E. Carpenter

Developmental changes in isolated rat type I carotid body cell K+ currents and their modulation by hypoxia.

1 Whole‐cell patch‐clamp recordings were used to investigate possible age‐related changes in K+ currents of type I carotid body cells isolated from the rat. K+ current density increased with age, as measured in cells isolated from 4‐day‐old, 10‐day‐old and adult rats (≥ 5 weeks old). 2 The proportion of current reversibly inhibited by high [Mg2+] (6 mm), low [Ca2+] (0.1 mm) solutions, indicative of the proportion of current attributable to activation of Ca2+‐sensitive K+ (KCa) channels, was significantly smaller in cells of 4‐day‐old rats compared with 10‐dayold rats, despite inward Ca2+ current densities being similar in these two age groups. Inhibition of K+ currents by high [Mg2+], low [Ca2+] solutions was similar in 10‐day‐old and adult type I cells. 3 Hypoxia (PO2, 16–23 mmHg) caused reversible reductions in type I cells from rats of all age groups. However, reductions seen in cells of 4‐day‐old rats were significantly smaller than those seen in cells of 10‐day‐olds and adults. The degree of hypoxic inhibition in these latter two groups was not significantly different. 4 In the presence of high [Mg2+], low [Ca2+] solutions, hypoxia (PO2, 16–23 mmHg) was without significant effect on residual K+ currents in cells from all age groups. 5 These observations indicate that K+ current density increases with postnatal age in the rat. Between days 4 and 10, there appears to be a predominant enhancement of KCa channels, and over the same age range hypoxic sensitivity of K+ currents increases. Our findings demonstrate that this latter observation arises because hypoxia selectively inhibits KCa channels in cells at all ages studied. These results suggest an important role for KCa channels in postnatal maturation of hypoxic chemoreception in the rat carotid body.

Swelling- and cAMP-activated Cl- currents in isolated rat carotid body type I cells.

1 In the whole‐cell configuration of the patch clamp technique, isolated rat carotid body type I cells exhibited reversible activation of Cl− currents during cell swelling effected by hypotonic extracellular solutions. 2 Hypotonic solutions evoked outwardly rectifying, non‐inactivating currents which showed time‐independent activation. The reversal potential (Erev) for the hypotonically evoked current was 1.6 ± 0.6 mV (n= 26). Reduction of extracellular Cl− from 133 to 65.5 mm caused a shift in Erev of ± 14.7 ± 0.4 mV (n= 5). 3 The swelling‐activated Cl− current could not activate when ATP was omitted from the patch pipette or when substituted for the non‐hydrolysable ATP analogues 5′‐adenylylimido‐diphosphate, AMP‐PNP (2 mm) or β,γ–methylene‐adenosine 5′‐triphosphate, AMP‐PCP (2 mm). The current also failed to activate in the absence of free intracellular Ca2+. 4 The swelling‐activated Cl− current was sensitive to blockade by the Cl− channel blockers niflumic acid (300 μm and 4,4′‐diisothiocyanatostilbene‐2,2′‐disulphonic acid (DIDS; 200 μm), although the blockade by DIDS was voltage dependent. 5 A similar, non‐inactivating, outwardly rectifying Cl− current was evoked by the inclusion of cAMP (200 μm) in the patch pipette. This current could be inhibited by niflumic acid (300 μm) DIDS (200 μm) and hypertonic solutions, and was virtually abolished in the absence of intracellular ATP. 6 In conclusion, carotid body type I cells possess Cl− currents activated by cell swelling and rises in intracellular cAMP concentration. These currents may be involved in cell volume regulation, blood volume and osmolarity regulation and the response of the type I cell to chemostimuli.

Effects of hypoxia and dithionite on catecholamine release from isolated type I cells of the rat carotid body

1 Amperometric recordings were conducted to investigate the ability of hypoxia and anoxia to evoke quantal catecholamine secretion from isolated type I cells of the rat carotid body. 2 Hypoxia (P  O 2 8–14 mmHg) consistently failed to evoke catecholamine secretion from type I cells, when cells were perfused either at room temperature (21‐24 °C) or at 35–37 °C, and regardless of whether Hepes‐ or HCO3−/CO2‐buffered solutions were used. 3 Elevating extracellular [K+] caused concentration‐dependent secretion from individual type I cells, with a threshold concentration of approximately 25 mM. In the presence of this level of extracellular K+, hypoxia (P  O 2 8–14 mmHg) caused a marked enhancement of secretion which was fully blocked by 200 μM Cd2+, a non‐specific blocker of voltage‐gated Ca2+ channels. 4 Anoxia (N2‐equilibrated solution containing 0·5 mM dithionite) evoked exocytosis from type I cells when extracellular [K+] was 5 mM. This secretion was completely inhibited by removal of extracellular Ca2+, but was not significantly affected by Cd2+ (200 μM), Ni2+ (2 mM), Zn2+ (1 mM) or nifedipine (2 μM). Secretion was also observed when 0·5 mM dithionite was added to air‐equilibrated solutions. 5 Anoxia also evoked secretion from chemoreceptive phaeochromocytoma (PC12) cells, which was wholly Ca2+ dependent, but unaffected by Cd2+ (200 μM). 6 Our results suggest that hypoxia can evoke catecholamine secretion from isolated type I cells, but only in the presence of elevated extracellular [K+]. This may be due to the cells being relatively hyperpolarized following dissociation. In addition, we have shown that dithionite evokes catecholamine release regardless of P  O 2 levels, and this release is due mainly to an artefactual Ca2+ influx pathway activated in the presence of dithionite.

