Channels

It is generally accepted that a balance between constitutively active K+ channels and voltage-dependent Ca2+ channels (VDCC) is an important control mechanism of arterial tone (33). Under physiological conditions, the pulmonary circulation is a high flow, low resistance and low pressure system with measured resting membrane potential between -60 and -50 mV that does not reveal spontaneous electrical activity (10). K+ channel blockers, e.g., 4-aminopyridine

(4-AP) or tetraethylammonium (TEA), depolarize the cell membrane, increase intracellular Ca2+ concentration ([Ca2*],) and induce contraction of intact pulmonary arteries (24, 32, 56, 57), indicating a key role for K+ channels in the maintenance of the negative resting potential in PASMCs. The first recording of K.+ currents in the pulmonary artery was performed by Buryi and Gurkovskaia in 1980 in a smooth muscle strip from the rabbit main pulmonary artery using the double sucrose gap method (12). Simultaneous development of the patch clamp technique, isolation of viable single vascular SMCs, and molecular biology techniques equipped investigators with potent experimental tools and enabled substantial progress in our understanding of the function and molecular nature of ion channels in vascular SMCs in general and in PASMCs in particular.

Functional K+channels are multimers of a-subunits which form the ionic pore. More than 70 a-subunits have now been identified which differ in structure, voltage-dependence, kinetics and pharmacology. Functional diversity of K+ channels is further increased by the ability of different a-subunits to form heteromultimers and by the presence of a-subunits splice variants and regulatory (auxiliary) p-subunits. Not all types of K+ channels are expressed in the vasculature. Currently, four main groups of K+ currents have been described in vascular SMCs: voltage-gated (Kv) and large conductance Ca2+-activated (BKCa) currents, which are encoded by a-subunits belonging to the six transmembrane segment and one pore domain class of K+channels, and ATP-sensitive (KATP) and inward rectifier (Kir) currents which belong to the two transmembrane segment and one pore domain class of K+ channels (see Ref. 7 for additional information about the molecular structure and properties of K+ channels in vascular SMCs). Kv, and BKCa, and KATP, currents have been identified in single SMCs isolated from rat (4, 48, 59), mouse (5), rabbit (14, 15), dog (41), and human (19, 39) PASMCs. Currently, there is no evidence that Kir channels are present in PASMCs. In addition, a novel non-inactivating K+current, termed /KN, which is proposed to be formed by TASK-1 channels (25), has been described in PASMCs (21).

The complexity and heterogeneity of the K+ channel expression pattern in PASMCs isolated from different species, and the lack of selective pharmacological tools, makes the investigation of the regulation of K+channels by oxygen and second messenger systems a challenging task. Our current understanding of these issues will now be discussed for each type of K+channel expressed in PASMCs.

2.1.KV Channels

The main Kv current found in most types of PASMCs belongs to the delayed rectifier type (referred here and thereafter as Kv current) which is characterised by a relatively slow rate of activation and inactivation, in contrast to the rapidly-inactivating A-type voltage-gated current, also found in some types of PASMCs (32). Post et al. (41) first demonstrated that Kv currents were reversibly inhibited by hypoxia in canine PASMCs (41). Therefore, it was proposed that these channels could act as both potential oxygen sensors and mediators of HPV (57). Since then, research in pulmonary K+ channel physiology has focused on determining the molecular identity of the Kv currents expressed in PASMCs, on whether molecular correlates of the Kv currents are modulated by oxygen, and on establishing the oxygen-sensing mechanism of Kv channels. Using RT-PCR and immunoblotting, the expression of mRNA and/or protein of Kvl.l, Kv1.2, Kv1.3, Kv1.4, Kv1.5 Kv1.6, Kv2.1, Kv3.1b a-subunits and a "silent" Kv9.3 a-subunit has been demonstrated in PASMCs (8, 35, 38, 65). Electrophysiological and pharmacological characteristics of the heteromeric Kv1.2/Kv1.5, Kv2.1/Kv9.3 orhomomeric Kv3.1b a-subunits have proven to be the closest match to those of the native Kv currents in PASMCs (8, 17, 28, 35, 51). The presence of Kv1.5 andKv2.1 isoforms inPASMCs was also confirmed by using specific antibodies to inhibit the native Kv currents (8, 26). Hence, a consensus is nowemergingthatKv1.2/Kv1.5 andKv2.1/Kv9.3 heteromultimers and Kv3.1b homomultimers are the primary candidates underlying native Kv channels in PASMCs (17).

