The calculated calcium equilibrium potential (E_Ca) was 137±0.74 mV. Hypoxia, induced by Na₂S₂O₄ 1 mM, reduced pO₂ to 10–14 Torr. This effect was accompanied by cell depolarization to −19.1 mV. Hypoxia increased [Ca²⁺]_i to 231 nM when detected with Ca-sensitive microelectrodes, but only to 130.2 nM when measured with Indo-1/AM. Calcium increases were preceded by decreases in [Ca²⁺]_i, which also were more pronounced with microelectrode measurements. CoCl₂ 1 mM blocked the hypoxic [Ca²⁺]_i increase and exaggerated the decreases in [Ca²⁺]_i. Correlations between ΔE_m and Δ[Ca²⁺]_i during hypoxia were significant (p<0.05) in 19% of the cells. But, in 29% of them significance was at the p<0.1 level. In the rest (52%), there was no correlation between these parameters. Thus, voltage-gated calcium channels are rare in mouse glomus cells. Their activation by depolarization cannot explain the two to threefold increase in [Ca²⁺]_i seen during hypoxia. More likely, [Ca²⁺]_i increase may be due to hypoxic inactivation of a Ca–Mg ATPase transport system across the cell membrane. The blunting of hypoxic [Ca²⁺]_i increase, seen in Indo-1/AM experiments, is probably due to its solvent (DMSO), which also depresses hypoxic cell depolarization.     

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 asE₂. The ratioE₂/V₁ or coupling coefficient (K_C, varied from 0.003 to 1.R₀ or input resistance (24.1–3,500 MΩ), was calculated from the voltage elicited in the injected cell by current injection (V₁/I₁). The coupling resistance (R_C) was estimated by using Bennett's model² and was inversely related toK_C. It ranged from 8.5 to 46,112 MΩ. Values forK_C are provisional since we may not have always recorded from immediately adjacent cells. Similarly, calculations ofR₀ andR_C 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.     

Action potential-like responses in B 104 cells with low Na channel densities

Action potential generation and Na⁺ currents were studied in B104 neuroblastoma cells in vitro using the whole-cell patch-clamp method in voltage-clamp and current-clamp mode. Action potential-like responses were elicited in 38 of 42 cells, with a threshold close to −55 mV for depolarizing stimuli, and −56 mV for anode-break stimuli. Response amplitudes were larger when cells were held at more negative prepulse potentials, and were well fit by a Boltzmann distribution with a midpoint of approx. −75 mV, close to theV_1/2 for Na⁺ current steady-state inactivation in these cells. Cells displaying action potential-like responses exhibited a peak Na⁺ current density of 133 ± 0.14 pA/pF (range, 10.2–296.2 pA/pF) and a lowg_K: g_Na ratio (0.0067 ± 0.0023). Exposure to 0.1 mM Cd²⁺ did not block the generation of action potential-like responses in B104 cells, while 1 μM TTX abolished the responses. We conclude that low densities of Na⁺ channels ( < 3/μm² and < 1/μm² in some cells) can support the generation of action potential-like responses in B104 cells if they are held at hyperpolarized levels to remove inactivation. The low leak and K⁺ conductance of these cells may contribute to their ability to generate action potential-like responses under these circumstances.     

Hypoxia induced by Na2S2O4 increases [Na]i in mouse glomus cells, an effect depressed by cobalt. Experiments with Na-selective microelectrodes and voltage-clamping

The intracellular sodium concentration ([Na⁺]_i) and resting potential (E_m) of cultured mouse glomus cells (clustered and isolated) were simultaneously measured with intracellular Na⁺-sensitive and conventional, KCl-filled, microelectrodes. Results obtained in clustered and isolated cells were similar. During normoxia (PO₂ 122 Torr), [Na⁺]_i was 12–13 mM corresponding to a Na⁺ equilibrium potential (E_Na) of about 58 mV. Em was about −42 mV. Hypoxia, induced by Na₂S₂O₄ 1 mM (PO₂ 10 Torr), depolarized the cells by about 20 mV, [Na⁺]_i increased by 21 mM and E_Na dropped to about 35 mV. One millimolar of CoCl₂ depressed, or blocked, the effects of Na₂S₂O₄ on [Na⁺]_i but did not affect hypoxic depolarization. Voltage-clamping at −70 mV, while delivering pulses of different amplitudes, produced only small (about 10 pA) and slow TTX-insensitive inward currents. Fast and large (TTX-sensitive) inward currents were not detected. The cell conductance (measured with voltage ramps) was less than 1 nS. It was not affected by hypoxia but was depressed by cobalt. Voltage ramps elicited small inward currents in control and hypoxic solutions that were much smaller than those induced by barium (presumably enhancing calcium currents). Also, normoxic and hypoxic currents had lower thresholds and their troughs were at more negative voltages than in the presence of Ba²⁺. All currents were blocked by 1 mM CoCl₂ suggesting that, at this concentration, cobalt exerted a nonspecific effect on glomus membrane channels. Hypoxia induced a large [Na⁺]_i increase (presumably through inflow), but very small voltage-gated inward currents. Thus, Na⁺ increases (inflow) probably occurred by disturbing a Na⁺/K⁺ exchange mechanism and not by activation of voltage-gated channels.     