Ca 2+ Channel Currents in Type I Carotid Body Cells of Normoxic and Chronically Hypoxic Neonatal Rats

Whole-cell patch-clamp recordings were used to study voltage-gated Ca2+ channel currents in type I carotid body cells of young rats born and reared in normoxia or in a chronically hypoxic (CH) environment (10% O2). Currents activated at potentials of -40 mV and more positive, and typically peaked at 0 mV in both groups of cells. Steady-state inactivation curves were similar in the two populations. Ca2+ currents were significantly larger in CH type I cells, but this was accounted for by the increased size of CH cells: current density was similar in both cell types. Nifedipine (5 microM) always partially inhibited currents and Bay K 8644 (2-5 microM) always enhanced currents, indicating the presence of L-type channels. In a small number of cells from each group, the N-type channel blocker omega-conotoxin GVIA caused partial, irreversible inhibition, but in most cells was without discernible effect. These results indicate that type I cells possess L-type Ca2+ channels, that N-type are expressed in some cells and that non-L, non-N-type channels are also present. Furthermore, chronic hypoxia does not appear to cause specific adaptive changes in the properties of Ca2+ channels in type I cells.

INHIBITION OF CA2+-DEPENDENT K+ CHANNELS IN RAT CAROTID BODY TYPE I CELLS BY PROTEIN KINASE C

1 Whole‐cell patch clamp recordings were used to investigate the effects of protein kinase C (PKC) activation on K+ and Ca2+ currents in type I cells isolated from the rat carotid body. 2 Pretreatment of cells for 10 min at 37 °C with 4α‐phorbol 12,13‐didecanoate (4α‐PDD, 200 nm), a phorbol ester which does not activate PKC, did not affect K+ current density as compared with cells pretreated with vehicle alone. By contrast, identical pretreatment with 200 nm 12‐O‐teradecanoylphorbol‐13‐acetate (TPA, a PKC activator) dramatically reduced K+ current density in type I cells. This effect was prevented by co‐incubation of cells with the PKC inhibitor bisindolylmaleimide (BIM, 3 μm). 3 The sensitivity of K+ currents to inhibition by 200 μm Cd2+ (indicative of the presence of Ca2+‐dependent K+ channels) was markedly reduced in TPA‐treated cells as compared with sham‐treated cells, cells treated with 4α‐PDD, and cells treated with both TPA and BIM. Cd2+‐resistant K+ current densities were of similar magnitude in all four groups of cells, as were the input resistances determined over the voltage range −100 mV to −50 mV. 4 Ca2+ channel current density was not significantly different in type I cells pretreated with 200 nm 4α‐PDD as compared with cells treated with the same concentration of TPA. 5 The degree of inhibition of K+ currents caused by hypoxia (Po2 15–20 mmHg) was unaltered by pretreatment of cells with 3 μm BIM. 6 The resting membrane potential of cells pretreated with TPA was depolarized as compared with controls, and the Ca2+‐dependent K+ channel inhibitor iberiotoxin (20 nm) failed to depolarize these cells further. 7 Our results suggest that activation of PKC causes a marked, selective inhibition of Ca2+‐dependent K+ currents in type I carotid body cells, but that PKC activation is unlikely to account for inhibition of these channels by acute hypoxia.

A standing Na+ conductance in rat carotid body type I cells.

Substitution of extracellular Na+ with N-methyl d-glucamine caused marked hyperpolarisation in rat isolated carotid body type I cells, suggesting the presence of a standing Na+ conductance. Choline substitution produced smaller hyperpolarisations, whilst Li+ was virtually without effect. This Na+ conductance was not blocked by amiloride, tetrodotoxin, Zn2+ or Gd3+ and did not arise from electrogenic Na-glucose co-transport, since substitution of glucose with sucrose could not mimic the effects of Na+ substitution. Hypoxia and acidosis did not modify the tonic Na+ influx. Our results suggest that Na+ influx provides a constant depolarising influence on type I cells which acts to shift membrane potential beyond that required for initiation of neurosecretion, an essential step in carotid body chemotransduction.

Ionic currents in carotid body type I cells isolated from normoxic and chronically hypoxic adult rats.

