Fundam Clin Phurmacol(1992) 6,279-293 0 Elsevier, Paris
Potassium channel openers: pharmacological and clinical aspects U Quast * Pre-clinical Reseurch, Sandoz Phurma Ltd, CH-4002 Basel. Switzerland (Received 21 September 1992; accepted 2 October 1992)
Summary - Opening of plasmalemmal K+ channels leads to cellular hyperpolarization which, in excitable tissues possessing voltagedependent Ca?+channels, prevents the opening of such channels and thus prevents excitation. In the last few years, an increasing number of compounds have been identified which elicit their effects by opening K+ channels, preferentially in smooth muscle, but also in other excitable tissues. These include the novel benzpyrans, cromakalim and bimakalim, the thioformamide aprikalim, and also well known antihypertensives such as minoxidil sulphate, diazoxide and pinacidil. After a short overview of the various families of K+ channel openers (KCOs), their basic pharmacological properties, including inhibition by the sulfonyl ureas (such as glibenclamide) are presented. The actual discussion concerning the type of K+ channel(s) opened by these compounds and their mechanism(s) of vasorelaxalion will be reported. The therapeutic potential of these compounds in the cardiovascular field (as antihypertensives and, in particular, as anti-ischemic agents in heart and skeletal muscle), and in asthma (where they reverse established airway hyperreactivity) will also be discussed. Improved tissue selectivity may be the essential pre-requisite for true clinical success of this class of compounds.
K+ channel opener / hyperpolarization and smooth muscle tone / K+ channels
Introduction K+ channels The direction of the movement of ions across the cell membrane is governed by the electrochemical gradient, which in turn, is determined by the difference in ion concentration inside and outside the cell and the potential difference across the membrane (at rest negative inside with respect to outside). The pumping action of the Na+/K+ ATPase generates a K+ gradient which is directed outward (intracellular [K+] =: 150 mM versus extracellular [ K+] =: 5 mM, fig 1 ). Opening of plasmalemmal K+ channels allows cytosolic K+ to flow out of the cell along its electrochemical gradient, thereby leading
to a loss of positive charges from the cytosol. This results i n a more negative membrane potential which reduces the driving force for K+ to leave the cell. At the K+ equilibrium potential (usually -80 to -90 mV), the electrical field over the membrane (which retains K+ inside the cell) matches the outward directed chemical gradient of K+ and K+ efflux is matched by K+ influx, ie the system reaches a stationary state. The opening of plasmalemma1 K+ channels therefore drives the membrane potential towards the K+ equilibrium potential, eg in the case of smooth muscle from -60 mV towards -80 mV. Plasmalemmal K+ channels determine the resting cell membrane potential and influence parameters as different as cell excitability and cell volume.
*Present address: Department of Pharmacology, University of Tiibingen, Wilhelmstr. 56, D-7400Tiibingen, Germany
Opening of K+ channel
Vin = - 8 5 mV
K+ Equilibrium Potential
Fig 1. Ionic gradients across the cell membrane and the effect of K + c h a n n e l o p e n i n g . T h e p u m p i n g actio n o f t h e sodiudpotassium ATPase (NKA) (which pumps 3 Na+ ions out of the cell and 2 K+ ions from outside into the cell at the expense of I molecule ATP) creates the ionic gradients indicated. Under these circumstances, the opening of K+ channels in the cell membrane leads to the efflux of K+ from the cell. This loss of positive charges renders the interior of the cell more negative (hyperpolarization). The ensuing electrical field over the membrane (the interior of the cell being at a more negative potential than the outside which is arbitrarily set at 0 ) increasingly opposes the further efflux of K+ until the electrical force compensates for the difference in K+ concentration. The potential at which the net electrochemical gradient for K+ over the membrane disappears is called the K+ equilibrium potential (Nernst equilibrium potential for K+) and lies around - 85 mV (inside negative).
