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. 1998 Jun 15;509(Pt 3):887–893. doi: 10.1111/j.1469-7793.1998.887bm.x

Oxygen-sensing mechanisms are present in the chromaffin cells of the sheep adrenal medulla before birth

G Y Rychkov 1, M B Adams 1, I C McMillen 1, M L Roberts 1
PMCID: PMC2231003  PMID: 9596807

Abstract

  1. The ability of the fetal adrenal medulla to respond directly to hypoxaemia and secrete catecholamines before the development of a functional innervation of the gland is essential for intrauterine survival. The cellular mechanisms involved in this response to low PO2 are not known, although the presence of oxygen-sensitive K+ channels in carotid body chemoreceptor cells and other sites suggests that these might underlie the chromaffin cell response.

  2. Whole-cell patch-clamp techniques have been used to study K+ currents during normoxia and hypoxia in chromaffin cells isolated from the adrenal glands of fetal sheep.

  3. Two types of chromaffin cells were observed, those with a fast inactivating K+ current and a larger capacitance and those with a delayed K+ current and smaller capacitance. No cell showed both types of current. The fast inactivating current showed voltage-dependent inactivation and was blocked by 1 mM 4-aminopyridine, characteristics of an IA-type current. The delayed current had two components, a TEA-sensitive, Ca2+-dependent current and a component with the kinetic behaviour of a delayed rectifier.

  4. Both types of current were oxygen sensitive. The IA-type current was reduced by 27.4 ± 3.2 % when the PO2 was reduced to about 15 mmHg. With the delayed current, hypoxia reduced the amplitude by 26.9 ± 2.4 %, largely by reduction of the Ca2+-dependent component.

  5. In the presence of hypoxia, reduction in the amplitude of these oxygen-sensitive K+ currents would increase the frequency and duration of action potentials, leading to increased activation of the L-type Ca2+ channels, influx of Ca2+ and the subsequent secretion of catecholamines.

In the human and sheep fetus, acute episodes of hypoxaemia stimulate the adrenal medulla to secrete catecholamines, which initiate and co-ordinate the metabolic and cardiovascular responses that are essential for intrauterine survival (Slotkin & Seidler, 1988). In late gestation, the secretion of catecholamines from the adrenal medulla in response to hypoxaemia is predominantly mediated by the cholinergic splanchnic nerves (Comline & Silver, 1961; Cheung, 1990). Before development of functional innervation, however, the fetal adrenal is able to secrete catecholamines in response to hypoxaemia through a direct, non-neurogenic process (Comline & Silver, 1961; Cheung, 1990; Adams, Simonetta & McMillen, 1996). The cellular mechanisms underlying the non-neurogenic secretion of catecholamines from the immature adrenal medulla in response to hypoxaemia are unknown.

Previously, we had shown that in the in vitro perfused adrenal gland of the fetal sheep, hypoxia stimulates non-neurogenic secretion of noradrenaline and adrenaline which is blocked by the L-type Ca2+ channel antagonist, nifedipine (Adams et al. 1996). This is clear evidence that the direct catecholamine secretory response to hypoxia is dependent on depolarization of the chromaffin cell membrane and the associated entry of Ca2+ through voltage-dependent Ca2+ channels. The mechanism by which hypoxia leads to depolarization of the adrenal chromaffin cells has not been defined.

Oxygen-sensitive cells occur in other sites, including the glomus cells of the carotid body, a major chemoreceptor, where hypoxia leads to closing of oxygen-sensitive K+ channels (Lopez-Barneo, Lopez-Lopez, Urena & Gonzales, 1988), and the resultant opening of voltage-dependent Ca2+ channels (Buckler & Vaughan-Jones, 1994). The glomus cell of the carotid body and the adrenal chromaffin cell share a common embryological origin and we have tested whether chromaffin cells also contain oxygen-sensitive K+ channels which would account for their ability to respond directly to hypoxia. During the course of these studies, the existence of oxygen-sensitive K+ currents in the adrenal chromaffin cells of neonatal rats was reported (Thompson, Jackson & Nurse, 1997).

