Cotransporter Blocker Research Articles

Exacerbation of Epilepsy by Astrocyte Alkalization and Gap Junction Uncoupling.

Seizures invite seizures. At the initial stage of epilepsy, seizures intensify with each episode; however, the mechanisms underlying this exacerbation remain to be solved. Astrocytes have a strong control over neuronal excitability and the mode of information processing. This control is accomplished by adjusting the levels of various ions in the extracellular space. The network of astrocytes connected via gap junctions allows a wider or more confined distribution of these ions depending on the open probability of the gap junctions. K+ clearance relies on the K+ uptake by astrocytes and the subsequent diffusion of K+ through the astrocyte network. When astrocytes become uncoupled, K+ clearance becomes hindered. Accumulation of extracellular K+ leads to hyperexcitability of neurons. Here, using acute hippocampal slices from mice, we uncovered that brief periods of epileptiform activity result in gap junction uncoupling. In slices that experienced short-term epileptiform activity, extracellular K+ transients in response to glutamate became prolonged. Na+ imaging with a fluorescent indicator indicated that intercellular diffusion of small cations in the astrocytic syncytium via gap junctions became rapidly restricted after epileptiform activity. Using a transgenic mouse with astrocyte-specific expression of a pH sensor (Lck-E2GFP), we confirmed that astrocytes react to epileptiform activity with intracellular alkalization. Application of Na+/HCO3- cotransporter blocker led to the suppression of intracellular alkalization of astrocytes and to the prevention of astrocyte uncoupling and hyperactivity intensification both in vitro and in vivo Therefore, the inhibition of astrocyte alkalization could become a promising therapeutic strategy for countering epilepsy development.SIGNIFICANCE STATEMENT We aimed to understand the mechanisms underlying the plastic change of forebrain circuits associated with the intensification of epilepsy. Here, we demonstrate that first-time exposure to only brief periods of epileptiform activity results in acute disturbance of the intercellular astrocyte network formed by gap junctions in hippocampal tissue slices from mice. Moreover, rapid clearance of K+ from the extracellular space was impaired. Epileptiform activity activated inward Na+/HCO3- cotransport in astrocytes by cell depolarization, resulting in their alkalization. Our data suggest that alkaline pH shifts in astrocytes lead to gap junction uncoupling, hampering K+ clearance, and thereby to exacerbation of epilepsy. Pharmacological intervention could become a promising new strategy to dampen neuronal hyperexcitability and epileptogenesis.

Renal Dysfunction Induced by Kidney-Specific Gene Deletion of Hsd11b2 as a Primary Cause of Salt-Dependent Hypertension.

Genome-wide analysis of renal sodium-transporting system has identified specific variations of Mendelian hypertensive disorders, including HSD11B2 gene variants in apparent mineralocorticoid excess. However, these genetic variations in extrarenal tissue can be involved in developing hypertension, as demonstrated in former studies using global and brain-specific Hsd11b2 knockout rodents. To re-examine the importance of renal dysfunction on developing hypertension, we generated kidney-specific Hsd11b2 knockout mice. The knockout mice exhibited systemic hypertension, which was abolished by reducing salt intake, suggesting its salt-dependency. In addition, we detected an increase in renal membrane expressions of cleaved epithelial sodium channel-α and T53-phosphorylated Na+-Cl- cotransporter in the knockout mice. Acute intraperitoneal administration of amiloride-induced natriuresis and increased urinary sodium/potassium ratio more in the knockout mice compared with those in the wild-type control mice. Chronic administration of amiloride and high-KCl diet significantly decreased mean blood pressure in the knockout mice, which was accompanied with the correction of hypokalemia and the resultant decrease in Na+-Cl- cotransporter phosphorylation. Accordingly, a Na+-Cl- cotransporter blocker hydrochlorothiazide significantly decreased mean blood pressure in the knockout mice. Chronic administration of mineralocorticoid receptor antagonist spironolactone significantly decreased mean blood pressure of the knockout mice along with downregulation of cleaved epithelial sodium channel-α and phosphorylated Na+-Cl- cotransporter expression in the knockout kidney. Our data suggest that kidney-specific deficiency of 11β-HSD2 leads to salt-dependent hypertension, which is attributed to mineralocorticoid receptor-epithelial sodium channel-Na+-Cl- cotransporter activation in the kidney, and provides evidence that renal dysfunction is essential for developing the phenotype of apparent mineralocorticoid excess.

Huqi San-Evoked Rat Colonic Anion Secretion through Increasing CFTR Expression.