Of all the types of ion channels investigated, K+ channels form the most heterogeneous group (Rudy, 1988; Cook, 1990). In general, K+ channels are classified according to their gating mechanism. One class (voltage-dependent K+ channels, K,) is opened by changes in membrane potential (most often depolarization), another by ligands such as intracellular Ca?+ (Ca2+-dependentK+ channels, Kca) or G-proteins. Many neurotransmitters and hormones (indirectly) open K+ channels v i a G-proteins: binding to the receptor activates a G-protein which then opens a K+ channel. This is the chain of events by which stimulation of the vagus is transduced into reduction of heart rate and force of (atrial) contraction. An interesting and heterogeneous family of (voltage-independent) K+ channels is closed by intracellular ATP (ATP-sensitive K+ channels, K,,,); the inhibition by ATP is modulated by other nucleotides such as MgADP. K,,, thus couple cellular excitability to the metabolic state of the cell. In general, these channels are sensitive to blockade by the sulfonylureas, eg glibenclamide (Quast and Cook, 1989a; Ashcroft and Ashcroft, 1990). In the pancreatic pcell, K,,, mediate insulin release in response to the level of plasma glucose. An increase in plasma glucose leads to an increase in intracellular ATP in the p-cell, which closes K,,, and depolarizes the cell, leading to Ca2+ entry and insulin secretion. KATPare also found in other excitable tissues such as brain, heart, skeletal and smooth muscle where they serve different functions. One of these is the protection of the tissue during and after ischemia and hypoxia. These conditions lead to an increase in the [ADP]/[ATP] ratio and to acidosis which both promote opening of K,,,, thus clamping the cells at the resting membrane potential and salvaging their compromised [ATP] (Quast and Cook, 1989a; Escande and Cavero, 1992). To date, only a few classes of K+ channels have been cloned. Most information is available on a superfamily of voltage-gated K+ channels (K,) related to the gene products of the Shaker locus of drosophila (Jan and Jan, 1992). Structurally, completely unrelated to these is the ‘mini’ K+ channel (also a K,) which is very well conserved across
Potassium channel openers
species and tissues (Philipson and Miller, 1992). In ventricular cells of the mammalian heart, these two classes of K+ channels are co-expressed to constitute the rapid and slow components of the delayed rectifier current which terminates the ventricular action potential (Sanguinetti and Jurkiewitz, 1990; HonotC et al, 1991). A member of the K,, group has also recently been cloned; it is a large conductance calcium-dependent K+ channel from drosophila with a sequence remotely related to the Shaker-type K+ channels (Atkinson et al, 1991). No sequence of any K,,, channel is known yet; the sulfonylurea receptor from pig brain and from a Pcell tumor has a molecular weight of = 150.000 (Bernardi et a f , 1988; Kramer et al, 1988). This review concentrates on a new class of agents, the K+ channel openers (KCOs) which act by opening K+ channels primarily in smooth muscle, but also in other excitable tissues, eg nervous tissue, heart and iuxtaglomerular cells.
The coronary vasodilator nicorandil (Chugai, for structure see fig 2) was the first drug shown to hyperpolarize smooth muscle by increasing the cell membrane permeability for K+, presumably by opening K+ channels (Furukawa et a f , 1981). Two years after this first report, it was also shown that the nitro group in its structure enables nicorandil to activate soluble guanylate cyclase, thereby increasing the concentration of cGMP in the smooth muscle cell (Holzmann, 1983), an effect also leading to relaxation. Nicorandil thus has two (independent) mechanisms of action and it depends on the experimental system as to which of the two predominates. Nicorandil is now under develop-
The different families of K+ channel openers The structure-activity relationships of the KCOs have recently been compiled (Edwards and Weston, 1990; Robertson and Steinberg, 1990) and the clinical pharmacology of the newer members of this class of compounds reviewed (Anderson, 1992).
Fig 2. Structures of the K+ channel openers and blockers discussed in the text.
ment in Japan and other states as a coronary vasodilator. Recently, more potent analogues of nicorandil have been synthesized (eg KRN 2391 Kirin (Edwards and Weston, 1990)). Benzpyrans
The prototype of the KCOs is cromakalim (BRL 34915, Beecham), the mechanism of which was elucidated by Hamilton et al (1986). In comparison to nicorandil, cromakalim is approximately 100 times more potent and seems to act exclusively by opening K+ channels. Chemically, this compound is a benzpyran with two optical centers in the trans configuration (fig 2). The development of cromakalim in hypertension and asthma has been suspended in favor of the active enantiomer levcromakalim (BRL 38227, formerly lemakalim),
which has the (-) (3S, 4R) conformation. Several related molecules have been synthesized, among them bimakalim (E Merck, Darmstadt; identical to Sanofi SR 44866) which lacks an asymmetric carbon (fig 2; for more detailed information on the cromakalim analogues under development (see Edwards and Weston, 1990; Robertson and Steinberg, 1990). Cyanoguanidines Pinacidil, an antihypertensive recently introduced in Denmark (Leo) and the US (Lilly), is also a KCO (Bray et al, 1987), Cook et al, 1988a). This molecule (fig 2), a pyridine with a cyanoguanidine-containing substituent in position 4, is again a racemate, with the KCO activity residing largely in the (-, R) enantiomer (Cook et al, 1989). There is, however, evidence that pinacidil possesses vasorelaxant mechanisms in addition to K+ channel opening which contributes at higher (= 20 x) concentrations, and lack stereoselectivity (Cook et al, 1989, Cook and Quast, 1990). Recent reports indicate that racemic pinacidil (2 1 pM) inhibits Ca2+release from intracellular stores or the refilling of these stores (Xiong et al, 1991), and that, at concentrations of 2 10 pM, it acts directly on the contractile apparatus to inhibit Ca2+-inducedcontractions (Itoh et al, 1991). The enantiomeric selectivity of these very interesting findings remains to be determined. Thioformamides Recently, a new class of KCOs has been described, the thioformamides, eg RP 49356 (fig 2) and its active (-) enantiomer RP 52891 (aprikalim, Aloup et al, 1990; Richer et al, 1990) which are essentially equipotent with cromakalim; the structurally related oxime RP 61674 (fig 2) is 500 times more potent than RP 49356 (Quast, 1992).