METHODS

Isolation of chromaffin cells

Fifteen pregnant Border Leicester Merino cross ewes between 137 and 144 days gestation (term is 147 ± 3 days) were used for these experiments, which were approved by the University of Adelaide Animal Ethics Committee. Ewes were killed with intravenous pentobarbitone (8.1 g), and the fetus was removed through a laparotomy incision and decapitated. The fetal adrenal gland was removed and the medulla, dissected free of the cortex, was minced and incubated for 45 min in a solution consisting of (mM): NaCl, 138; KCl, 5; Na2HPO4, 0.7; Hepes, 25; pH 7.4, supplemented with collagenase (Yakault, Japan; 1 mg ml−1) and deoxyribonuclease (Sigma, Type IV; 150 U ml−1). The tissue was mechanically dispersed by repeated pipetting and the cells were washed twice with Dulbecco's modified Eagle's-F12 medium (Gibco) containing 10 % charcoal-absorbed fetal bovine serum (Trace, New South Wales, Australia), 100 U ml−1 penicillin and 0.5 mg ml−1 streptomycin. The cells were then resuspended in the same medium and either used immediately for patch-clamp experiments or plated on collagen-coated coverslips and maintained in culture.

Patch-clamp studies

Chromaffin cells, in a bath of approximately 400 μl volume, were superfused continually at 3 ml min−1 with a solution containing (mM): NaCl, 135; KCl, 5; CaCl2, 1.8; MgCl2, 2; Hepes, 10; with pH adjusted to 7.3 with NaOH. The bath solution was equilibrated with either room air or 100 % N2. Whole-cell currents were recorded using pipettes filled with a solution containing (mM): KCl, 70; potassium glutamate, 60; MgCl2, 2; Na2ATP, 2; EGTA, 2; Hepes, 10; adjusted to pH 7.0 with N-methylglucamine, and having a resistance of between 2 and 4 MΩ. Series resistance did not exceed 10 MΩ and was 75-85 % compensated. All experiments were carried out at room temperature. Currents were recorded with an EPC7 amplifier (List), data acquisition and analysis were performed on an IBM compatible computer using pCLAMP software (version 6.0, Axon Instruments) and leakage was subtracted using the P/4 protocol. The voltage protocols used are described in the figure legends and corrections for the liquid junction potential between the bath and electrode solutions were estimated by JPCalc (Barry, 1994). Leakage currents were not subtracted from the currents used to construct the inactivation curves and a least squares method was used to fit a Boltzmann equation to the normalized currents from the inactivation protocol, giving estimates of the half-inactivation potential, V½, and the slope factor.

Data are presented as means ± s.e.m. Student's unpaired t test was used to test for the significance of differences in capacitance. The significance of differences in currents in control and hypoxic cells was determined by a two-way ANOVA with replicate measures. In both tests, P < 0.05 was taken as the minimum level of significance.

RESULTS

Characterization of adrenomedullary K+ currents

Freshly isolated adrenal chromaffin cells studied in normoxic solutions showed either a fast inactivating K+ current (Fig. 1A) or a delayed K+ current which showed little inactivation over the course of a 75 ms pulse (Fig. 2A). No cell had both types of outward current. The chromaffin cells with the fast inactivating currents had a larger capacitance (12.3 ± 1.8 pF; n = 20) compared with the cells with the delayed currents (4.8 ± 0.5 pF; n = 19) (P < 0.01). The current density, measured at +50 mV, was smaller in the fast inactivating cells (81.4 ± 18.5 pA pF−1; n = 20) than in cells with delayed outward currents (283.2 ± 14.6 pA pF−1; n = 19). Resting membrane potential, measured in the whole-cell current clamp mode, was -58.3 ± 2.7 mV (n = 20) for cells with the fast inactivating K+ current and -56.1 ± 2.1 mV (n = 19) for cells with delayed K+ currents. About 40 % of cells had a fast inactivating outward current, although the proportion of cells with the different K+ currents varied from preparation to preparation. When cells were maintained in culture, the currents present depended on the culture conditions. Cells left at room temperature for 18-24 h showed the same two types of outward current, but when incubated at 37°C for 24 h no cells showing fast inactivating currents were found. The results presented in this paper were obtained in cells maintained and studied at room temperature within 24 h of isolation.

Figure 1. Characteristics of the fast inactivating outward current recorded from freshly isolated fetal sheep adrenal chromaffin cells.

