Channel as an Effector

In 1986, we hypothesized that HPV related to inhibition ofK+ conductance (7). Hasunuma et al. found that the Kv channel blocker 4-aminopyridine (4-AP) caused pulmonary vasoconstriction that could, like HPV, be prevented by nifedipine in the isolated rat lung model (40). In 1992, studies done by Post et al. showed that hypoxia inhibit whole cell IK in canine PASMCs and depolarize the membrane of canine PASMCs (93). This work initiated extensive research to evaluate the role of K+channels in HPV.

K+ channels are proteins consisting of four transmembrane-bound pore-forming a subunits, often associated with four regulatory P subunits (2). The K+ channel pore is conferred by the formation of tetramers of a subunits. The subunits determine the intrinsic conductance and voltage sensitivity of the channel. On the other hand, P-subunits are small subunits that associate with the a subunits and alter the channel's activation and inactivation kinetics (92). There are several types of K+ channel a-subunits, including Kv channels, inward rectifier K+ channels (Kjr), ATP-sensitive K+ channels (KATP), and Ca2+-activated K+ channels (KCa) (92). Despite the existence of different kinds ofK+ channels, the Kv channels in particular have emerged as a participants in HPV, in large part because they control resting membrane potential and are redox-sensitive (opening when oxidized and closing when reduced) (97).

Katp channels are sensitive to changes in intracellular ATP and therefore are regulated by the cell's metabolic status (76). Because of this, KATP channels have been proposed as ideal candidates for HPV. However, it was quickly established that Katp channels do not contribute to HPV because glibenclamide, a KATP inhibitor, had no effect on pulmonary perfusion pressure in hypoxia (40). Similarly, the role of Kir channels in HPV is less clear, as its presence in PASMC (as opposed to the endothelium where it is abundant) is still questionable (76). KCa, on the other hand, seem to act as a brake on HPV. Big conductance KCa (BKCa) channels control the arterial smooth muscle membrane in a negative feedback mechanism (76). At less negative or positive potentials, when the arterial bed is constricted and intracellular Ca2+ is increased, BKCa channels are activated, leading to K+ efflux and restoration of membrane potential toward more negative values (hyperpolarization) (96). In cerebral arteries, hypoxia activates BKCa channels (37). Thus, although many different K+ channels exist, the Kv channels are the only most likely candidates for HPV.

Kv channels are important determinants of membrane potential (Em) of vascular SMCs. When Kv channels close and the tonic efflux of K+ is decreased, the cell interior becomes relatively less negative (depolarized). At these potentials (i.e., -30 to -10 mV) the opening probability of L-type voltage-gated Ca2+ channels increases. This increases Ca2+ influx and activates Ca2+-induced Ca2+ release, effectively increasing total Ca2+ levels inside the cell. This increase in cytosolic Ca2+ levels not only activates contraction via the actin-myosin apparatus but, in chronic settings, also increases the activation of immediate early genes to induce a proliferative response. Thus, regulation of K+ channel activity and the subsequent regulation of Ca2+ is important to maintain vascular tone and the morphology of the PASMCs (75).