laxation of its active metabolite, minoxidil sulphate (Meisheri et al, 1988; Newgreen et al, 1990; Bray and Quast, 1991a) and the antihypertensive effect of the parent drug (references in Quast, 1992) show the characteristic traits of the KCOs. Minoxidil sulphate is equipotent with cromakalim in relaxing the tonic contraction of the rat aorta to noradrenaline, but has only a poor efficacy in producing 42K+ or 86Rb+ efflux from this preparation (Newgreen et al, 1990; Bray and Quast, 1991a) and induces only a minor hyperpolarization in rat portal vein (Newgreen et al, 1990). There is some evidence that the compound may act as a partial agonist on K+ channels which are opened with much higher efficacy by other KCOs (Bray and Quast, 1991a). Diazoxide is an antihypertensive whose chronic administration has been precluded by its marked hyperglycemia, an effect due to reduced insulin secretion (Gilman et al, 1985) via the opening of ATP-sensitive K+ channels (KATp) in the pancreas (Ziinkler et al, 1988). K, in pancreatic &cells are opened by diazoxide with an EC,, of 20 /& inIthe presence of 0.3 mh4 ATP (Ziinkler et al, 1988). Evidence that its vasodilator activity may be due to the opening of K+ channels has been reported (Quast and Cook, 3989b; Newgreen et al, 1990). The vasorelaxant effects in vitro occur around l e 5 M , ie at concentrations similar to those effective in the & cell. In the anaesthetized rat, the effects of diazoxide on plasma insulin and glucose levels are detected at the same concentrations as the blood pressure lowering effects suggesting that, also in vivo, diazoxide discriminates poorly between the ATP-sensitive K+ channel in the pancreas and the K+ channel(s) opened in smooth muscle (Quast and Cook, 1989b), thus corroborating the clinical experience with diazoxide (Gilman et al, 1985).
Pharmacological properties of the KCOs
Minoxidil sulphate and diazoxide (fig 2) Effects in smooth muscle
Minoxidil and diazoxide, two established antihypertensives, have recently been shown to act via K+ channel opening. Minoxidil itself is inactive as a vasorelaxant in vitro. However, the in vitro vasore-
Hyperpolarization and tracer efslux Since the membrane potential of smooth muscle cells is generally in the order of -50 to -60 mV
Potassium channel openers
(and thus substantially more positive than the K+ equilibrium potential), opening of K+ channels will lead to a hyperpolarization of the tissue due to the efflux of K+. Micro-electrode experiments have indeed shown that the KCOs hyperpolarize smooth muscle of various origins and elicit an outward current (reviews Cook and Quast, 1990; Edwards and Weston, 1990; Robertson and Steinberg, 1990). In tissues labelled with 4*K+ or 86Rb+ the KCOs induce a concentration-dependent increase in the efflux of these tracers, thereby allowing inferences on some properties of the K+ channel(s) opened by these drugs (Quast, 1992). For technical reasons (in particular the convenient half life), 86Rb+ has been widely used as a substitute for 42K+ in studies involving the KCOs and yields generally qualitatively similar results (Quast and Baumlin, 1988). Detailed comparison of the cromakaliminduced increases in the permeabilities for Rb+ vs K+ suggests heterogeneity of the K+ channels opened by this agent in rat aorta (Bray and Quast, 1991a). Smooth muscle relaxation
The vasodilator profile of cromakalim shows characteristic features which distinguish it from other vasodilators. Most importantly, cromakalim is able to relax contractions to low (< 25 mM) but not high (> 30 mM) KCl in various vascular preparations (Hamilton et al, 1986; further references in Cook and Quast, 1990), and the presence of elevated KCI abolishes the vasorelaxant effect of cromakalim against other stimuli (eg noradrenaline and angiotensin I1 (Cook et al, 1988b). This profile of action is expected for an agent acting solely by opening K+ channels. In the presence of high KCI concentrations, the membrane potential approaches the K+ equilibrium potential, thus reducing the degree of hyperpolarization achievable by opening K+ channels (Furukawa et al, 1981). Furthermore, in the presence of elevated extracellular K+, the K+ equilibrium potential may be more positive than the threshold potential at which the voltage-dependent Ca2+ channels close. Thus, in high K+ solution, the ability of the KCO’s