Figure 1

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A, currents obtained in control conditions. B, in the presence of 0.8 mM 4-AP. C, the concentration- response curve for 4-AP (n = 4). To obtain the results in panels A, B and C, cells were held at -90 mV and outward currents were elicited by 75 ms steps to potentials which ranged from -50 to +50 mV at 20 mV intervals. The time between steps was 30 s as intervals shorter than this led to incomplete recovery from inactivation. D, currents obtained in response to a step to +50 mV for 20 ms following a 1 s prepulse to potentials which ranged from -90 to -45 mV with 5 mV increments. The cell was held at -90 mV for 30 s between pulses. Leakage current has not been subtracted from these recordings. E, inactivation curve obtained from cells tested with the protocol described in D. The peak current measured at +50 mV following each prepulse was expressed as a proportion of the current measured following a prepulse to -90 mV and the line for the inactivation curve represents the fit of a Boltzmann function to the normalized currents (n = 11).

Figure 2. Characteristics of delayed outward current in fetal sheep adrenal chromaffin cells.

Figure 2

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Currents measured in control conditions (A), during exposure to 5 mM Co2+ (B), and during exposure to 0.8 mM TEA (C). The cells were held at -90 mV and outward currents elicited by 75 ms steps to potentials which ranged from -40 to +70 mV at 10 mV intervals. D, I-V relationships in control conditions (○), during exposure to 5 mM Co2+ (♦), following removal of Co2+ from the bath (•), and during the subsequent exposure to 0.8 mM TEA (⋄). E, concentration-response curve for the block of this current by TEA. The peak current in the presence of TEA is plotted as a proportion of that measured in control conditions. (n = 4)

The fast inactivating current had the characteristics of an IA-type K+ current. Addition of 4-aminopyridine (4-AP) to the extracellular solution rapidly blocked this current with an EC50 of 0.21 ± 0.03 mM (n = 4) and with almost complete block at 0.8 mM 4-AP (Fig. 1). The effects of 4-AP could be readily reversed by washing the cells. Addition of TEA to the bath at concentrations up to 10 mM had no effect on the inactivating current (results not presented). This current showed voltage-dependent inactivation (Fig. 1D and E), with a V½ of -56.9 ± 0.4 mV (n = 10) and a slope factor of -4.0 ± 0.4.

The delayed outward current had an I-V relationship that showed a peak at about +30 mV (Fig. 2D), suggesting that the outward current in this group of cells contains a component that is Ca2+ dependent. The existence of Ca2+-dependent and -independent components was supported by the effect of 5 mM Co2+, where 79 ± 4 % (n = 5) of the peak current was blocked and the N shape of the I-V plot was completely eliminated (Fig. 2B and D). The effect of Co2+ was fully reversible on washing (Fig. 2D). TEA (0.8 mM) had virtually the same effect on the current as Co2+, reducing the current and eliminating the N shape of the I-V curve (Fig. 2B and D), the EC50 for TEA block being 0.10 ± 0.03 mM (n = 4). The delayed K+ currents were not altered by 4-AP at concentrations up to 1.6 mM while 6.4 mM 4-AP blocked approximately 40 % of this current (n = 3). The delayed current showed some inactivation when command potentials of longer duration were used.

The effects of hypoxia on adrenomedullary K+ currents

Both types of outward currents were oxygen sensitive (Fig. 3). In those cells expressing the fast inactivating current, when the PO2 was reduced to approximately 15 mmHg there was a reduction in peak current (Fig. 3A and B) which, at all potentials, averaged 27.4 ± 3.2 % (n = 12). A two-way ANOVA of the data in the I-V curve showed that the reduction produced by hypoxia was significant (P < 0.01, n = 5). Digital subtraction of the current during hypoxia from that recorded in the control, normoxic period demonstrated that the effect of hypoxia was most obvious during the rising phase of the current (Fig. 3A), indicating an effect of hypoxia on activation of the channel. This was supported by the change in the time constant of activation during a step to +50 mV, from 0.78 ± 0.11 ms in normoxia to 1.45 ± 0.17 ms in hypoxia (n = 7, P < 0.01). The time course of decay of the current at +50 mV increased from 17.2 ± 1.9 ms in normoxia to 26.8 ± 3.5 ms during hypoxia (n = 6, P < 0.05). The effect of hypoxia developed within 1 min of replacement of the external solution and was completely reversible in these cells (Fig. 3). The inactivation curve for this current did not shift when they were exposed to hypoxia, with a V½ of -56.0 ± 1.3 mV (n = 6).

Figure 3. The effect of hypoxia (PO2≈ 15 mmHg) on the two types of outward current recorded from the isolated adrenal chromaffin cells.