Moreover, when expressed in heterologous systems, both Kv1.2/Kvl.5, Kv2.1 (26), Kv2.1/Kv9.3 (28, 38) andKv3.1b (35) channel currents are inhibited by acute hypoxia, making these Kv isoforms suitable candidates for oxygen-sensitive K+ channels in PASMCs. Since Kv3.1b single channel currents are inhibited in inside-out patches, it has been proposed that Kv3. lb a-subunits can sense oxygen directly (35). However, the dependence of oxygen sensitivity of the Kv1.2/Kv1.5 and Kv2.1/Kv9.3 heteromultimeric channels depends on the expression system, suggests that oxygen sensing by these Kv a-subunits depends on another as yet unidentified mechanism(s) (17). A potential role of auxiliary P-subunits, which interact primarily with a-subunits and are expressed in pulmonary arteries, has been suggested (17), but has not been directly confirmed. How oxygen inhibits homomeric Kv2.1 and heteromeric Kv2.1/Kv9.3 channels also remains unknown.

Hypoxic alteration in the cellular redox state defined by reactive oxygen species (ROS) or by the ratio GSH/GSSG or NAD(P)H/NAD(P)+ may also affect Kv channel activity. Indeed, whole-cell K+ currents in rat PASMCs are inhibited by the mitochondrial electron transport chain inhibitors rotenone and antimycin A (2), by the mitochondrial uncouplers FCCP (61), deoxyglucose and reduced glutathione (GSH) (62), and by cytochrome P-450 inhibitors (63) in PASMC. However, direct effects of diphenyleneiodonium, an NADPH-oxidase inhibitor, on K+ and VDCC channels, as well as the presence of HPV in an NADPH oxidase deficient mice (lacking the gp 91 phox subunit) (6) argues against the involvement of NADPH oxidase in hypoxia-mediated inhibition of Kv channels and HPV. Hence, the role of the cellular redox state in the modulation of the pulmonary Kv channels remains to be established.

Despite the fact that pulmonary vasoconstriction is strongly modulated by a variety of humoral agents (10), our understanding of pulmonary Kv channels' regulation by vasoactive mediators and intracellular second messenger systems remains sparse. Activation of the Kv current by nitric oxide (NO), mediated by cGMP-dependent protein kinase, occurs in extrapulmonary arterial SMCs (3), while another report has proposed a direct effect of NO on Kv currents (64). Although no modulation via a protein kinase A (PKA)-dependent mechanism has yet been demonstrated, another serine/threonine protein kinase C (PKC) can alter Kv currents in PASMCs. Endothelin 1 (ET-1) causes PKC-dependent inhibition of the Kv current and accelerates its inactivation (45). We have previously demonstrated that activators of PKC, diacylglycerol and arachidonic acid (AA), produce a dual effect on Kv currents in rat intrapulmonary SMCs: an initial PKC-dependent increase in the current amplitude followed by a predominant PKC-independent inhibition of the Kv current amplitude. The latter prevailed overall since AA caused membrane depolarisation in these cells (50). It is noteworthy that PKC-dependent phosphorylation may be required for the association of Kv1.5 a-subunits and auxiliary Kvpi.3 subunits which modulates the kinetics of the Kv current in a heterologous expression system (30). Whether a similar mechanism exists in PASMCs remains to be established. The information about the regulation of Kv currents by protein tyrosine kinases is practically absent except for one negative report (49).

Direct inhibition of the Kv currents by intracellular divalent cations, calcium and magnesium, has also been proposed in canine PASMCs (23). However, perfusion of rat single PASMCs with high (-0.5 fiM) [Ca2+]f does not significantly affect the Kv current amplitude (48, 53), suggesting that, at least in this preparation, the Kv current is not sensitive to intracellular Ca2+.