to indirectly close these channels by hyperpolarizing the cell membrane is abolished. An important observation made by many authors in various types of smooth muscle (blood vessels, uterus, bladder and trachea) is that 8sRb+ efflux or membrane hyperpolarization are only observed at KCO concentrations where relaxation is essentially complete (for references see Cook and Quast, 1990). This paradox is only partially resolved by the use of 42K+as the tracer (Quast and Baumlin, 1988) and one may have to invoke vasorelaxant mechanisms unrelated to the opening of plasmalemma1 K+ channels to explain this discrepancy. On the other hand, we have found an excellent correlation between the potencies of the KCOs as stimulators of 86Rb+efflux and as vasorelaxants in rat aorta and portal vein or as blood pressure lowering agents in the anesthetized rat (Cook and Quast, 1990; Quast, 1992). Only the ‘atypical’ KCO minoxidil sulphate (Newgreen et al, 1990; Bray and Quast, 1991a) appears as an outlier. Seemingly, these excellent correlations support the hypothesis that the vasorelaxant effect of the KCOs relies on their ability to open plasmalemmal K+ channels in vascular smooth muscle. Inhibition by glibenc lamide and tedisamil
The hypoglycemic sulfonylurea, glibenclamide, first line treatment in diabetes type 11, is a potent blocker of the ATP-sensitive K+ channel(s) in the pancreatic p-cell and related cell lines (SchmidAntomarchi et af, 1987; Zunkler et al, 1988) as well as in heart, skeletal muscle, brain and smooth muscle (reviews Quast and Cook, 1989a; Ashcroft and Ashcroft, 1990). Glibenclamide inhibits the effects of the KCOs on tracer efflux and/or contraction at concentrations around 0.1 pM and inhibits the blood pressure lowering effect of cromakalim and other KCOs in the rat at doses of 2 10 m g k g iv (Quast and Cook, 1989b; further references in Quast, 1992). Inhibition by glibenclarnide of cromakalim’s effects in vitro generally has a competitive appearance. However, the limited concentration range in which this agent can be safely used (< 1 pM) pre-
cludes a definitive assessment of the type of inhibition (Quast and Cook, 1989b). In contrast to the above, the vasorelaxant effects of minoxidil sulphate in rat aorta are inhibited by glibenclamide in a noncompetitive manner (Newgreen et al, 1990). The fact that the effects of all KCos reported so far are inhibited by similar concentrations of glibenclamide, suggests that these compounds may have a common target in smooth muscle, and that this target may be an ATP-sensitive K+ channel (Quast and Cook, 1989a). However, the second part of this contention must be considered with caution since, at concentrations exceeding 1 pM (frequently used in work with the KCOs), glibenclamide is no longer a specific blocker of these channels (Panten et al, 1989; Quast and Cook, 1989b; Bray and Quast, 1992a). Tedisamil (KC 8857), a sparteine derivative which blocks several K+ channels i n heart and smooth muscle with IC,, values around 10 pM (Dukes and Morad, 1989; Pfrunder and Kreye, 1991, exhibits bradycardic and antiarrhythmic properties (Beatch et al, 1990). This molecule is a potent blocker of cromakalim- and minoxidil-induced tracer efflux from rat aorta (ICs0 = 50 nM), but it is a surprisingly weak inhibitor of the vasorelaxant effects of these KCOs (Bray and Quast, 1991b; Bray and Quast, 1992a). This differential inhibition of plasmalemma1 K+ channel opening and vasorelaxation (which is in contrast to the block by glibenclamide) suggests that cromakalim has mechanisms of vasorelaxation in addition to the opening of plasmalemma1 K+ channels; these mechanisms are sensitive to inhibition by glibenclamide but not tedisamil. Heart
The KCOs, at concentrations 30-1 00 times higher than those used in vascular smooth muscle, shorten action potential duration in various regions of the heart and decrease contractile force, effects reversed by K+ channel blockers such as glibenclamide, ICS 205-930 and sotalol (reviews Cook and Quast, 1990; Robertson and Steinberg, 1990). Experiments at the single channel level show that the KCOs