Effects of ginsenosides on Ca channels and membrane capacitance in rat adrenal chromaffin cells

We investigated the effects of ginseng total saponins (GTS) and five ginsenosides on voltage-dependent Ca²⁺ channels and membrane capacitance using rat adrenal chromaffin cells. In this study, cells were voltage-clamped in a whole-cell recording mode and a perforated patch-clamp technique was used. The inward Ca²⁺ currents (I_Ca) was elicited by depolarization and the change in cell membrane capacitance (ΔC_m) was monitored. The application of GTS (100 μg/ml) induced rapid and reversible inhibition of the Ca²⁺ current by 38.8 ± 3.6% (n = 16). To identify the particular single component that seems to be responsible for Ca²⁺ current inhibition, the effects of five ginsenosides (ginsenoside Rb₁, Rc, Re, Rf, and Rg₁) on the Ca²⁺ current were examined. The inhibitions to the Ca²⁺ current by Rb₁, Rc, Re, Rf, and Rg₁ were 15.3 ± 2.2% (n = 5); 36.9 ± 2.4% (n = 7); 28.1 ± 1.9% (n = 12); 19.0 ± 2.5% (n = 10); and 16.3 ± 1.6% (n = 15), respectively. The order of inhibitory potency (100 μM) was Rc > Re > Rf > Rg₁ > Rb₁. A software based phase detector technique was used to monitor membrane capacitance change (ΔC_m). The application of GTS (100 μg/ml) induced inhibitory effects on ΔC_m by 60.8 ± 9.7% (n = 10). The inhibitions of membrane capacitance by Rb₁, Rc, Re, Rf, and Rg₁ were 35.3 ± 5.5% (n = 7); 41.8 ± 7.0% (n = 8); 40.5 ± 5.9% (n = 9); 51.2 ± 7.6% (n = 9); and 35.9 ± 5.1% (n = 10), respectively. The inhibitory potencies of the ginsenosides on ΔC_m were Rf > Rc > Re > Rg₁ > Rb₁. Therefore, we found that GTS and ginsenosides exerted inhibitory effects on both Ca²⁺ currents and ΔC_m in rat adrenal chromaffin cells. These results suggest that ginseng saponins regulate catecholamine secretion from adrenal chromaffin cells and this regulation could be the cellular basis of antistress effects induced by ginseng.     

8-Azido-Nucleotides as Substrates of Torpedo Electric Organ Apyrase. Effect of Photoactivation on Apyrase Activity

Ecto-apyrase is a widespread enzymatic activity that hydrolyses tri- and diphosphonucleotides and consequently controls the amount of available extracellular ATP and ADP. In the nervous system, purines have important neuromodulatory actions, acting at pre- and postsynaptic sites, and consequently, ecto-apyrase may play an indirect role in the modulation of nucleotide- and nucleoside-mediated processes. The azido-nucleotides have been largely employed to characterize the nucleotide binding sites of several proteins. In the present work the azido-nucleotides are described as putative substrates for apyrase activity in a presynaptic plasma membrane preparation (PSPM) from the Torpedo electric organ. Both 8-N₃-ATP and 8-N₃-ADP were hydrolyzed in a calcium-dependent manner showing V_max of 23.8 ± 4.8 and 14.5 ± 3 U/mg of protein, and K_m values (in μM) of 116 ± 39 and 119 ± 4, respectively. V_max for calcium-dependent hydrolysis of ATP and ADP were significantly higher: 59.2 ± 3.9 and 32.9 ± 3.5 U/mg of protein respectively, while K_m values did not show any significant differences regarding azido-nucleotides: 83.8 ± 12 μM for Ca²⁺-ATP and 121 ± 34 μM for Ca²⁺-ADP. The photoactivation of the PSPM in the presence of the azido-derivatives results in an irreversible inactivation of apyrase activity, showing an IC₅₀ of 10 μM and a maximal inhibitory effect of 38 and 60% on Ca²⁺-ATPase and Ca²⁺-ADPase activities. Apyrase was protected from inactivation by nucleotides that are natural substrates for this enzymatic activity and also by AMP while adenosine did not protect from apyrase inhibition.     