It is worth mentioning that a number of factors could be responsible for the lack of information about the regulation of Kv channels not only in PASMCs, but in vascular SMCs in general (54). Broadly speaking, the native Kv channel can be regulated via changes in its permeability and/or voltage-dependent gating. To monitor these changes, the choice of the experimental protocol and elimination of other conductances, particularly BKCa and KATP currents, are crucial (discussed by Beech et al. in Chapter 23 in Ref. 7). Another important reason is that, despite the progress in molecular biology, it is still not clear whether multiple Kv channel isoforms functionally coexist in the same SMC. This issue is complicated by the absence of selective pharmacological tools allowing us to distinguish between hetero- and homo-multimeric Kv channels expressed in PASMCs; the most effective experimental pharmacological tools are still 4-AP and TEA. Nevertheless, the use of these two inhibitors has proven useful in functional discrimination between different Kv currents in the rat pulmonary arterial tree where at least three cell subtypes, distinguished electrophysiologically on the basis of the expressed Kv currents, have been identified (51). Two cells subtypes, termed IK1 and cells because their characteristics closely resemble those of the members of the KV1 (Shaker) and Kv2 (Shab) subfamilies respectively, are found in the main PA. The third cell subtype, identified in resistance PASMCs, therefore termed also has properties of the KV1 type current which are different from those described for IK1 cells in the main PA. The key pharmacological features of IK1 and ^currents are their different sensitivity to 4-AP and TEA, whereas currents are blocked by TEA and are relatively insensitive to high concentrations of 4-AP (51). It is noteworthy that the properties of the Kv current in adult rat aortic SMCs are similar to those of IK2 cells, while the Kv currents in newborn animals strongly resemble those in IK1 cells (11). Moreover, chronic exposure of adult rats to carbon monoxide increases the sensitivity of the whole cell current to 4-AP and, reciprocally reduces its sensitivity to TEA in coronary arterial SMCs, although no thorough characterization of Kv currents has been given in this study (9). These results could suggest that Kv channel expression is dynamically regulated during vascular development and in pathological conditions. Understanding the mechanisms which control this process in PASMCs is particularly important in chronic hypoxia (CH) which causes the downregulation of Kv channel genes (34, 40, 42, 53) and may represent an intriguing area of the future research.

The recent evidence discussed above, suggests that the existence of "IK1" and "Im" cell subtypes may be a more general phenomenon in the vasculature, and may be partly responsible for the variation in responses to hypoxia and modulation of Kv currents by vasoactive agents (10). Since mice are rapidly gaining popularity as an experimental model, we have compared some properties of Kv currents in mouse small intrapulmonary arterial SMCs (Fig. 1 A) with those of rat main PASMC IK1 (an example of the TEA-sensitivity of which is shown in Fig.IB) and currents. The electrophysiological characteristics and sensitivity to TEA of the mouse Kv currents (Fig. 1C) revealed a remarkable similarity to those described in "IK2" type cells in rat main PA (51) and adult rat aorta (11). Although it is premature to rule out the contribution of TEA-sensitive Kv3.1b channels, mRNA expression of which has been demonstrated in mouse lungs (5), it is noteworthy that a component of current with the pharmacological properties ofKv3.1b channels (i.e., blocked by TEA with an IC50 in the |iM range (7), was not detected (Fig. 1C). The pharmacological characteristics of " W type current include a moderate sensitivity to TEA (IC50 ranged between 2 and 3 mM, Fig. 1C, open symbols) and sensitivity to 4-AP in the mM range (11, 51), currently match only the characteristics of Kv2 channels (see references in Ref. 51). Thus, although a small contribution of other Kv channels cannot be entirely excluded, it is likely that mouse PASMCs predominantly express Kv2.1 channels. This is also supported by the Kv2.1 mRNA expression in the mouse lung (5). It is noteworthy that the presence of a Kv2-type current in mouse PASMCs may explain, at least in part, the lack of the inhibition of acute hypoxic vasoconstriction in NADPH oxidase deficient mice (6), since these channels probably sense oxygen via a different mechanism. Therefore, rat and mouse small intrapulmonary arterial SMCs can be a useful experimental model for investigation of mechanisms of the regulation of the KV1 and Kv2 channel currents, respectively.

A Mouse Intrapulmonary Artery B Rat Resistance Pulmonary Artery

A Mouse Intrapulmonary Artery B Rat Resistance Pulmonary Artery

Figure 1. "IK," and "IK2" cells types in rat and mouse PASMCs. Family of Kv currents shown in A and B were recorded between -40 and +80 mV in 20 mV increments from a holding potential of-80 mV using the same experimental conditions and protocols described in Refs. 11,51. P&G-PSS is abbreviation for physiological saline solution (PSS) containing 1 nM paxilline and 10 nM glibenclamide. C Summary of TEA-sensitivity of Kv currents. TEA-sensitive ("IK2" subtype) and TEA-insensitive ("IKl" subtype) are shown by open and filled symbols, respectively. Smooth lines were drawn according to the equation described previously in Refs. 11 and 51, giving IC50 values of 2.3 (mouse PA) and 2,6 (for IK2 in main PASMCs) mM.

Figure 1. "IK," and "IK2" cells types in rat and mouse PASMCs. Family of Kv currents shown in A and B were recorded between -40 and +80 mV in 20 mV increments from a holding potential of-80 mV using the same experimental conditions and protocols described in Refs. 11,51. P&G-PSS is abbreviation for physiological saline solution (PSS) containing 1 nM paxilline and 10 nM glibenclamide. C Summary of TEA-sensitivity of Kv currents. TEA-sensitive ("IK2" subtype) and TEA-insensitive ("IKl" subtype) are shown by open and filled symbols, respectively. Smooth lines were drawn according to the equation described previously in Refs. 11 and 51, giving IC50 values of 2.3 (mouse PA) and 2,6 (for IK2 in main PASMCs) mM.