increase the open probability of K,, in the cardiac myocyte by decreasing their affinity for ATP, thus relieving the blockade of K,, by physiological levels of ATP in the cell (Thuringer and Escande, 1989). The opening of K,, by pinacidil is noncompetitively inhibited by (intracellular) ATP (Nakayama et al, 1990). Interestingly, diazoxide is a blocker of K,, in the heart (Faivre and Findlay, 1989), pointing at important pharmacological differences between K,, in different tissues. In ventricular myocytes, the KCOs linearize the N-shaped steady-state current-voltage relationship by removing rectification at membrane potentials more positive than the K+ equilibrium potential and, i n some cases, by decreasing P, at more negative potentials (Osterrieder, 1988; McCullough et al, 1990). The latter may reflect inhibition of the inwardly rectifying background K+ current, I,, (McCullough et al, 1990). In Purkinje fibres, cromakalim (5 pM) antagonizes abnormal pacemaker activity, conferring potentially antiarrhythmic properties to the drug (Liu et al, 1988). Cardioprotective effects
In vitro and in vivo investigations show that several KCOs (benzpyrans, thioformamides, nicorandil and, to a lesser degree, pinacidil) are able to protect the myocardium against the sequelae of transient ischemia (Gross, 1991 ; Grover, 1991 ; Escande and Cavero, 1992). When given before and during the ischemic insult, they improve recovery of contractile function upon reperfusion, reduce infarct size, preserve adenine nucleotides (Escande and Cavero, 1992) and attenuate the inflammatory process in the ischemic area, eg by inhibition of neutrophil infiltration (Pieper and Gross, 1992). Interestingly, these effects are sensitive to inhibition by glibenclamide, suggesting that they may be mediated by an opening of K,,, (Gross and Auampach, 1992). In addition, some KCOs are able to selectively increase coronary collateral blood flow in the ischemic area at non-hypotensive doses (Gross, 1991, but see Grover, 1991), thus avoiding steal, reflex tachycardia and other problems arising in vasodilator therapy of cardiac
Potassium channel openers
ischemia. This may be of particular importance for therapeutic applications.
Skeletal muscle High concentrations of cromakalim (10-100 pM), pinacidil or RP 49356 increase the membrane conductance for K+ in skeletal muscle biopsies from patients suffering from myopathies with depolarized resting membrane potentials (Spuler et (11, 1989; Quasthoff et al, 1989). This effect is sensitive to inhibition by tolbutamide suggesting that the K+ channel opened in this tissue may be K,,, (Spuler, 1989). In fibres from patients suffering from hypokalemic periodic paralysis, cromakalim (100 p M ) strongly improves twitch force in a glibenclamide-sensitive manner suggesting that KCOs with increased specificity for K,, in skeletal muscle may have therapeutic potential in cases where myopathies are accompanied by membrane depolarization (Grafe et al, 1990). Experiments at the single channel level show that high concentrations (> 100 p M ) of the KCOs cromakalim, pinacidil and RP 49356 increase the open-probability of K,, in inside-out patches from mouse skeletal muscle, whereas diazoxide is (essentially) ineffective (Weik and Neumcke, 1990). Nervous system Peripheral newous system The KCOs interfere with neurotransmission in the airways. In this tissue, cromakalim (> 0.1 p M ) inhibits more potently contractions elicited by preganglionic vagal stimulation than those to exogenous acetylcholine, suggesting a presynaptic effect of the drug (Hall and MacLagan, 1988). Cromakalim also inhibits contractions to NANC excitatory nerve stimulation in the guinea-pig airways in vivo (Ichinose and Barnes, 1990) and in vitro (Burka et ril, 1991), the latter at concentrations below those which exert a direct relaxant effect on airway smooth muscle (Burka et a l , 1991). This may explain the ability o f cromakalim and SDZ PCO 400 to reverse induced airway hyperreactivity at doses which have a negligible bronchodilator effect in v i w (Chapman et al, 1992).
