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C Eyzaguirre

Publications and source records attributed to C Eyzaguirre.

At least 19 recordsLinked to original sources
PubMed2006

Effects of prolonged hypobaric hypoxia on carotid nerve endings and glomus cells. Changes in intercellular coupling.

Carotid bodies were removed from anesthetized rats kept under normobaric (640 Torr) and hypobaric conditions (380 Torr for 2-3 weeks). Slices (100-150 microm) 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 Em, high input resistance (Ro) and time-dependent rectification in response to negative current pulses. Also, when nerve endings had an Em more negative than -40 mV showed spontaneous activity in the form of mini-receptor potentials (mrps). Glomus cells had less negative Em and lower Ro. Prolonged hypobaric hypoxia did not change the Em of nerve endings and glomus cells. However, in both structures, Ro 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.

Animals↗
PubMed2005

Calcium channels of cultured rat glomus cells in normoxia and acute hypoxia.

Glomus cells harvested from Wistar rat carotid bodies were cultured for 4 to 7 days. Inward calcium currents elicited by voltage ramps (0.24 V/s) or pulses were recorded during voltage-clamping in the whole-cell and perforated patch configurations. Currents were enhanced by an excess of [Ca(2+)](o), barium and BayK 8644, and depressed or eliminated by cobalt or nifedipine. Single calcium channels were studied by patch-clamping in the cell-attached configuration with voltage clamp pulses ranging from 0.5 to 50 s. Channel conductances (g) decreased and open times (OT) increased as clamp pulses increased in duration. For comparisons, conductances and OTs obtained with short (0.5-1 s) and long (6-12 s) pulses were grouped as SVH and LVH, respectively. SVH conductances were higher and OTs shorter when compared to LVH. BayK 8644 increased conductances and OT during SVH but this agonist decreased g during LVH. Nifedipine either eliminated channel activity, had no effects or depressed g and OT. Hypoxia (pO(2) 30 Torr) induced by 100% N(2) significantly increased calcium currents in normal bathing solutions and during exposure to 110 mM BaCl(2) in whole-cell and perforated patch recordings. Sodium dithionite (Na(2)S(2)O(4)), lowering pO(2) to 10 Torr, also increased the amplitude of calcium currents, but shifted to more positive voltages the onset and trough (maximum) of calcium currents. N(2)-induced hypoxia increased g and reduced OT during SVH but had opposite effects with longer pulses: conductance decreased and open times increased. N(2)-induced hypoxia increased the numbers of active channels (from 1 to 35) over a mean normoxic level of 47 per cell. It is suggested that increased calcium currents accompany calcium inflow in glomus cells, but calcium influx may not depend exclusively on this mechanism.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed2004

Effects of hypoxia and putative transmitters on [Ca2+]i of rat glomus cells.

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: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. 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.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.ACh, nicotine and pilocarpine potentiated the excitatory effect of Na2S2O4 on [Ca2+]i. Bethanechol depressed this excitation whereas muscarine had inconsistent effects. Atropine and C6 depressed [Ca2+]i increases elicited by Na2S2O4 but the effects of D-TC were variable. 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.

Animals↗
PubMed2002

Behavior of junction channels between rat glomus cells during normoxia and hypoxia.