To date, nine families of Kv channel have been identified. These channels activate in a nonlinear fashion with depolarization and many are inhibited by 4-AP (100). Out of these, Kv1.2, Kv1.5, Kv2.1, Kv3.1b and Kv9.3 are present in the pulmonary vasculature. One or more of them may form the PASMC 02 sensitive Kv channel (27). The identification of the Kv channels involved in the Em of PASMCs has been difficult, due to the lack of specific Kv channel blockers and to the fact that the whole-cell K+ current is an ensemble of currents conducted by many types of K+ channels. The fact that glyburide and charybdotoxin, inhibitors of KATP and BKCa channels, respectively, do not cause much increase in PVR whereas 4-AP causes similar constriction to hypoxia, suggests that Kv channels are key to HPV. Indeed the 02 sensitive IK in PASMCs is voltage sensitive (11, 16, 27, 132), To determine the molecular basis for the 02-sensitive IK, we "dissected" the whole cell current in rat PASMCs by making whole-cell patch clamp current recordings with or without antibodies directed againstKv channels in the intracellular solution (17). When anti-Kv2.1 antibody was added to patch pipette, the outward K+ current was inhibited and the membrane depolarized, suggesting that Kv2.1 plays a role in setting the resting membrane potential in rat resistance PAs. When anti-Kv 1.5 antibody was added to the patch pipette, whole cell K+ currents significantly decreased and the ability of both hypoxia and 4-AP to increase Ca2+ in isolated PASMCs was attenuated. Thus, we showed thatKv1.5 andKv2.1 are important components of whole-cell K+current in rat PASMCs. Moreover, it was found that although Kv2.1 sets the resting membrane potential, Kv1.5 is instrumental in mediating HPV (Fig. 8) (17). Subsequent studies have confirmed this, although it also suggests a role for Kv1.2/1.5 heterotetramers (44). Moreover, heteromeric channels comprised of Kv2.1/KV9.3 have also been implicated in the mechanism of HPV (84), although, evidence ofheteromeric channels existing in vivo is lacking (116). Recently it has been demonstrated that Kv channels are involved in 02 sensing, although the specific populations involved have yet to be definitively established. Interestingly, Kv1.5 and Kv2.1 appear to be involved in the human DASMCs' response, although the sensitivity to H202 is reversed compared to the PA (74). In a model for the NEB cell (lung carcinoma line H146), acute hypoxia inhibits IK and causes cell depolarization by inhibiting the tandem P domain family of K+ channels, hTASKl or hTASK3 (132). In NEB cells, K+ channel inhibition re suits in serotonin secretion as a consequence of membrane depolarization and a rise in cytosolic Ca2+(35). In the carotid body, it appears likely that several K+ channels maybe involved in 02 sensing, including Kv3.x and Kv4.x channels (89, 105), and possibly TWIK or TASK, TASK-1, acid sensitive channels with four transmembrane segments and two pore domains (83).

Figure 8: The role of Kv1.5 in HPV. A: Resistance PA rings from mice lacking Kv1.5 (Kv1.5"'') have diminished HPV compared to wild-type mice. HPV was elicited from these rings without priming with a vasoconstrictor. B: PASMCs from Kv1.5"'' mice have decreased 02-sensitive current compared to wild-type mice. C: Antibodies against Kv1.5 and Kv2.1 (but not against GIRK1) channels inhibit K* current when applied intracellular^ via the patch pipette (Reproduced

Figure 8: The role of Kv1.5 in HPV. A: Resistance PA rings from mice lacking Kv1.5 (Kv1.5"'') have diminished HPV compared to wild-type mice. HPV was elicited from these rings without priming with a vasoconstrictor. B: PASMCs from Kv1.5"'' mice have decreased 02-sensitive current compared to wild-type mice. C: Antibodies against Kv1.5 and Kv2.1 (but not against GIRK1) channels inhibit K* current when applied intracellular^ via the patch pipette (Reproduced

Exposure to hypoxia for 1-2 weeks elicits chronic hypoxic pulmonary hypertension (CH-PHT). Rodent CH-PHT is a relevant model for PHT that occurs in humans living at high altitude and in patients with chronic lung diseases. Paradoxically, acute hypoxic pulmonary vasoconstriction is blunted in CH-PHT, whilst the response to other vasoconstrictors is preserved or enhanced (71, 90). This is associated with inhibition ofPASMC K+ current (113) and a selective downregulation ofmRNA expression of some, but not all, PASMC K+ channels (e.g., Kv1.5, Kv2.1, Kv4.3 andKv9.3) (97, 135). CH also selectively decreases function/expression of these channels in cultured PASMCs (18). Mice lacking Kv1.5 also have depressed HPV (16). Furthermore, anorexigens which have precipitated outbreaks of human PHT inhibit K+ channels (11), including Kv1.5 (88). These data point strongly toward a role for Kv1.5, a slowly inactivating, 4-AP-sensitive Kv channel, in the mechanism of HPV. We recently utilized gene transfer to directly test the importance of Kv 1.5 to the loss of HPV that occurs with chronic hypoxia. We hypothesized that restoration of Kv1.5 using gene transfer could be accomplished by nebulization of an adenoviral vector carrying the gene for Kv1.5 under a CMV promoter, thereby restoring Kv1.5 expression, reducing CH-PHT, and restoring HPV (94). In this recent study, we confirmed that administration ofKv1.5 to the pulmonary circulation via an aerosol is feasible and effective in eliciting transgene expression in resistance PASMCs. Furthermore, expression of cloned human PA Kv1.5 reduced PVR in experimental PHT. Furthermore, Kv gene therapy with an insensitive channel restored HPV and 02 sensitive IK in rats with established CH-PHT. This study supports a central role for Kv 1.5 in the mechanism of HPV and is consistent with the hypothesis that a K+ channel deficiency state is involved in the pathogenesis of PHT (5, 133).

