Elsevier

Neuropharmacology

Volume 69, June 2013, Pages 62-74
Neuropharmacology

Invited review
Cation-chloride cotransporters NKCC1 and KCC2 as potential targets for novel antiepileptic and antiepileptogenic treatments

https://doi.org/10.1016/j.neuropharm.2012.05.045Get rights and content

Abstract

In cortical and hippocampal neurons, cation-chloride cotransporters (CCCs) control the reversal potential (EGABA) of GABAA receptor-mediated current and voltage responses and, consequently, they modulate the efficacy of GABAergic inhibition. Two members of the CCC family, KCC2 (the major neuron-specific K–Cl cotransporter; KCC isoform 2) and NKCC1 (the Na–K–2Cl cotransporter isoform 1 which is expressed in both neurons and glial cells) have attracted much interest in studies on GABAergic signaling under both normal and pathophysiological conditions, such as epilepsy. There is tentative evidence that loop diuretic compounds such as furosemide and bumetanide may have clinically relevant antiepileptic actions, especially when administered in combination with conventional GABA-mimetic drugs such as phenobarbital. Furosemide is a non-selective inhibitor of CCCs while at low concentrations bumetanide is selective for NKCCs. Search for novel antiepileptic drugs (AEDs) is highly motivated especially for the treatment of neonatal seizures which are often resistant to, or even aggravated by conventional AEDs. This review shows that the antiepileptic effects of loop diuretics described in the pertinent literature are based on widely heterogeneous mechanisms ranging from actions on both neuronal NKCC1 and KCC2 to modulation of the brain extracellular volume fraction. A promising strategy for the development of novel CCC-blocking AEDs is based on prodrugs that are activated following their passage across the blood–brain barrier.

This article is part of the Special Issue entitled ‘New Targets and Approaches to the Treatment of Epilepsy’.

Highlights

► Cation-chloride cotransporters (CCCs) modulate the efficacy of GABAergic inhibition. ► Two members of the CCC family, KCC2 and NKCC1, are of particular importance. ► Both transporters are potential targets for novel antiepileptic and antiepileptogenic treatments. ► The NKCC1 inhibitor bumetanide has been widely studied in this respect. ► This review critically discusses the pros and cons of CCC blockers for epilepsy therapy.

Introduction

Epilepsy, one of the most common disorders of the brain, is characterized by recurrent, usually unprovoked epileptic seizures, and there is a wide spectrum of cognitive, psychosocial, and social consequences of this condition. Ictogenesis, the rapid process of initiation and propagation of a seizure in time and space, is symptomatic of the underlying pathology.

A variety of genetic and developmental abnormalities as well as brain insults, including traumatic brain injury, ischemic stroke, intracerebral hemorrhage, status epilepticus (SE) and encephalitis, have the potential to induce the development of epilepsy in humans and rodent disease models (Cepeda et al., 2006; Chang and Lowenstein, 2003; Löscher and Brandt, 2010; Pitkänen and Lukasiuk, 2009). The mechanisms underlying this gradual process, termed epileptogenesis, whereby brain is altered becoming susceptible to spontaneous recurrent seizures, are only incompletely understood, but they include inflammation, neurodegeneration, blood–brain barrier (BBB) disruption, and alterations in expression and function of diverse receptors and ion channels (Dichter, 2009; Heron et al., 2007; Löscher and Brandt, 2010; Pitkänen and Lukasiuk, 2009; Timofeev et al., 2010).

During the recent years, changes in plasmalemmal ion-transport mechanisms, which are instrumental for ion channel function and volume regulation, have been suggested to play pivotal roles in epileptic processes (Blaesse et al., 2009).

Seizures occur more often during the neonatal period than at any other age (Annegers et al., 1995; Hauser et al., 1993). Neonatal seizures are deleterious events and they can have profound long-term consequences, often leading to chronic epilepsy and significant cognitive and motor disabilities (Rakhade and Jensen, 2009). A well-recognized, major clinical problem is that neonatal epileptic seizure activity shows only limited response to commonly used GABA-mimetic and GABA-modulating antiepileptic drugs (AEDs; also known as anticonvulsant, anti-ictal or anti-seizure drugs) such as phenobarbital and benzodiazepines (Bonifacio et al., 2011; Painter et al., 1999; Rennie and Boylan, 2007), which enhance the inhibitory actions mediated by GABAA receptors in adults (Rogawski and Löscher, 2004). AEDs used in the treatment of epilepsy in adults are often ineffective in neonates and can even potentiate seizure episodes. Hence, the search for novel AEDs and other therapeutic strategies (Helmy et al., 2011) is particularly important in the case of neonatal seizures. In fact, with regard to neonatal and pediatric seizures, controlling neuronal pHi might turn out to be a very successful way to manipulate neuronal excitability (Helmy et al., 2011; Schuchmann et al., 2006). A decrease in pHi of 0.05 units was induced in a quantitatively identical manner by various membrane-permeant weak organic acids as well as isohydric hypercapnia, and the intraneuronal acidosis had a pronounced suppressing action on neuronal network activity (Ruusuvuori et al., 2010). This effect has nothing to do with mitochondrial energy metabolism (see Holmgren et al., 2010; and Bregestovski and Bernard, 2012) as is evident from the fact that quantitatively similar suppression of giant depolarizing potential (GDP) generation took place regardless of whether the weak acid applied was (l-lactate) or was not (d-lactate, propionate) an effective substrate of mitochondrial ATP production (Ruusuvuori et al., 2010). Moreover, CO2 is an end-product of energy metabolism. Mitochondrial membrane potential within the neurons was not affected by any of these weak acids, but a strong dependence on the availability of glucose was evident (Ruusuvuori et al., 2010).