Whole-cell recordings were used to investigate the effects of a 3-week period of hypoxia (10% O2) on the properties of K+ and Ca2+ currents in type I cells isolated from adult rat carotid bodies. Chronic hypoxia significantly increased whole-cell membrane capacitance. K+ current amplitudes were not affected by this period of hypoxia, but K+ current density was significantly reduced in cells from chronically hypoxic rats as compared with normoxically maintained, age-matched controls. K+ current density was separated into Ca2+-dependent and Ca2+-independent components by bath application of 200 microM Cd2+, which blocked Ca2+ currents and therefore, indirectly, Ca2+-dependent K+ currents. Ca2+-dependent K+ current density was not significantly different in control and chronically hypoxic type I cells. Cd2+-resistant (Ca2+-insensitive) K+ current densities were significantly reduced in type I cells from chronically hypoxic rats. Acute hypoxia (Po2 15-22 mmHg) caused reversible, selective inhibition of Ca2+-dependent K+ currents in both groups of cells and Ca2+-insensitive K+ currents were unaffected by acute hypoxia. Ca2+ channel current density was not significantly affected by chronic hypoxia, nor was the degree of Ca2+ channel current inhibition caused by nifedipine (5 microM). Acute hypoxia did not affect Ca2+ channel currents in either group. Our results indicate that adult rat type I cells undergo a selective suppression of Ca2+-insensitive, voltage-gated K+ currents in response to chronic hypoxia in vivo. These findings are discussed in relation to the known adaptations of the intact carotid body to chronic hypoxia.

Adult human epidermal melanocytes for neurodegeneration research

Neuronal models for Alzheimers disease research frequently have limitations as a result of their nonhuman origin and/or transformed state. Here we examined the potential of readily accessible neural crest-derived human epidermal melanocytes isolated from elderly individuals as a model system for Alzheimers disease research. The amyloidogenic isoforms of amyloid precursor protein (APP; isoforms APP751/770) and amyloid beta (A&bgr;)1–40 were detected in epidermal melanocytes using immunocytochemistry and western blotting. Incubation of epidermal melanocytes with aggregated A&bgr;1–40 peptide caused a concentration-dependent reduction in cell viability, whereas age-matched dermal fibroblasts remained unaffected. These findings suggest that epidermal melanocytes from elderly donors are capable of amyloidogenesis and are sensitive to A&bgr;1–40 cytotoxicity. Thus, these cells may provide a readily accessible human cell model for Alzheimers disease research.

Opioid receptor independent inhibition of Ca2+ and K+ currents in NG108-15 cells by the kappa opioid receptor agonist U50488H.

The whole-cell patch clamp technique was used to investigate the actions of the opioid agonist U50488H on Ca2+ and K+ currents in differentiated NG108-15 cells. U50488H (5-50 microM) caused a concentration-dependent, reversible inhibition of high voltage-activated Ca2+ currents which persisted in the presence of nifedipine (2 microM), indicating a blockade of N-type Ca2+ channels. The actions of U50488H were also observed in the presence of 30 microM naloxone, which fully abolished current inhibition caused by a selective delta opioid receptor agonist. U50488H also inhibited Ca(2+)-insensitive, voltage-gated K+ currents in NG108-15 cells in the presence of naloxone. Our results indicate that U50488H can inhibit neuronal ionic channels via a mechanism which does not involve activation of kappa opioid receptors.

Ca2+ Channel Currents in Type I Carotid Body Cells from Normoxic and Chronically Hypoxic Rats

Rats born and reared under chronically hypoxic (10% O2) conditions do not respond to acute hypoxia with an increased ventilation. Their carotid bodies undergo hyperplasia and hypertrophy and we have recently shown that K+ channels recorded in type I carotid body cells isolated from normal and chronically hypoxic (CH) rats show marked differences (Wyatt et al, 1995): normoxic type I cells express Ca2+-activated K+ (KCa) channels which are inhibited by acute hypoxia, leading to cell depolarization, opening of voltagegated Ca2+ channels (VGCCs) and the consequent influx of Ca2+ to trigger neurotransmitter release (Peers & Buckler, 1995). In type I cells from CH rats, there is far less expression of KCa channels, and, whilst the remaining K+ channels are inhibited by hypoxia, this does not lead to cell depolarization, which may explain the lack of ventilatory response to acutely inspired hypoxia in intact CH rats (Wyatt et al, 1995). An important factor in the response of normal type I cells to hypoxia is the activation of VGCCs, of which numerous sub-types exist in various tissues. It is known that L-type VGCCs are involved in hypoxic chemotransduction (Buckler & Vaughan-Jones, 1994), but very little is known about the possible presence of other VGCCs, whether they may be involved in chemotransduction, and whether they are affected by chronic hypoxia. We have therefore compared the properties of VGCCs in type I carotid body cells isolated from normoxically-reared and CH rats.