M. A. Delpiano

Evidence for a PO2‐sensitive K+ channel in the type‐I cell of the rabbit carotid body

Type‐I cells of rabbit carotid bodies were studied with the patch‐clamp technique in the whole‐cell and on the cell‐attached configuration. Cells exhibiting resting potentials of about −40 mV under normoxic conditions (P O2: 20 kPa), depolarized during hypoxia (P O2: 3.7 kPa). Hypoxia did not affect inward Ca2+ currents but inactivated outward K+ currents in voltage‐clamp experiments. Single‐channel currents recorded for the cell‐attached mode showed a slope conductance of about 137 pS and a 0 mV reversal potential under symmetrical K+ concentration (140 mM). The open‐probability (P o) of the single channel was dependent on the extracellular P O2. These data demonstrate the existence of a P O2‐sensitive K+ channel in type‐I cells, which may account for cell depolarization and the resulting chemosensory response.

Modulatory effect of extracellular Mg2+ ions on K+ and Ca2+ currents of capillary endothelial cells from rat brain

Using whole‐cell patch‐clamp recording, we demonstrate that exposure of single rat brain capillary endothelial cells to different extracellular Mg2+ concentrations (0.3, 4.8 and 9.6 mM) affects the conductance of K+ and Ca2+ currents elicited under control conditions (1.2 mM). Extracellular Mg2+ concentrations ([Mg2+]o) of 4.8 and 9.6 mM reversibly depress outward K+ currents by about 30 ± 12% (n =10) and 3% ± 13%(n = 10), at all activating potentials, respectively. Using identical concentrations reversibly depressed the Ca2+ current by about 40 ± 16% (n = 8) and 46 ± 18% (n = 6), respectively. Using a low Mg2+ concentration of 0.3 mM, the K+ current activation was unexpectedly and mildly increased by about 15 ± 5% (n = 5), and the inward Ca2+ current was attenuated. When studying this effect of low [Mg2+]o on ‘pure’ Ca2+ currents, free of outward currents, we found that this inward current was depressed by about 38 ± 16% (n = 8), and its threshold for activation, in the current‐voltage relationship, was shifted to more negative potentials. It is concluded that high [Mg2+]o hinders the entry of Ca2+ through low‐voltage activated Ca2+ channels and thereby attenuates a Ca2+‐regulated K+ conductance. At a low [Mg2+]o (0.3 mM), Mg2+ shifts the steady‐state inactivation of the voltage‐activated Ca2+ channel to more negative potentials by about 8 mV (n = 6), probably due to a negative screening effect, i.e. a reduction of positive charges on the cell membrane. This may contribute to an apparent increase in K+ conductance by an, as yet, unknown mechanism.

Extracellular pH Changes in the Superfused Cat Carotid Body During Hypoxia and Hypercapnia

Extracellular pH changes were measured in the superfused cat carotid body with double barreled pH glass microelectrodes, under constant pH (7.45 +/- 0.02), temperature (35 degrees C) and flow (3.6 ml/min) of the superfusion medium. Changes of pO2 in the medium from about 188 Torr (30% O2) to 35 or 12 Torr (5% and 2% respectively) called hypoxia, induced a change of the pH signal of about 0.1 units indicating acidification of the tissue. Medium pH monitored with a pH macroelectrode did not change during hypoxic stimulation. An increase of pCO2 in the medium from about 20 Torr (3% CO2, pH 7.45 +/- 0.02) to 70 Torr (12% CO2, pH 6.98 +/- 0.01) called hypercapnia, under constant pO2 (188 +/- 2 Torr), temperature (35 degrees C) and flow (3.6 ml/min) resulted in acidification of the tissue of about 0.3 pH units. Extracellular pH changes during hypoxia did not occur when the superfusion medium had no glucose; however, pH changes during hypercapnia persisted under these conditions. The hypoxic and hypercapnic chemosensory response of the sinus nerve were decreased or abolished during glucose deprivation in a time-dependent manner. Replacement of glucose with 2-deoxyglucose in the medium led to a similar pattern, i.e. inhibition of the hypoxic and hypercapnic chemosensory nerve response and of the extracellular hypoxic pH changes. These results indicate that glycolysis takes place and contributes to O2 and CO2-chemoreception in the carotid body.