The activity of gap junction channels between cultured and clustered carotid body glomus cells of the rat was studied with dual voltage clamping during normoxia (PO(2) 300 Torr) and hypoxia induced by sodium dithionite (Na(2)S(2)O(4)) or 100% N(2). Na(2)S(2)O(4) reduced the saline PO(2) to approximately 10 Torr, whereas 100% N(2) reduced ambient O(2) to approximately 60 Torr. The following observations were made. 1) In normoxia, the intercellular macroconductance (G(j) = 3.0 +/- 1.01 ns, mean +/- SE) was changed unevenly (increased and decreased) under hypoxic conditions by either agent, although N(2) produced the largest changes. 2) The intercellular microconductances of the channels (g(j) = 104.44 +/- 10.16 pS under normoxic conditions) significantly decreased in 100% N(2) but showed depressions and enhancements in Na(2)S(2)O(4). 3) The conductance of single-junction channels (SChs), calculated as g(j) variance/mean g(j), yielded a mean of approximately 17.6 pS. Larger values were obtained with manual measurements of the data (approximately 34 pS). Hypoxic hypoxia (induced by 100% N(2)) significantly depressed the conductance of SChs when calculated from digitized records or from manual measurements. Hypoxia induced by Na(2)S(2)O(4) did not significantly change junctional conductance. 4) The number of intercellular channels, calculated as g(j)/SCh g(j), had a mean of approximately 452 (range 1 to 2,471). During N(2)-induced hypoxia, this number significantly decreased to approximately 84 but remained unchanged during Na(2)S(2)O(4) hypoxia. 5) The mean open time of junction channels varied from 4 to 30 ms in different experiments, having an overall mean of mu = 11.33 +/- 0.33 ms. This value was significantly reduced by 100% N(2) but was not changed by Na(2)S(2)O(4). 6) Intracellular calcium ([Ca(2+)](i)), 46.2 +/- 4.84 nM under normoxia, significantly increased to 77.32 +/- 11.27 nM with Na(2)S(2)O(4) and to 66.39 +/- 11.64 nM with 100% N(2). It is concluded that 100% N(2) uncouples glomus cells by significantly reducing intercellular macro- and microconductances. Hypoxia induced by Na(2)S(2)O(4) had variable effects. The coupling effects of hypoxia may depend on, or be aided by, increases in [Ca(2+)](i) and/or intracellular pH changes. However, secreted transmitters and ATP plus the effects of hypoxia on second messengers and other cytoplasmic components may also play an important role in this phenomenon.

Animals↗
PubMed2001

Acidic regulation of junction channels between glomus cells in the rat carotid body. Possible role of [Ca(2+)](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 [Ca(2+)](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 (DeltaV(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 [Ca(2+)](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 [Ca(2+)](i) did not change significantly (there were increases and decreases). However, when pH(o) was lowered to 4.4, [Ca(2+)](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 [Ca(2+)](i). However, there are no studies correlating changes of [Ca(2+)](i) and intercellular coupling in glomus cells. Stronger acidification (pH(o) 4.4), producing much larger changes in [Ca(2+)](i), may enhance this synergism. But, again, there are no studies correlating these effects.

Acid-Base Equilibrium↗
PubMed2000

Carotid body gap junctions: secretion of transmitters and possible electric coupling between glomus cells and nerve terminals.

It is proposed that intercellular coupling between glomus cells and carotid nerve terminals form an integral part of the chemoreceptor process. Coupling is possible because gap junctions occur between these elements. At rest, most glomus cells would be coupled. Stimuli uncouple (or reduce coupling) most glomus cells that extrude their contents toward the nerve terminals. However, other glomus cells do not secrete but recharge and intercellular coupling increases. These phenomena would allow for sustained chemoreceptor activity during prolonged stimulation. Coupling between glomus and sustentacular cells may explain why the behavior of glomus cells in the intact carotid body and when clustered in cultures (when their sustentacular envelope is preserved) is different from that of isolated cells where sustentacular cells are destroyed. The presence of electric synapses between glomus cells and nerve terminals may explain the poor performance of synaptic blockers on natural (hypoxia, hypercapnia, acidity) carotid body stimulation.

Animals↗
PubMed2000

Short- and long-term regulation of rat carotid body gap junctions by cAMP. Identification of connexin43, a gap junction subunit.