Figure 3

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A, fast inactivating current recorded during control, hypoxic and recovery periods. The bottom trace is obtained by digital subtraction of the current during hypoxia from the control current. The cell was held at -90 mV and stepped to +50 mV for 20 ms. B, I-V curves for cells with the fast inactivating current during normoxic (○) and hypoxic (•) conditions. C, delayed current recorded during control, hypoxic and recovery periods. The cell was held at -90 mV and stepped to +20 mV for 75 ms. D, I-V curves for cells in normoxia (○, n = 8), hypoxia (•, n = 8), in the presence of 5 mM Co2+ during normoxia (⋄, n = 4) and during hypoxia in the presence of 5 mM Co2+ (♦, n = 4).

Those cells with delayed outward currents showed a 26.9 ± 2.4 % (n = 14) reduction in the amplitude of the currents averaged over all command potentials when exposed to a PO2 of about 15 mmHg (Fig. 3C). The biggest absolute change in the amplitude of the delayed current during hypoxia occurred at the peak of the I-V curve (Fig. 3D), suggesting that the Ca2+-dependent component of this current was oxygen sensitive. This was confirmed by the lack of change in the currents during hypoxia if this Ca2+-dependent component was suppressed by the prior addition of Co2+ (Fig. 3D). As with the fast inactivating current, the effect of hypoxia on the delayed current developed within 1 min of changing solutions, and was reversed on return to normoxic solution, but in this case, the recovery was not usually complete (Fig. 3), with the mean current after return from hypoxic treatment being 89.0 ± 1.6 % (n = 8) of the pre-hypoxic current.

The fetal adrenal chromaffin cells showed inward currents on depolarization which were small compared with the outward currents (Figs 1A and 2A). The peak inward current did not change during hypoxia, which, taken with the small relative size of these currents, means that they would not account for the changes which hypoxia produced in the net outward currents.

DISCUSSION

In this study we have shown that chromaffin cells isolated from the adrenal medulla of fetal sheep have K+ currents which are reduced by hypoxia. The fast inactivating current observed in almost 50 % of these chromaffin cells had characteristics typical of an IA-type K+ current, including its time course, voltage dependence of inactivation, and sensitivity to 4-AP. This type of current has not been described previously in the adrenal medulla, although a fast inactivating K+ channel, BAK4, has been cloned from the adult bovine adrenal medulla and expressed in a neuroblastoma cell line (Garcia-Guzman, Ceña & Criado, 1992). These authors suggested that BAK4 has not been observed in chromaffin cells because the gene product would exist as part of multimeric K+ channels with modified characteristics. It seems more likely, however, that this channel has been downregulated during the culture period at 37°C which is routinely used before chromaffin cells are studied by patch-clamp techniques. It is also possible that the fast inactivating channel is expressed only in the immature adrenal medulla, although in a recent study of the chromaffin cells from neonatal rats, which are equivalent developmentally to those from the late gestation sheep fetus, Thompson et al. (1997) did not observe fast inactivating currents.

There are a number of mechanisms by which hypoxia could reduce the amplitude of the fast inactivating current. In heart cells, metabolic inhibition shifts the inactivation curve for an IA-type current to more negative potentials, apparently as a result of an increase in [Ca2+] in the region immediately below the plasma membrane (Pike, Bretag & Roberts, 1993). In the fetal adrenal medullary chromaffin cells, however, V½ is unaltered by hypoxia and there must be a different explanation of the reduction in peak current that is produced by exposure to low PO2. In cloned neuronal fast inactivating K+ channels, RCK4, the reduced state of a cysteine residue in the N-terminal region of the channel leads to a reduction in current through increased inactivation (Ruppersberg, Stocker, Pongs, Heinemann, Frank & Koenen, 1991; Vega-Saenz de Miera & Rudy, 1992). BAK4, the inactivating K+ channel cloned from the bovine adrenal, has an N terminus which is homologous to RCK4, including the cysteine residue at position 13 (Garcia-Guzman et al. 1992). The fast inactivating current in the fetal sheep adrenal chromaffin cells, however, shows slower activation during hypoxic episodes rather than more rapid inactivation, and so the channels responsible for this current may not be homologous to BAK4.