Relationship between tissue po2 and chemoreceptor activity of the carotid body in vitro.

Tissue pO2 (pgO2) and sinus nerve activity were recorded in the carotid body in vitro under hypoxic conditions produced either by interrupting the superfusion flow or by lowering the pO2 of the medium (pmO2). The pgO2 gradient is the steeper the higher pmO2 is. These findings point to a pO2-dependent oxygen consumption. Under hypoxia produced by interrupting the superfusion flow, pgO2 declines slowly down to final values and, concomitantly, the chemoreceptor discharge increases. Under hypoxia produced by lowering pmO2, pgO2 decreases rapidly down to values of about 3 torr, whereas the chemoreceptor discharge at first increases and then decreases, in spite of a maintained low pgO2. The pO2 threshold where the chemoreceptor starts firing under hypoxia, varies between 9 and 90 torr.

Hypoxic and hypercapnic responses of [Ca2+]0 and [K+]0 in the cat carotid body in vitro

Measurements of extracellular Ca2+ and K+ activities [( Ca2+]o, [K+]o) in the superfused cat carotid body in vitro with triple-barrelled ion-selective electrodes have shown that hypoxia induced a decrease in [Ca2+]o of 0.035 +/- 0.17 mM (mean +/- S.D.; n = 17) and a biphasic change in [K+]o which consisted of an increase of 2.3 +/- 1.8 mM followed by an undershoot of -0.52 +/- 0.34 mM (mean +/- S.D.; n = 17). Hypercapnia induced a monophasic upward deflection increase of both [Ca2+]o and [K+]o of about 0.037 +/- 0.013 mM and 0.33 +/- 0.15 mM, respectively (n = 17). During hypoxia, lowering [Ca2+] in the medium to 0.1 mM resulted in a reversed [Ca2+]o response, attenuated [K+]o increase and absence of chemosensory nerve discharges. TTX generally did not affect the hypoxic and hypercapnic induced ionic changes, although the [K+]o undershoot was reduced by 30%. Co2+ competitively blocked the changes in [Ca2+]o and the increase in the sensory nerve discharge elicited by hypoxia and, not competitively, the changes of [K+]o. The ionic changes to hypercapnia were less affected by Co2+. Ouabain inhibited the [K+]o undershoot induced by hypoxia, as did the removal of Na+ from medium. It is concluded that changes in extracellular free Ca2+ and K+ ions concentration induced by hypoxia and hypercapnia represent ionic fluxes related to the transduction process of carotid body cells (glomus and/or sustentacular).

O2 chemoreception of the cat carotid body in vitro.

Since the fundamental research of de Castro (1926) and of Heymans and Bouckaert (1930), the carotid body has been considered to be a peripheral chemoreceptor which transduces changes in arterial Po2 and arterial PCO2/PH into nerve signals. These signals predominantly regulate ventilation via the respiratory center and thus help to control the arterial blood gas level. However other organs such as the heart and the kidneys can also be influenced by nerve signals from the carotid body (Daly and Scott, 1958; Korner, 1963). Recently it has been found that the carotid body seems to be involved in very common diseases such as hypertension (Honig et al., 1981; Trzebski et al., 1982) as well as in sudden infant death syndrome (Naeye et al., 1976). The transducing process enabling the chemoreceptor to respond to changes in blood gases is unknown. If one regards the different cell types in the carotid body tissue as a complex of cells which collaborate in the chemoreceptive process, it can be postulated that there exists a Po2 or PCO2/PH dependent transmitter release from these cells which excites nerve fibres connected to these cells (Hayashida et al., 1981). It is our intention, to support this idea with our measurements of the tissue Po2, extracellular Ca2+ activity and cyclic AMP content during hypoxia and hypercapnia in the cat carotid body in vitro.