Intact and cultured carotid bodies (CBs) of the rat were used in this study. Applications of membrane-permeant db-cAMP to cultured carotid bodies increased electric coupling between most glomus cells (increasing junctional conductance) probably by opening preformed intercellular channels. This a short-term effect of the nucleotide, increasing gating between glomus cells. When cultures and intact carotid bodies were treated with membrane-permeant 8Br-cAMP for 3 h or more (to increase cytosolic cAMP), there was enhanced gap junction formation and better dye spread between carotid body cells. Connexin43 (CX43) was identified by immunocytochemical methods as forming part of the intercellular channels between carotid body cells, and the expression of Cx43 increased by cAMP. This is a long-term effect, inducing the formation of gap junctions. Thus, cAMP had short and long-term effects on the intercellular junctions of the carotid body. Long-term formation of gap junctions may be important in modulating carotid body functions during stimulation by chronic hypoxia.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1999

Carotid body glomus cells: chemical secretion and transmission (modulation?) across cell-nerve ending junctions.

Glomus cells of the carotid body contain and secrete chemicals during 'natural' stimulation (hypoxia, hypercapnia and acidity), thus, the birth of the 'transmitter hypothesis of chemoreception'. Released chemicals would cross the synaptic cleft between glomus cells and carotid nerve terminals to depolarize the nerve ending membrane during excitation and hyperpolarize the membrane during inhibition. The main problem with this hypothesis is that specific synaptic blockers modify but do not block the effects of natural stimulation, while blocking the effects of the putative transmitters. It is proposed in this review that the secretion of chemicals is modulated by changes in electric coupling between glomus cells and that glomus cell-nerve ending transmission is not blocked by specific blockers for two reasons. One is that multiple transmitters are released. The other, and more the likely explanation, is that there are electric connections between these elements allowing the flow of currents that are unaffected by the blockers.

Acetylcholine↗
PubMed1999

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 (Em). In another series, [Ca2+]i was measured with Indo-1/AM, dissolved in DMSO. Normoxic cells had a mean Em of -42.4 mV and [Ca2+]i was about 80 nM (measured with both methods). The calculated calcium equilibrium potential (ECa) 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 DeltaEm and Delta[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.

Animals↗
PubMed1998

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 (Em) 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 (ENa) 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 ENa 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.

Animals↗
PubMed1998

Modulation of junctional conductance between rat carotid body glomus cells by hypoxia, cAMP and acidity.

Short-term cultures of glomus cells (up to seven days), were employed to study intercellular electrical communications. Bidirectional electric coupling was established under current clamping after impaling two adjacent glomus cells with microelectrodes, and alternate stimulation and recording. Their resting potential (Vm) and input resistance (Ro) were thus measured. Both coupled cells were then voltage clamped at a level between their Vms. Current pulses applied to either cell elicited a transjunctional voltage (Vj) and current (Ij), used to calculate the junctional conductance (Gj). Gj was 1.52+/-0.29 nS (mean+/-S.E.; n=147). Vj linearly influenced Gj, suggesting ohmic junctions. Gj was not affected by Vm in 50% of the cases. However, there was Vm-dependence in the others, but voltage changes had to be large (>+/-40 mV from the Vm). Therefore, physiologically or pharmacologically induced glomus cell depolarization or hyperpolarization may not significantly affect intercellular coupling unless there are large variations in Vm. Hypoxia (induced by Na2S2O4 1 mM or 100% N2) decreased Gj in 60-80% of the pairs while producing tighter coupling in the rest. Similar effects were obtained when the medium was acidified with lactic acid 1-10 mM. Cobalt chloride (3 mM) prevented, diminished or reversed the changes in Gj observed during low PO2, suggesting that [Ca2+]i changes are important in hypoxic uncoupling. However, non-specific cationic effects of Co2+ have not been ruled out. Applications of the membrane-permeant dB-cAMP 1 mM tightened coupling in almost all cell pairs. This is important because endogenous cAMP increases during hypoxia. Our results suggest that multiple factors modulate junctional conductance between glomus cells. Changes in Gj by 'natural' stimuli and/or cAMP may play an important role in chemoreception, especially in titrating the release of transmitters toward the carotid nerve terminals.