Effect of the removal of extracellular Ca on the response of cytosolic concentrations of Ca to ouabain in carotid body glomus cells of adult rabbits

Effect of the removal of extracellular Ca²⁺ on the response of cytosolic concentrations of Ca²⁺ ([Ca²⁺]_i) to ouabain, an Na⁺/K⁺ exchanger antagonist, was examined in clusters of cultured carotid body glomus cells of adult rabbits using fura-2AM and microfluorometry. Application of ouabain (10 mM) induced a sustained increase in [Ca²⁺]_i (mean±S.E.M.; 38±5% increase, n=16) in 55% of tested cells (n=29). The ouabain-induced [Ca²⁺]_i increase was abolished by the removal of extracellular Na⁺. D600 (50 μM), an L-type voltage-gated Ca²⁺ channel antagonist, inhibited the [Ca²⁺]_i increase by 57±7% (n=4). Removal of extracellular Ca²⁺ eliminated the [Ca²⁺]_i increase, but subsequent washing out of ouabain in Ca²⁺-free solution produced a rise in [Ca²⁺]_i (62±8% increase, n=6, P<0.05), referred to as a [Ca²⁺]_i rise after Ca²⁺-free/ouabain. The magnitude of the [Ca²⁺]_i rise was larger than that of ouabain-induced [Ca²⁺]_i increase. D600 (5 μM) inhibited the [Ca²⁺]_i rise after Ca²⁺-free/ouabain by 83±10% (n=4). These results suggest that ouabain-induced [Ca²⁺]_i increase was due to Ca²⁺ entry involving L-type Ca²⁺ channels which could be activated by cytosolic Na⁺ accumulation. Ca²⁺ removal might modify the [Ca²⁺]_i response, resulting in the occurrence of a rise in [Ca²⁺]_i after Ca²⁺-free/ouabain which mostly involved L-type Ca²⁺ channels.     

Glutamine transport in cerebellar granule cells in culture

In the present study, uptake of glutamine by rat cerebellar granule cells, a predominantly glutamatergic nerve cell population, has been investigated. Glutamine is taken up by granule cells via at least three transport systems, A, ASC and L. The L-type low affinity system (K_m=2.6 mM) is the major transport system in the absence of Na⁺. The systems A and ASC represent the Na⁺-dependent transport routes, both with almost identical high affinity for glutamine (K_m=0.26 mM). Similar transport systems for glutamine are also found in cerebral cortical neurons, a predominantly GABAergic nerve cell population, and cerebral cortical astrocytes. The glutamine transport properties in granule cells, however, show a series of differences from that of cortical neurons and astrocytes: (1) uptake of glutamine by granule cells is primarily mediated by system A (54%), while contributions by system A in cortical neurons and astrocytes are less than 30%; (2) granule cells exhibit strikingly higher transport efficiency for glutamine (V_max/K_m=20 min⁻¹ for system A as compared to the V_max/K_m ratio of 5 min⁻¹ in cortical neurons and astrocytes), and (3) the initial uptake rates and the steady-state accumulation levels of glutamine are two- to threefold higher in granule cells than that of cortical neurons and astrocytes. These results taken together suggest that in accordance with the important need to replenish the neurotransmitter pool of glutamate, glutamatergic neurons exhibit highly efficient transport systems to accumulate glutamine, one of the major precursors of glutamate.     