Central nervous system Evidence for the existence of K,, in several areas of the brain has been obtained (reviews Miller, 1990). In rat substantia nigra, sulfonylurea-sensitive K,, couple plasma glucose levels to neuronal excitability and inhibit GABA release (SchmidAntomarchi et al, 1990). They are opened by the KCOs with a rank order of potency quite different from that found in smooth muscle (Schmid-Antomarchi et al, 1990) and appear to play an important role in brain ischemia (Miller, 1990). In addition, high concentrations of cromakalim (= 100 pM) reduce stimulated seizure-like activity in guinea-pig hippocampal slices and activate (or potentiate) an inwardly rectifying K+ conductance (Alzheimer et al, 1989). In several animal models of epilepsy, intracerebroventricular administration of KCOs prevents or reduces seizure-like activity (Gandolfo et al, 1989). Secretory systems Renin In cultured rat juxtaglomerular cells, cromakalim (0.1-10 pM) concentration-dependently increases renin release (Fenier et al, 1989). In the pithed rat, where reflex activation of the renin-angiotensin system is prevented, cromakalim (0.1 m g k g ) augments plasma renin activity, indicating a direct effect on juxtaglomerular cells at this dose (Richer et al, 1990). Pre-treatment with glibenclamide prevents this effect. It is known that juxtaglomerular cells are derived from vascular smooth muscle cells and that renin secretion is inversely related to the intracellular Ca2+ concentration (Kurtz, 1986). Hence, membrane hyperpolarization, by reducing the entry of Ca*+ into these cells, could account for the stimulation of renin secretion by cromakalim. Insulin In general, the KCos do not decrease plasma insulin levels at blood pressure lowering doses (Quast and Cook, 1989b), the exception being diazoxide, which discriminates poorly between K,, in the pancreatic p-cell (which regulate insulin release)
and the target K+ channels in smooth muscle. In mouse isolated pancreatic islets, the KCos inhibit glucose-induced insulin release in the rank order diazoxide >> pinacidil > cromakalim 2 nicorandil (Garrino er al, 1989). Patch clamp experiments in the insulin-producing cell line, RINmSF, show that high concentrations (> 100 pM) of the KCOs pinacidil, RP 49356, nicorandil and cromakalim open K,, in the presence of ATP. However, these effects are much weaker than those of diazoxide (Dunne et al, 1990). It is clear that the pharmacological properties of K, in the pancreatic p c e ll are quite different from those in heart and skeletal muscle, as well as from the target channel of the KCOs in the smooth muscle cell.
Mechanistic considerations Mechanism(s) of vasorelaxation The original working hypothesis on the mechanism of KCO-induced vasorelaxation stated that the opening of plasmalemmal K+ channels by the KCOs leads to hyperpolarisation of the cell membrane, thus preventing Ca2+entry through voltagedependent Ca2+channels (Hamilton et al, 1986). However, the observation that cromakalim is able to inhibit the contraction of the rabbit aorta to noradrenaline, which occurs without modification of the membrane potential, and where dihydropyridine-sensitive Ca2+ channels provide only a small fraction of the Ca2+ for contraction (Cook et al, 1988b), requires an update of this original postulate (Cook and Quast, 1990; Edwards and Weston, 1990). In agreement with the classical pharmacological profile of the KCos, this vasorelaxant effect of cromakalim is abolished in high KCI (Cook et al, 1988b) and is sensitive to inhibition by glibenclamide (Bray er al, 1991). In addition, cromakalim has been shown to inhibit contractions of several blood vessels in Ca2+-free medium, in a depolarization- and glibenclamide-sensitive manner (see eg Quast and Baumlin, 1991). Furthermore, the (-) enantiomer of cromakalim, BRL 38227, lowers resting [Cali in both Ca2+- containing and Ca2+-free medium i n a glibenclamide-sensitive
manner (Ito et al, 1991). Finally, the fact that tedisamil differentially inhibits the vasorelaxant and Rb+ efflux stimulating properties of cromakalim suggests that cromakalim has effects in addition to the opening of plasmalemmal K+ channels. ‘Additional’ mechanisms of vasorelaxation (fig 3) Recently, it has been shown that BRL 38277 (0.1 pM) is able to inhibit noradrenaline-induced Ca2+release from intracellular stores and to reduce the noradrenaline-stimulated increase in IP, production (It0 et al, 1991). Since IP, is the second messenger which mobilizes Ca2+from intracellular stores, it is reasonable to assume that the latter is the cause of the former. The inhibitory effect of BRL 38277 on agonist-induced IP, production is blocked by glibenclamide and greatly reduced in 128 mM KCl, suggesting that it may be linked to
Id l b1 II
Relllllng of store
Ca”, Na’ Fig 3. Ways in which the KCOs interfere with excitationcontraction coupling in the smooth muscle cell. When a contractile agonist binds to its receptor on the smooth muscle cell membrane, phosholipase C is activated, leading to the formation of IP, (inositol 1, 4, 5 triphophate), which, in turn, releases Ca+ from intracellular stores. In addition, the activated receptor opens receptor-operated channels (ROCs) allowing ion movements (influx of Na+ and Ca2+ o r efflux of CI-) which depolarize the cell and thus lead to Ca*+ entry via voltage-operated Ca2+ channels (VOCs). Opening of K+ channels by the KCOs leads to hyperpolarization which prevents the opening of VOCs. In addition, the KCOs inhibit agonist-induced IP, production so that Ca+ cannot be released and inhibit refilling of the stores. Other mechanisms (eg inhibition of ROCs etc) are still entirely speculative.