The delayed outward current observed in fetal sheep adrenal chromaffin cells resembles that seen in bovine and rat adrenal chromaffin cells and in the chemoreceptor cells of the neonatal rat carotid body (Marty & Neher, 1985; Peers & Green, 1991; Neely & Lingle, 1992). A similar oxygen-sensitive K+ current in the chromaffin cells from the adrenal gland of the neonatal rat has been reported recently (Thompson et al. 1997). The delayed outward current we have observed in the fetal sheep chromaffin cells consists of at least two components, a Ca2+-dependent and a Ca2+-independent component. Hypoxia reduces the amplitude of the TEA-sensitive, Ca2+-dependent current with no effect on the Ca2+-independent component. When the adrenal chromaffin cells with the delayed current were returned to normoxic conditions following hypoxia, the current did not regain its full pre-hypoxia amplitude, which probably reflects the gradual rundown of Ca2+ currents and the subsequent decrease of the amplitude of the Ca2+-activated K+ current (Marty & Neher, 1985) rather than an irreversible effect of hypoxia.

In addition to the demonstration of oxygen-sensitive K+ currents in neonatal rat (Thompson et al. 1997) and fetal sheep adrenal chromaffin cells, an oxygen-sensitive K+ current has recently been described in a phaeochromocytoma (PC-12) cell line (Zhu, Conforti, Czyzyk-Krzeska & Milhorn, 1996). This current differs from the fast inactivating current measured in the chromaffin cells of the fetal sheep adrenal medulla in that it is not inactivated at a holding potential of -30 mV, and on stepping to positive potentials it shows little decay. The current in the PC-12 cell line appears to differ from the delayed current found in the fetal adrenal medulla in that, while it declines only slightly over the period of a 800 ms pulse, it is not a Ca2+-dependent K+ current.

It has been previously shown that noradrenaline and adrenaline are synthesized and stored in separate adrenomedullary cells in the adult and fetal sheep. In the fetal sheep adrenal gland perfused in vitro, the noradrenaline and adrenaline cells are equally responsive to hypoxia. It is not known whether the existence of these two classes of hypoxia-sensitive, catecholamine-containing cells accounts for the two populations of cells with different oxygen-sensitive K+ currents, although the larger size of the cells with the fast inactivating current is compatible with them being adrenaline-secreting cells (McMillen, Mulvogue, Coulter, Browne & Howe, 1988).

The effect of both of these currents would be to increase catecholamine secretion during hypoxic periods by increasing the duration of the action potentials, and in the case of the IA current, modulating the firing frequency (Hille, 1992). Considering the voltage range in which they are activated, it is unlikely that they are responsible for the depolarization to threshold that is responsible for initiating secretion. Buckler (1997) showed the presence in rat carotid body type I cells of an oxygen-sensitive K+ current with the characteristics of a leak current, which accounts for the depolarization to threshold that occurs during hypoxia in this cell type. It is not known whether such a current exists in fetal sheep adrenal chromaffin cells.

Functional splanchnic innervation of the fetal lamb adrenal has developed by 130 days gestation and previous in vivo studies have demonstrated that innervation suppresses the direct effect of hypoxia on adrenal chromaffin cells (Comline & Silver, 1961; Cheung, 1990). We have shown, however, that hypoxia stimulates catecholamine release from isolated perfused adrenals collected from fetal sheep after 135 days gestation, i.e. after innervation has occured in vivo. Similarly, Seidler & Slotkin (1986) demonstrated that the adult adrenal secretes catecholamines after denervation of the gland. These data suggest that the oxygen-sensing mechanisms are suppressed in vivo by signals arriving via the splanchnic nerves. In contrast to these results, Thompson et al. (1997) found that chromaffin cells isolated from the innervated adrenal medulla of juvenile rats do not show oxygen-sensitive K+ currents. It is not clear whether this lack of response to acute denervation is a species difference or relates to conditions under which the cells are prepared and cultured.

In summary, we have demonstrated that there are two populations of cells in the fetal adrenal medulla which are directly chemosensitive, responding to hypoxia with a reduction in K+ current. The presence of oxygen-sensitive K+ currents in adrenal chromaffin cells that are not innervated indicates that cellular O2-sensing mechanisms may play a critical role in the physiological response to hypoxia before the emergence of neurogenic chemoreflex mechanisms.

Acknowledgments

This work was supported by a grant from the National Health and Medical Research Council to I. C. McM and an Australian Postgraduate Research Award to M. B. A. The authors also acknowledge the skilled assistance of Tim Butler in the preparation of the adrenomedullary cells.

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