Metabolic inhibitors affect the conductance of low voltage-activated calcium channels in brain capillary endothelial cells.

The brain capillary endothelium plays an essential role in the preservation of the blood-brain barrier and in the control of blood microcirculation. Since hypoxia (Po2: 20 Torr) increases K+ conductance in brain capillary endothelial cells (BCECs) (Delpiano, 1994), the question arises whether chemical agents that impair cell metabolism may produce similar changes in Ca2+ conductance. To better understand the signal transduction mechanism that operates on BCECs during hypoxia and which may be of physiological relevance for the control of brain blood flow microcirculation, these cells were investigated in voltage-clamp experiments using the perforated nystatin patch-clamp technique (Horn & Marty, 1988).

Nicotine-evoked cytosolic Ca2+ increase and cell depolarization in capillary endothelial cells of the bovine adrenal medulla

Endothelial cells are directly involved in many functions of the cardiovascular system by regulating blood flow and blood pressure through Ca(2+) dependent exocitosis of vasoactive compounds. Using the Ca(2+) indicator Fluo-3 and the patch-clamp technique, we show that bovine adrenal medulla capillary endothelial cells (B AMCECs) respond to acetylcholine (ACh) with a cytosolic Ca(2+) increase and depolarization of the membrane potential (20.3+/-0.9 mV; n=23). The increase in cytosolic Ca(2+) induced by 10microM ACh was mimicked by the same concentration of nicotine but not by muscarine and was blocked by 100 microM of hexamethonium. On the other hand, the increase in cytosolic Ca(2+) could be depressed by nifedipine (0.01 -100 microM) or withdrawal of extracellular Ca(2+). Taken together, these results give evidence for functional nicotinic receptors (nAChRs) in capillary endothelial cells of the adrenal medulla. It suggests that nAChRs in B AMCECs may be involved in the regulation of the adrenal glands microcirculation by depolarizing the membrane potential, leading to the opening of voltage-activated Ca(2+) channels, influx of external Ca(2+) and liberation of vasoactive compounds.

ATP-dependent K+ and voltage-gated Ca2+ channels in endothelial cells of brain capillaries

The energy demand of mammalian cells in order to maintain life is closely related to the presence of molecular oxygen. Deficiency in oxygen delivery to tissue therefore activates protective mechanisms to restore the state of high order that characterises life. Endothclial cells, like many other cells of the cardiovascular system, play an important role in this complex machinery that prevent and protect cells of lack in oxygen supply. Capillary endothelial cells per se are able to sense changes in environmental The cellular permeability and ATPase activity of brain capillary endothelial cells is affected when exposed to low or chemically induced hypoxia, respectively (E. Dux et al., 1984; N. Kawai et al., 1996a). Since information is lacking concerning the participation of ion channels and membrane potential in brain capillary endothelial cells during hypoxia, the aim of this study was designed to investigate their ion channel properties and to see whether these channels are involved in the transductory pathway of the protective response to hypoxia, as already known in other cells like type I cells of the carotid body, smooth muscle cells and neuroepithelial cells of the lung (J. Lopez-Barneo, 1996).

Ionic Currents on Endothelial Cells of Rat Brain Capillaries

It is generally accepted that endothelial cells lining the inner surface of the vascular tree release vasoactive substances in response to low critical values of the arterial oxygen partial pressure. Although much is known about the electrical membrane properties of endothelial cells (Revest & Abbott, 1992) less is known about the intimate transduction mechanism which initiates O2-chemoreception. Ionic currents of endothelial cells from rat brain capillaries were studied in voltage-clamp experiments to find out whether the response to hypoxia could be also related to changes in the membrane conductance as known on carotid body type-I cells (Lopez-Barneo et al., 1988; Hescheler et al., 1989). Here, it is shown that endothelial cells from rat brain capillaries contain PO2-modulated K+ channels.