Potassium channel openers
the plasmalemma-hyperpolarizing effect of the KCO (Ito et al, 1991). In addition to this, evidence has been presented that cromakalim is able to interfere with the refilling of intracellular Ca2+ stores (Bray et al, 199 1). This will finally result in store depletion and thereby (indirectly) impair the ability of an agonist to release Ca+ from such stores (fig 3 ) . Other possibilities (eg the inhibition of Ca2+ entry via dihydropyridine-insensitive voltage- sensitive calcium channels or receptor-operated/second messenger-operated cation channels (ROCs, fig 3)) are entirely hypothetical at present (Cook and Quast, 1990).
Quast, 1992). Experiments at the single channel level show clearly that the KCOs (in particular cromakalim) are able to open different K+ channels i n vascular smooth muscle, i n particular various types of ATP- and glibenclamide-sensitive K+ channels (Weston and Edwards, 1991; Weston and Edwards, 1992; Quast, 1992). Recently, a novel small conductance (15 pS) K+ channel which is dependent on GDP for opening and which is blocked by ATP and glibenclamide has been shown to be opened by pinacidil (Kajioka et a l , 1991). As discussed above, there is ample evidence that the KCOs open K,, in heart, brain, skeletal muscle and pancreatic pcells.
Dependence on an intact endothelium In vitro, the vasodilator effects of the KCOs are not dependent on the presence of an intact endothelium ( s e e e g Cook e t a l , 1988b). In the conscious dog, the dilator effect of cromakalim and pinacidil in a large coronary artery (A circumflexa) but not in arterioles was found to be largely flow-dependent (Giudicelli et a l , 1990). After endothelial denudation, the dilator effect of the KCOs in the large coronary (but not in coronary arterioles) was abolished, whereas the effects of nicorandil and hydralazine were unchanged (Drieu la Rochelle et al, 1992). This clearly shows the involvement of the coronary endothelium in the vasodilator effect of cromakalim and pinacidil in the large coronary artery. With regard to the mechanism of this effect, it is noted that the KCos enhance Ca2+ influx into endothelial cells (possibly by opening an intermediate conductance Ca2+dependent voltage-independent K+ channel), thus promoting the Ca2+-dependent formation of endothelium-derived relaxing factor (Liickhoff and Busse, 1990).
Binding studies In view of the structural diversity of the KCOs (fig 2) one will immediately ask the question whether the different families of compounds open the same channel and, if so, whether they bind to the same site of the channel. Obviously, a binding assay for the KCOs in smooth muscle would be the method of choice to answer this question; however, no such assay has yet been described. By synthesizing a tritiated derivative of the pinacidil analogue P1075 (fig 2) we have recently been able to identify a specific binding site for the KCOs in intact rings of rat aorta (Bray and Quast, 1992b). The site labelled by 3H-P 1075 recognizes binding of the different families of KCOs in a 'pharmacologically relevant manner, thus providing evidence that the different KCOs bind to the same target (although not necessarily to the same site of this target). The blocker glibenclamide binds to a site negatively allosterically coupled to a site(s) for the openers (Bray and Quast, 1992b). It is hoped that studies of this type will also allow the biochemical characterization of the target of the KCOs.
Nature of the K + channel opened by the KCOs in vascular smooth muscle A detailed discussion of this still controversial subject is beyond the scope of this review; the reader is referred to recent reviews for this matter ( E d w a r d s and Weston, 1990; Weston and Edwards, 1991; Weston and Edwards, 1992;
Therapeutic potential of the KCOs The existing KCOs (with the exception of diazoxide) show some specificity for vascular smooth muscle and peripheral nerves (eg in the airways); central effects may be hampered by poor penetration of the blood brain barrier. Accordingly, the
major applications appear to be in the cardiovascular field and in asthma. Cardiovascular applications Hypertension. The KCOs are effective in controlling high blood pressure (reviewed in Edwards and Weston, 1990; Richer et al, 1990; Robertson and Steinberg, 1990; Anderson, 1992). As with other peripheral vasodilators, acute studies show (in addition to flush and headache) reflex tachycardia, increase in the plasma levels of noradrenaline and renin, and fluid retention. Co-administration of angiotensin converting enzyme inhibitors, diuretics or Padrenoceptor blockers may be beneficial. Levcromakalim and another benzpyran KCO from Yoshitomi are currently in development as antihypertensives in Japan. Zschemias. In a rat model of occlusive arterial disease (intermittant claudication), the KCOs cromakalim, pinacidil and nicorandil (in contrast to calcium antagonists or hydralazine) selectively enhance blood cell flux and the PO, in collateralized hypoxic skeletal muscle (Angersbach and Nicholson, 1988). In addition, in collaterized rat hind limbs, they accelerate the recovery of high energy phosphates after a period of transient is-chemia at doses lower than those that lower blood pressure (Cook et al, 1992). A recent study in the isolated rabbit ear has shown that levcromakalim (but not verapamil or sodium nitroprusside) selectively improve collateral perfusion following arterial occlusion (Randall and Griffith, 1992). Cardioprotection. The beneficial effects of KCOs in the ischemic myocardium (Gross, 1991; Grover, 1991; Escande and Cavero, 1992) have been discussed above. A very interesting point is the similarity of the cardioprotective effect of some KCOs with the protection against infarction afforded by ‘myocardial preconditioning’ (Escande and Cavero, 1992). The preconditioning phenomenon denotes the protective effect of a short period of ischemia followed by reperfusion against the cardiac damage caused by subsequent prolonged ischemia (review Murry et al, 1991). Preconditioning