About 30% of adult patients with epilepsy do not respond to currently used AEDs. Such AED resistance is associated with serious comorbidity and increased mortality, warranting an urgent need for more effective AEDs (Löscher and Schmidt, 2011). In addition, prevention of epilepsy in patients at risk after brain insults remains an unmet medical need, therefore, development of drugs that target the mechanisms of epileptogenesis is important (Löscher and Brandt, 2010; Pitkänen and Lukasiuk, 2009). A modest acid load has been shown to be effective in suppressing drug-resistant seizures in adults (Tolner et al., 2011). However, we will focus below on drugs acting on Cl regulation in the control of neonatal, pediatric and adult seizures.

Recently, inhibitors of plasmalemmal cation-chloride cotransporters (CCCs) such as furosemide and, especially, bumetanide have attracted much interest as putative AEDs as will be described in detail in this review. Considering the multitude of overlapping mechanisms and endpoints of major central nervous system (CNS) diseases, it is perhaps not surprising that the above drugs have been also implicated in the treatment of conditions such as cerebral edema and swelling-related neurodegeneration after ischemic and traumatic brain injury (Kahle et al., 2008; Kintner et al., 2007; Walcott et al., 2012), chronic pain (Price et al., 2005), as well as autism (Lemonnier and Ben-Ari, 2010) and conditioned anxiety (Krystal et al., 2012).

Section snippets

Physiology and pathophysiology of cation-chloride cotransporters in the brain

The electrochemical gradient of Cl across the neuronal plasma membrane is controlled by CCCs (for review, see Blaesse et al., 2009). CCCs are secondary active transporters that drive net Cl extrusion (K–Cl cotransporters, KCCs) or uptake (Na–K–2Cl cotransporters, NKCCs) by using the K+ and Na+ gradients which, in turn, are generated by the Na–K ATPase (the “sodium pump”). The neuron-specific KCC isoform, KCC2, is the main Cl extruding mechanism in hippocampal and neocortical principal

Basic mechanisms of GABAergic inhibition: hyperpolarization and shunting

GABAA-mediated transmission is not globally suppressed in epileptic tissue (Avoli et al., 2005; Avoli and de Curtis, 2011; Köhling et al., 1998; Mann and Mody, 2008), but the neuronal Cl extrusion capacity, which is an important factor in controlling the efficacy of postsynaptic inhibition (see below) may play a role in setting the susceptibility of neurons to epileptiform activity, at least under certain conditions (Ben-Ari et al., 2007; Blaesse et al., 2009; Briggs and Galanopoulou, 2011).

Pharmacological targeting of NKCC1 and KCC2

Based on the assumption that abnormal functional expression of KCC2 and NKCC1 might promote ictal activity (Kahle et al., 2008), these Cl transporters have been suggested as targets for antiepileptic and antiepileptogenic treatments. As will be explained below, pharmacological inhibition of not only NKCC1 but also of KCC2 in central neurons might have antiepileptic effects.

Epileptogenesis in neonates

Most of the available data does not suggest that GABA would be overtly depolarizing in the healthy newborn human cortex (Section 2.). However, the situation might be different in preterm babies as well as in full term neonates with epilepsy. Neonatal epilepsy is often caused by birth trauma, such as asphyxia or hemorrhages (Ronen et al., 1999), which is expected to lead to neuronal damage and de-differentiation and, consequently, to a high neuronal [Cl]i.

Recent experimental work by the groups

Ictogenesis in neonates

As discussed above, the depolarizing actions of GABA in immature neurons are a consequence of the high [Cl]i. In theory, inhibition of NKCC1, by reducing [Cl]i, could reduce GABA-mediated excitation or even potentiate the well-known inhibitory modes of GABA signaling in neonatal neurons (see Section 3.; and Lamsa et al., 2000; Wells et al., 2000).

Dzhala et al. (2005) reported that bumetanide (0.1–0.2 mg/kg i.p.) attenuated kainate-induced electrographic seizures in neonatal rats (but see

Conclusions

The work on the antiepileptic and antiepileptogenic effects of NKCC1 and KCC2 inhibitors have revealed at least four distinct mechanisms of action which may or may not show overlap under various experimental and clinical conditions.

  • (1)

    The most common working hypothesis in studies of diuretic agents on epilepsy posits that a major epilepsy-promoting mechanism is an NKCC1-mediated increase in neuronal [Cli] which diminishes the efficacy of GABAergic inhibition or even reverts it to excitation. A

Acknowledgments

We thank Daryl Hochman for helpful discussion and for providing details on his monkey experiments, as well as Peter Blaesse, Mohamed Helmy and Eva Ruusuvuori for comments on an early version of this paper.

The authors' original research work was supported by grants from the Letten Foundation, the Academy of Finland, the Sigrid Juselius Foundation, the Jane and Aatos Erkko Foundation (K.K.) and the Deutsche Forschungsgemeinschaft (Bonn, Germany; Lo 274/11; W.L.). K.K. is a member of the Finnish

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