Ionic currents on type-I cells of the rabbit carotid body measured by voltage-clamp experiments and the effect of hypoxia

Type-I cells (from rabbit embryos) in primary culture were studied in voltage-clamp experiments using the whole cell arrangement of the patch-clamp technique. With a pipette solution containing 130 mM K⁺ and 3 mM Mg-ATP, large outward currents were obtained positive to a threshold of about −30 mV by clamping cells from −50 mV to different test pulses (−80 to 50 mV). Negative to −30 mV, the slope conductance was low (outward rectification). The outward currents were blocked by external Cs⁺ (5 mM) and partially blocked by TEA (5 mM) and Co²⁺ (1 mM). The initial part of the outward currents during depolarizing voltage pulses exhibited a transient Ca²⁺ inward component partially superimposed to a Ca²⁺-dependent outward current. Inward currents were further characterized by replacing K⁺ with Cs⁺ in the intra- and extracellular solution in order to minimize the outward component and by using 1.8 mM Ca²⁺ or 10.8 mM Ba²⁺ as charge carrier. Slow-inactivating inward currents were recorded at test potentials ranging from −50 to 40 mV (holding potential −80 mV). The maximal amplitude, measured at 10 mV in the U-shaped I–V curve, amounted to 247 ± 103pA(n = 3). This inward current was insensitive to 3 μM TTX, but blocked by 1 mM Co²⁺ and partially reduced by 10 μM D600 and 3 μM PN 200-110. In contrast to outward currents, the inward currents exhibited a ‘run-down’ within about 10 min. Lowering the pO₂ from the control of 150 Torr (air-gassed medium) to 28 Torr had no apparent effect on inward currents, but depressed reversibly outward currents by 28%. In conclusion, it is suggested that type-I cells possess voltage-activated K⁺ and Ca²⁺ channels which might be essential for chemoreception in the carotid body.     

Kinetic analysis ofl-leucine transport across the blood-brain barrier

Unidirectionall-leucine influx across cerebral capillaries was measured at different concentrations with an in situ rat brain perfusion technique, which has been several advantages over presently-used methods such as the Brain Uptake Index (BUI) technique. The maximal influx rate (V_max) and half-saturation concentration (K_m) equaled1.07 ± 0.02 × 10^3- μmol·s⁻·⁻¹and0.026 *+- 0.002 μmol·ml⁻¹, respectively, for the saturable component, and the constant for non-saturable equaled6.8 ± 1.4 × 10⁻⁵s⁻¹. These values differ by 3–4-fold from respective values obtained with the BUI technique.     

Anoxia-induced depolarization in CA1 hippocampal neurons: role of Na-dependent mechanisms

We have previously shown that (1) removal of extracellular sodium (Na⁺) reduces the anoxia-induced depolarization in neurons in brain-slice preparations and (2) amiloride, which blocks Na⁺-dependent exchangers, prevents anoxic injury in cultured neocortical neurons. Since anoxia-induced depolarization has been linked to neuronal injury, we have examined in this study the role of Na⁺-dependent exchangers and voltage-gated Na⁺ channels in the maintenance of membrane properties of CA1 neurons at rest and during acute hypoxia. We recorded intracellularly from CA1 neurons in hippocampal slices, monitored V_m and measured input resistance (R_m) with periodic injections of negative current. We found that tetrodotoxin (TTX, 1 μM) hyperpolarized CA1 neurons at rest and significantly attenuated both the rate of depolarization (ΔV_m/dt) and the rate of decline of R_m (ΔR_m/dt) by about 60% during the early phase of hypoxia. The effect of TTX was dose-dependent. Amiloride (1 mM) decreased V_m and increased R_m in the resting condition but changed little the effect of hypoxia on neuronal function. Benzamil and 5-(N-ethyl-N-isopropyl)-2′,4′-amiloride (EIPA), two specific inhibitors of Na⁺ dependent exchangers, were similar to amiloride in their effect. We conclude that neuronal membrane properties are better maintained during anoxia by reducing the activity of TTX-sensitive channels and not by the action of Na⁺-dependent exchangers.     

Ca-dependent adaptive properties in the solitary olfactory receptor cell of the newt

The time-dependent decay of the olfactory receptor potential was analyzed with a solitary cell preparation by using the whole-cell patch clamp technique. During prolonged stimulation by 10 mMN-amylacetate under standard conditions, 17 out of 63 isolated olfactory cells responded with slow depolarization. Of these 17 cells, response amplitudes in 14 cells (‘phasic/tonic’ response) gradually decayed within 9 s, with a half-decay time of1.71 ± 1.10s(mean±S.D.). The relative amplitude (ratio of tonic component to peak amplitude,V_tonic/V_max) was0.29 ± 0.10. The response decay was attributed to the inactivation of the odorant-activated conductance. The recovery after inactivation, which was determined with double pulse experiments, was dependent on the resting interval. The inactivation of the odorant-activated conductance was found to be observed only when the external medium contained Ca²⁺. In addition, it was found that the odorant-activated conductance was capable of permeating Ca²⁺ (P_Ca/P_Na= 6.5), and a rise in the internal EGTA concentration (to 50 mM) inhibited the inactivation. These observations suggest that the decay of the olfactory response to prolonged stimulation is mediated by Ca²⁺ influx.     