seems to be mediated by adenosine released during ischemia (Liu et al, 1991) and is abolished by pretreatment with glibenclamide, suggesting that K,, may be involved (Gross and Auchampach, 1992). Electrophysiological studies show that under conditions of reduced intracellular ATP (as occurs in ischemia), adenosine indirectly opens K,, in ventricular myocytes via binding to an adenosine A , receptor and activation of a G,-protein (Kirsch et al, 1990). The KCOs may thus be viewed as exogenous ‘preconditioners’ which help the heart to survive during limited periods of ischemia. There is, however, controversy over the doses needed to elicit these cardiac effects in comparison to blood pressure lowering doses (Gross, 1991; Grover, 199 1). For the therapeutic exploitation of the KCOs as cardioprotective agents, it is important that the cardioprotective effects occur at doses lower than those that induce generalized vasodilation. High doses of KCOs induce a redistribution of blood flow from inner to outer layers of the heart (Hof et a / , 1988) which may lead to subendocardial necroses upon prolonged treatment. Angina pectoris. The ultimate usefulness of the KCOs in this indication depends on their ability to improve perfusion of the ischemic area. To this end, it seems important that they dilate the large coronaries with atherosclerotic lesions more than resistance vessels in order to avoid steel phenomena and that they do not induce tachycardia. The fact that the vasodilator effect of cromakalim and pinacidil in large coronary arteries depends critically on an intact endothelium (Drieu la Rochelle et al, 1992). may cast some doubt on the value of the KCOs in the treatment of angina pectoris. I n addition, minoxidil is known for its propensity to aggravate ischemic symptoms in susceptible patients even after topical application (Leenen et al, 1988). On the other hand, it has been shown that the hypoxic dilation of coronary arteries is mediated by the opening of K,, (Daut et al, 1990). This may be mediated, at least in part, via the release of adenosine (Daut et a / , 1990) so that the KCOs may again exploit an endogenous mechanism in dilating the coronaries.
Potassium channel openers
Antiarrhythmic properties. The action potential
duration shortening action of the KCOs may be considered as a pro-arrhythmic effect. However, a case may also be made for the opposite, since cromakalim can inhibit abnormal automaticity i n canine Purkinje fibers (McCullough et al, 1990). This issue requires more work and careful consideration (Grover, 199 1 ). Non-cardiovascular indications Asthma. Clinical trials have shown that cromaka-
lim alleviates the drop in early morning lung function in patients with nocturnal asthma at a dose of 0.5 mg orally per day (Williams et al, 1990). This is lower than typical antihypertensive doses; however the KCOs are more potent relaxants in vascular than in airway smooth muscle (Cook and Quast, 1990). This discrepancy suggests that the presynaptic effects of cromakalim on nerves may be more important clinically than direct bronchodilator effects, thus corroborating animal studies. The pivotal question is whether the tissue specificity of the existing KCOs (nerves vs arterioles) is sufficient to provide protection against asthma attacks without cardiovascular side effects (eg tachycardia, headache, etc); problems encountered in a similar manner with Padrenoceptor agonist therapy. Urogenital tract and miscellaneous. Applications of the KCOs have been proposed in urinary incontinence (due to unstable bladder, eg after outflow obstruction (Malmgren et al, 1989)) and in penile erectile dysfunction (Holmquist e f a l , 1990). Again, the question of tissue selectivity is the cmcia1 one; the same applies to indications as divergent as male pattern baldness (androgenic alopecia), gastrointestinal disorders and certain skeletal muscle myopathies.
Concluding remarks Despite considerable progress in the field of KCOs, major questions remain controversial (eg the type(s) of K+ channels opened by these compounds in vascular smooth muscle, intracellular vs plasmalemmal sites of action, the multiplicity of drug receptor sites
for the different families of KCOs, etc). The recognition that the KCOs also have presynaptic sites of action and, after intracerebroventricular administration or in brain slices, elicit interesting neuronal effects, gives a new twist to the story of these 'smooth muscle relaxants'. For the therapeutic application of the KCOs in the very different indications discussed above, the problem of (tissue) selectivity is a crucial one and, considering the KCOs actually known, there seems to be ample room for improvement.
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