The K+ channel pore is formed by coordinated alignment of key amino acids in the S5-S6 region of each of the a-subunits. Some (but not all) K+ channels are well suited to 02 sensing by virtue of possessing key cysteine and methionine groups. Reduction or oxidation of these residues by a redox mediator, such as an AOS, could cause a conformational change in the channel and thus alter pore function (17). Studies with AOS have shown that they can regulate protein function by five possible ways: i) modification of proteins by oxidation of cysteine residues (e.g., S-glutathionylation), ii) formation of intra-molecular disulfide linkages (e.g., the oxidative stress-responsive protein OxyR), iii) protein dimerization by inter-molecular disulfide linkages (e.g., activation by various protein kinase), iv) di-tyrosine formation by H202-dependent reactions (e.g., gp91phox regulation of extracellular matrix), v) metal-catalyzed oxidation of protein by "Fenton-like" chemistry (e.g., protease action and ubiquitination) (119). In this regard, certain K+ channels, including Kv1.5, respond to reduction and oxidation by changing their gating characteristics and open state probability. In the PASMCs, oxidants (e.g., H202, diamide oxidized glutathione) increase IK whereas reducing agents (e.g., reduced glutathione and duroquinone) and agents that facilitate electron shuttling (e.g., the synthetic co-enzyme Q mimetic) inhibit IK(99). In accordance with their electrophysiological effects, oxidants dilate the pulmonary circulation (mimicking 02), while reducing agents mimic hypoxia (Fig. 9). Hypoxia and redox agents may alter the function of ion channels, specifically K+ channels, directly (47) or by modulating levels of a diffusable redox mediator, such as an AOS. The existence of redox sensitive K+ channels does not verify whether the electrophysiological effects of 02 are direct or mediated via a remote sensor that produces a mediator which ultimately alters channel activity. However, research is warranted in this area to fully elucidate the precise mechanism behind AOS-mediated Kv inhibition.

Figure 9: Redox control of PA tone. A: The sulfhydryl oxidant, diamide, reverses HPV in anesthetized dogs. Preincubation of diamide with reduced glutathione prevents this effect. B: Endothelium-denuded PA rings constrict in response to the electron shuttling agent duroquinone and this is reversed by diamide (Reproduced from Ref. 99 with permission).

Thus the Kv channels themselves are redox-sensitive, which leaves the question: to what signal are they responding. While we propose that this signal is mitochondria-derived H202, other hypotheses have been advanced: altering of a membrane-bound 02-sensitive regulatory moiety that is adjacent or coupled to the channel subunits, activating mitochondrial NADPH oxidase and increasing 02'~, inhibiting cytochrome P450, decreasing intracellular pH, releasing Ca2+ from intracellular stores and, recently, affecting directly the Kv channel protein (116). In this regard, Marshall and Marshall have suggested a gp91phox containing NADPH oxidase is involved in HPV (making more AOS during hypoxia) (66). In contrast, we confirmed that HPV and 02-sensitive IK are preserved in mice lacking a functional NADPH oxidase (15).

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