Sensitivity of Glomus Cells in Carotid Body Thin Slices and Secretory Responses to K Channel Blockers

The model of acute 02 sensing based on the regulation of membrane K+ channels described in the carotid body has been demonstrated to operate in other neurosecretory systems, such as cells in the lung neuroepithelial bodies (43), chromaffin cells of the adrenal medulla (38), or PC-12 cells (44). There are, however, controversies on whether 02-sensitive membrane electrical events in glomus cells are directly involved in the chemosensing process. The major argument supporting this notion is that inhibitors of the potassium current, like tetraethylammonium (TEA), 4-aminopyridine (4-AP), or charybdotoxin (CTX), do not enhance either action potential firing frequency of the afferent sensory fibers or secretory activity in the whole carotid body preparations used in these experiments (8, 16, 29). Although it was shown in a study that CTX can depolarize dialyzed rat glomus cells (42) it has been reported that neither TEA nor 4-AP influence the membrane potential of the same cells (3). To investigate the reasons for the discrepancies among laboratories using dispersed rat glomus cells and the contradictions between findings in isolated cells and the whole organ, we have developed a carotid body slice preparation to study the 02-sensitivity of glomus cells in the best possible physiological conditions (30, 31). We also attempted to obtain a preparation of glomus cells with consistent properties since cellular 02-sensitivity is a labile phenomenon easily destroyed by uncontrolled variables during the enzymatic treatment and mechanical disruption of the tissue (32).

Figure 3. Responses to hypoxia of carotid body glomus cells in slices. A: Morphological appearance of a typical glomerulus within a cultured rat carotid body thin slice maintained in culture for 72 h. A well-defined single cell is indicated by the arrow. B: Carotid body slice immunostained with antibodies against tyrosine hydroxylase. The carotid body was fixed, then sliced and stained. Note the typical appearance of glomus cells with large nuclei and a thin layer of stained cytoplasm. The organization of type I cells in glomeruli is similar to that seen in fresh slices. C: Superimposed K+ currents from a glomus cell elicited by depolarizing pulses from -80 mV to 0 (left) or +20 mV (right) in the three experimental conditions (control, hypoxia and recovery). Note the reversible reduction of the current by low P02 («20 mmHg). D: Top, Amperometric recordings from an Oj-sensitive glomus cell illustrating the increase of secretory activity elicited by hypoxia (Po2 «20 mmHg), Note also the typical response of the cell to high extracellular K+. Bottom, Secretory activity recorded from an 02 sensitive glomus cell to illustrate the reversible abolishment of the response to hypoxia during the blockade of Ca2+ channels by addition of 0.2 mM cadmium to the extracellular solution (Modified from Refs. 30, 32).

Figure 3. Responses to hypoxia of carotid body glomus cells in slices. A: Morphological appearance of a typical glomerulus within a cultured rat carotid body thin slice maintained in culture for 72 h. A well-defined single cell is indicated by the arrow. B: Carotid body slice immunostained with antibodies against tyrosine hydroxylase. The carotid body was fixed, then sliced and stained. Note the typical appearance of glomus cells with large nuclei and a thin layer of stained cytoplasm. The organization of type I cells in glomeruli is similar to that seen in fresh slices. C: Superimposed K+ currents from a glomus cell elicited by depolarizing pulses from -80 mV to 0 (left) or +20 mV (right) in the three experimental conditions (control, hypoxia and recovery). Note the reversible reduction of the current by low P02 («20 mmHg). D: Top, Amperometric recordings from an Oj-sensitive glomus cell illustrating the increase of secretory activity elicited by hypoxia (Po2 «20 mmHg), Note also the typical response of the cell to high extracellular K+. Bottom, Secretory activity recorded from an 02 sensitive glomus cell to illustrate the reversible abolishment of the response to hypoxia during the blockade of Ca2+ channels by addition of 0.2 mM cadmium to the extracellular solution (Modified from Refs. 30, 32).

The procedures followed to make carotid body slices are described in Pardal et al. (30) and Pardal and Lopez-Barneo (32). In healthy slices, clusters of glomus cells were clearly distinguished from the surrounding tissue (Fig. 3A). These clusters, of similar appearance to the glomeruli described in histological preparations of the carotid body, contained numerous ovoid cells of «10 to 12 |Lim of diameter. The similarity between fresh and fixed carotid body preparations can be appreciated by observing slices immunostained with antibodies against tyrosine hydroxylase (TH), where glomus cells appear in clusters with intensely stained thin cytoplasmic layers and big clear nuclei (Fig. 3B). For the experiments, a slice was transferred to a recording chamber mounted on the stage of an upright microscope, where it was continuously perfused by gravity (flow

1 to 2 ml/min) with a solution containing (in mM) 117 NaCl, 4.5 KC1, 23 NaHCOj, 1 MgCl2,2.5 CaCl2, and 10 glucose. The recording electrodes (either patch pipette or amperometric carbon fiber) were placed adjacent to a well-identified cell within a glomerulus, such as the one indicated by the arrow in Figure 3 A. In experiments designed to test the effect of hypoxia, the control (normoxic) solution was bubbled with a gas mixture of 5% C02, 20% 02, and 75% N2 (Po2 «150 mmHg), and the hypoxic solution with 5% C02) and 95% N2 (Po2 in the chamber« 20 mmHg). After switching from normoxia to hypoxia, complete equilibration of the new solution in the chamber required between 1 and 2 min.