Animals↗
PubMed1996

Effects of calcium on the electric coupling of carotid body glomus cells.

Pairs of electrically coupled glomus cells from rat carotid bodies were impaled with microelectrodes. In the current clamp mode, intracellular stimulation and recording established the coupling coefficient (KC), across the intercellular junctions. About 80% of 26 pairs uncoupled during exposure to 9.45 mM [Ca2+]o, and about 72% of 18 pairs showed the same effect during applications of ionophore A23187. During superfusion with zero [Ca2+]o and EGTA, about 73% of 40 pairs of cells became more tightly coupled. Similar results (71%) were obtained during exposure of 42 cell pairs to BAPTA/AM, a membrane-permeant calcium chelator. Thus, [Ca2+]i seemed to play a significant a role in glomus cell intercellular communication. A23187 and BAPTA/AM, dissolved in DMSO, tended to reduce intercellular coupling during prolonged exposures of the preparations to this solvent. Consequently, the effects elicited by A23187 and BAPTA/AM were superimposed on a coupling effect produced by DMSO.

Animals↗
PubMed1996

Effects of hypoxia on the intercellular channel activity of cultured glomus cells.

Dual voltage clamp experiments have shown that hypoxia induced by Na-dithionite or N2 reduced junctional macroconductance (Gj) in about 70% of cultured and coupled glomus cell pairs while increasing it in the rest. To explore possible mechanisms for these effects, we studied the activity of gap junction channels under similar conditions. The calculated single channel conductances (gj) fell into two categories. A low-conductance group, which was most frequently observed, had a mean gj of 27.8 +/- 0.29 pS (mean +/- SEM; n = 968 events). The other group had higher conductances (47.6 +/- 0.35 pS; n = 528). When PO2 was reduced (hypoxia), the low conductances did not change significantly in any of the junctions. The high-conductance units appeared less frequently in some junctions whereas in others they remained unaltered. Thus, rapid channel flickering during hypoxia may not be the only mechanism determining Gj during coupling or uncoupling. It is possible that slow (seconds) opening and closing of the channels could play an important role in this phenomenon.

Animals↗
PubMed1995

Effects of hypoxia on the intracellular K+ of clustered and isolated glomus cells of mice and rats.

Carotid bodies of rats and mice were used to measure the intracellular potassium activity, ai(K), of clustered and isolated glomus cells normally oxygenated (pO2 102-139 Torr), and during hypoxia (pO2 2-82 Torr) induced by Na-dithionite. ai(K) was measured with intracellular ion-selective microelectrodes, and the resting potential (EM) with KCl-filled micropipettes. Under normoxia, the ai(K) of clustered cells in both species was higher than that of isolated cells. This resulted in more negative potassium equilibrium potentials (EK's). There was no correlation between ai(K) and EM in clustered cells, but this correlation was significant in isolated cells. Hypoxia significantly decreased ai(K) in clustered and single mouse cells, and in clustered rat cells, although its effects on single rat cells were variable. ai(K) decreases were accompanied by cell depolarization and positive shifts in EK. During hypoxia, there were significant correlations between ai(K) and EM in all cells. It is suggested that ai(K) did not influence the EM of clustered cells under normoxia because of interference by K+ pumping mechanisms toward glomus cells from surrounding sustentacular processes. This hindrance is not present when glomus cells are isolated. During hypoxia K+ pumping from sustentacular cells is disrupted, allowing the EM of clustered glomus cells to follow their ai(K) and behave like isolated cells. The different effects of hypoxia on isolated rat and mouse cells may be due to activation of different types of glomus cells.

Animals↗
PubMed1994

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 (Kc) 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 EM. Pulses of either polarity, delivered to Cell 1, produced current flow through the intercellular junction and allowed direct measurements of junctional currents (Ij) and total conductances (Gj). Ij had a mean value of about 12.5 pA and Gj of 391 pS. Unitary (presumably single channel) conductance (gj) was about 78 pS.

Action Potentials↗