2.2 BKc, Channels

Another ubiquitous type of K+channel, BKCa channels, are activated by both voltage and intracellular Ca2+. Although they are expressed in PASMCs (32) the relative contribution of the BKCa current to the whole cell current varies greatly in different species and different regions of the pulmonary arterial tree (1, 4, 14, 39, 41). The expression of a BKCa a-subunit has been demonstrated in rat PA (42). The single channel conductance of BKCa channels has been reported to lie in the range between 170 and 270 pS in symmetrical K+ conditions. It is generally agreed that afunctional BKCa channel in vascular SMCs exists as complex ofa-subunits and regulatory p-subunits, which enhance Ca2+-sensitivity of the channel (7). Additionally, BK^ channels, which originate from a single gene, undergo an extensive alternative splicing that may further add to the diversity of BK^ currents in vascular SMCs. Technically, therefore, regulation of the BKq, channels could occur via modulation of individual a- and/or P-subunits, or via functional uncoupling between a- and p-subunits in native BK^ channels. Thus, for example, the expression of different BKCa splice isoforms could explain the presence of high-conductance (245 and 185 pS) intracellular Mg2+- and ATP-activated BKCa currents in rat PASMCs (1). The sensitivity of the BK^ current to [Ca2+]i is apparently also low in rat intrapulmonary arterial SMCs (48, 53). It is not yet clear whether this is due to a low level of expression ofthe BKq, a-subunit or to decreased Ca2+ sensitivity of the expressed channels.

Owing to their Ca2+-sensitivity, BKCa channels are activated due to the increased [Ca2+]i caused by vasoconstricting mediators, resulting in vasodilatation. In PASMCs, BKCa channel activity is directly enhanced by membrane stretch and arachidonic acid (29, 52). Changes in the cell redox potential also affect BKCa currents. In rabbit small PASMCs, reducing agents such as dithiothreitol, GSH and NADH decreased, while an oxidizing agent (DTNB) increased the activity of BKq, channels (36). However, in SMC isolated rabbit conduit PA, BKCa channels were not affected by either reducing (GSH and NADH) or oxidizing (GSSG and NAD+) agents except DTNB which enhanced the BKCa channel activity (55), similar to the effect of redox agents on BKCa currents in rabbit ear artery SMCs (36). The reason for such differences is not clear. However, it is possible that various BKq, isotypes, which respond differently to changes in the cellular redox state, are present in different regions of the pulmonary arterial tree. Since selective BKca channel inhibitors do not affect acute HPV, it is unlikely that redox modulation of BK^ channels play a key role in HPV. However, their role can be enhanced in chronic hypoxia when Kv channel expression is reduced (46). Chronic hypoxia also decreases BKCa Ca2+ sensitivity and the ability of cGMP and NO to activate BKq, channels in cultured human PASMCs (39).

2.3.Katp Channels

The Katp channel is composed of four pore-forming inward rectifier K+ channel subunits and four sulphonylurea receptors (SUR). Some voltage-independent Katp channels are inhibited by cytosolic ATP, while others are activated by nucleotide-diphosphates (NDP). In vascular SMCs, it is believed that native KATP and NDP-activated KATP channels (KNDP) are formed by SUR2B/Kir6.2 and SUR2BKir6.1, respectively. Both KATP and KNDP are selectively blocked by sulphonylurea compounds such as glibenclamide, and are activated by levcromakalim (see Ref. 7 for details). Although the presence of Katp currents in PASMCs was demonstrated almost a decade ago (15), their molecular identity has only now become evident. Recently, the expression of Kjr6.1, and not Kir6.2, and SUR2B, but not SUR1, has been detected in cultured human PASMCs (19), consisted with the presence of NDP-gated KATP currents in native cells. Interestingly, the reduction in cytosolic ATP enhanced the stimulatory effect of levcromakalim in PASMCs but not in HEK293 cells transfected with SUR2B/Kir6.1, indicating the presence of specific regulatory mechanisms controlling native KATP currents in PASMCs (19). Although KATP channels can contribute to the control of the resting potential in isolated PASMCs (15, 53), it is generally believed that these channels are mostly inactive under normal conditions, but may be opened by changes in the intracellular ATP/ADP and/or GTP/GDP ratios, therefore providing a link between cellular metabolism and membrane excitability. In vascular SMCs, the activity of KATP channels is regulated via cAMP-dependent activation of protein kinase A (54). However, mechanisms of regulation, heterogeneity and molecular isoforms of Katp channels in various PASMCs, remains to be elucidated.

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