As described in dispersed rabbit (11, 13, 20, 34) and rat (23, 33, 36) glomus cells the amplitude of macroscopic voltage-dependent currents recorded from cells in rat carotid body slices is reduced when exposed to low Po2 (Fig. 3C). However, reversible reduction of K+ current amplitude by hypoxia is a response seen less consistently in our slices than the increase of secretory activity monitored by amperometry. This could mean that in patch clamped rat glomus cells the 02-sensing mechanism is altered and the sensitivity to low Po2 decreased possibly due to the intracellular dialysis. It is also possible that, besides voltage-gated K+ channels, other conductances, mediated by voltage-gated Ca2+ (37) or K+-selective leaky (3) channels, not studied so far in slices, also contribute to mediate the low Po2-induced secretory response. In slices with well-defined glomeruli, low Po2 induces consistently a progressive increase in the frequency of secretory events (Fig. 3D), reaching values of 48.6±19 spikes/min (n=24 cells). At the peak of the response to hypoxia the secretory events normally fuse into a broad concentration envelope that quickly declines after switching to the control, normoxic, solution. As expected from electrically excitable cells, all the glomus cells that respond to hypoxia are also activated by solutions with high external K+ (Fig. 3D, top). Interestingly, glomus cells unresponsive to hypoxia but activated by depolarization with high external K+ are occasionally observed. The neurosecretory response to hypoxia ofrat glomus cells in slices is completely abolished by the addition of the voltage-dependent calcium channel blocker cadmium (Fig. 3D, bottom) or the removal of extracellular calcium with EGTA (30). These observations confirm previous data on dispersed rabbit carotid body cells (26, 39).

The major contributors to the voltage-gated 02-sensitive macroscopic K+ currents in rat glomus cells are the Ca2+-dependent maxi-K+ channels (23, 33, 42). Because these channels are blocked by TEA or iberiotoxin (IbTX), we have studied whether, like hypoxia, these agents induce entry and secretion in glomus cells. In most cells studied (33 of 34 cells), application of 5 mM TEA to the bath solution elicits an increase in the secretory activity similar to that triggered by hypoxia (Fig. 4A). In response to the K+ channel blocker the frequency of secretory events (42±17 spikes/min, n=6 cells) is similar to that obtained in low Po2 (Student's t test, P<0.05). The average quantal charge of events induced by TEA is 43±30 fC (n=275 spikes in 6 cells). This value and the distribution of quantal events are also similar to those estimated with events elicited by hypoxia (43±26 fC; n=576 spikes in 14 cells), suggesting that both stimuli can trigger the release ofthe same type of secretory vesicle (Fig. 4B). The effect of TEA can be observed even in quiescent cells, without any measurable spontaneous quantal release, as well as in 02-insensitive glomus cells. We have also tested the effect of IbTX, a selective blocker of Ca2+- and voltage-activated maxi K+ channels (10). Figure 4C illustrates the increase of secretory activity in a glomus cell exposed to 200 nM IbTX. The response is similar to the ones obtained with TEA or hypoxia, although the recovery phase seems to be somewhat longer possibly due to slower wash-out of IbTX. These observations indicate that direct blockade of the K+ channels with TEA or IbTX can elicit secretion from rat glomus cells in the slices.

Figure 4. Secretory responses of intact glomus cells to K+ channel blockers. A: Amperometric recording from a glomus cell illustrating the similar effects elicited by low Po2 and the application of 5 mM TEA to the external solution. B: Frequency histograms ofthe quantal charge of events elicited by hypoxia and TEA. Note that the parameters of the distributions are the same in the two experimental conditions. C: Secretory activity induced in a glomus cell by hypoxia and 200 nM IbTX (Modified from Ref. 30).

Figure 4. Secretory responses of intact glomus cells to K+ channel blockers. A: Amperometric recording from a glomus cell illustrating the similar effects elicited by low Po2 and the application of 5 mM TEA to the external solution. B: Frequency histograms ofthe quantal charge of events elicited by hypoxia and TEA. Note that the parameters of the distributions are the same in the two experimental conditions. C: Secretory activity induced in a glomus cell by hypoxia and 200 nM IbTX (Modified from Ref. 30).

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