Ryanodine receptor-mediated [Ca]i release in glomus cells is independent of natural stimuli and does not participate in the chemosensory responses of the rat carotid body

The hypothesis that intracellular calcium ([Ca²⁺]_i) release in glomus cells via ryanodine receptor (RyR) activation by caffeine may be independent of natural stimuli and chemosensory discharge was tested in the rat carotid body (CB). CB type I cells were isolated, plated and preloaded with calcium-sensitive fluorescent probe, Indo-1AM. With the increase of caffeine dose (0–50 mM) cytosolic calcium ([Ca²⁺]_c) increased from 85±15 nM to 1933±190 nM (n=6) at normoxia (P ₂=125–130 Torr, P ₂=25–30 Torr, pH 7.30–7.35). Hypoxia (P ₂=10–15 Torr) increased and hypocapnia (P ₂=7–9 Torr) decreased the cytoplasmic calcium [Ca²⁺]_c levels, independent of caffeine. Caffeine-related [Ca²⁺]_c increase was the same in the presence and the absence of extracellular calcium ([Ca²⁺]_o), indicating the source of Ca²⁺ ions is the cellular store. Permeabilization of the cell membrane with saponin (25 μg/ml) retained the caffeine response. Additional treatment of the cells with 50 μM ryanodine (an inhibitor of the caffeine-activated RyR site) abolished caffeine-stimulated response. In vitro CB chemosensory (carotid sinus nerve, CSN) responses to hypoxia (P ₂=35–40 Torr) were not altered by caffeine. These results suggest that [Ca²⁺]_i stores in CB cells, mobilized by RyR activation, do not participate in the CSN responses to natural stimuli.     

Cardiorespiratory effects produced by application ofl-glutamic and kainic acid to the ventral surface of the cat hindbrain

The purpose of our study was to determine the cardiorespiratory effects of exciting cell bodies at the rostral (area M), intermediate (area S), and caudal (area L) chemosensitive sites on the ventral surface of the medulla. To do this,l-glutamic and kainic acid were applied bilaterally to each chemosensitive site while monitoring tidal volume (V_T), respiratory rate (f), mean arterial pressure (BP), and heart rate (HR) in chloralose-anesthetized cats. Application of solutions (5 μl) ofl-glutamic acid ranging from 62.5 to 2000 mM to the intermediate area produced concentration-dependent increases inV_T (from2.0 ± 0.6ml to14 ± 1.1ml) andBP(from2.0 ± 1.7mm Hg to39 ± 5.3mm Hg). No significant changes in f and HR were noted. Similar effects were observed with application of kainic acid. Application ofl-glutamic acid to the caudal area produced hypotension−24 ± 5.4mm Hg) with no accompanying changes in V_T and f. No responses were observed after application ofl-glutamic acid to the rostral area. These data suggest that activation of cell bodies on the intermediate area produces simultaneous stimulatory effects on BP and V_T, whereas activation of cell bodies at the caudal area produces selective depressant effects on BP.     

Intracellular potassium activity, potassium equilibrium potential and membrane potential of carotid body glomus cells

The intracellular potassium activity (a_i(K)) of glomus cells in isolated rabbit carotid bodies was measured with potassium ion selective microelectrodes (K⁺ electrodes). The measurements yielded a mean a_i(K) value of 33.0 mM in Hepes-buffered Tyrode's solution equilibrated with 100% O₂. This value is significantly higher than that predicted by assuming that K⁺ ions are passively distributed across the membrane of glomus cells. The relationship between the membrane potential (E_M) and the equilibrium potential of K⁺ ions (E_K) at various extracellular potassium activities (a_o(K)) suggests that K⁺ ions are not involved in the maintenance of the carotid body glomus cell E_M.     

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

The hypothesis that suppression of O₂-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 [Ca²⁺]_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 CO₂-HCO₃^- buffer) and recording [Ca²⁺]_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 [Ca²⁺]_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 [Ca²⁺]_i by 4-AP are not consistent with the implied meaning of the suppressant effect on K⁺ current of glomus cells.