Huqi San (HQS) is a Chinese herbal preparation of eight medicinal herbs that promote diuresis, detoxification, blood circulation, and cholestasis. Defects in transporter expression and function can cause cholestasis and jaundice. However, the mechanism of the cholestasis underlying HQS effects, especially on the gastrointestinal tract ion secretion, has not been elucidated. Real-time RT-PCR and Western blotting were used to study the expression and localization of cystic fibrosis transmembrane conductance regulator (CFTR) and α-ENaC in rat alimentary tract, and then the effect of HQS on the ion transport in rat distal colon mucosa was investigated using the short-circuit current (I SC) technique. The results showed that pretreatment with HQS significantly enhanced mRNA transcripts and protein content of CFTR in liver and distal colon but not α-ENaC in alimentary organs. HQS increases I SC and decreases the transepithelial resistance. Pretreatment with epithelial Na+ channel blocker did not affect the I SC responses elicited by HQS, but removal of extracellular Cl− or pretreatment with Cl− channel or Na+-K+-2Cl− cotransporter blocker inhibited HQS-elicited I SC responses. These findings demonstrated that HQS, RA, and RP can stimulate Cl− secretion in the distal colon by increasing the mRNA transcripts and protein content of CFTR in liver and distal colon.

Acute and chronic efficacy of bumetanide in an in vitro model of posttraumatic epileptogenesis.

Seizures triggered by acute injuries to the developing brain respond poorly to first-line medications that target the inhibitory chloride-permeable GABAA receptor. Neuronal injury is associated with profound increases in cytoplasmic chloride ([Cl(-)]i) resulting in depolarizing GABA signaling, higher seizure propensity and limited efficacy of GABAergic anticonvulsants. The Na(+)-K(+)-2Cl(-) (NKCC1) cotransporter blocker bumetanide reduces [Cl(-)]i and causes more negative GABA equilibrium potential in injured neurons. We therefore tested both the acute and chronic efficacy of bumetanide on early posttraumatic ictal-like epileptiform discharges and epileptogenesis. Acute hippocampal slices were used as a model of severe traumatic brain injury and posttraumatic epileptogenesis. Hippocampal slices were then incubated for 3 weeks. After a 1-week latent period, slice cultures developed chronic spontaneous ictal-like discharges. The anticonvulsant and anti-epileptogenic efficacy of bumetanide, phenobarbital, and the combination of these drugs was studied. Bumetanide reduced the frequency and power of early posttraumatic ictal-like discharges in vitro and enhanced the anticonvulsant efficacy of phenobarbital. Continuous 2-3 weeks administration of bumetanide as well as phenobarbital in combination with bumetanide failed to prevent posttraumatic ictal-like discharges and epileptogenesis. Our data demonstrate a persistent contribution of NKCC1 cotransport in posttraumatic ictal-like activity, presumably as a consequence of chronic alterations in neuronal chloride homeostasis and GABA-mediated inhibition. New strategies for more effective reduction in posttraumatic and seizure-induced [Cl(-)]i accumulation could provide the basis for effective treatments for posttraumatic epileptogenesis and the resultant seizures.

Chloride channel blockade relaxes airway smooth muscle and potentiates relaxation by β-agonists.

Severe bronchospasm refractory to β-agonists continues to cause significant morbidity and mortality in asthmatic patients. We questioned whether chloride channels/transporters are novel targets for the relaxation of airway smooth muscle (ASM). We have screened a library of compounds, derivatives of anthranilic and indanyloxyacetic acid, that were originally developed to antagonize chloride channels in the kidney. We hypothesized that members of this library would be novel calcium-activated chloride channel blockers for the airway. The initial screen of this compound library identified 4 of 20 compounds that relaxed a tetraethylammonium chloride-induced contraction in guinea pig tracheal rings. The two most effective compounds, compounds 1 and 13, were further studied for their potential to either prevent the initiation of or relax the maintenance phase of an acetylcholine (ACh)-induced contraction or to potentiate β-agonist-mediated relaxation. Both relaxed an established ACh-induced contraction in human and guinea pig ex vivo ASM. In contrast, the prevention of an ACh-induced contraction required copretreatment with the sodium-potassium-chloride cotransporter blocker bumetanide. The combination of compound 13 and bumetanide also potentiated relaxation by the β-agonist isoproterenol in guinea pig tracheal rings. Compounds 1 and 13 hyperpolarized the plasma cell membrane of human ASM cells and blocked spontaneous transient inward currents, a measure of chloride currents in these cells. These functional and electrophysiological data suggest that modulating ASM chloride flux is a novel therapeutic target in asthma and other bronchoconstrictive diseases.

Body Fluid Expansion in Acromegaly Is Related to Enhanced Epithelial Sodium Channel (ENaC) Activity

Soft-tissue swelling and increased extracellular volume, two features of acromegaly, are related to the antinatriuretic effects of excess GH/IGF-I, but the precise pathophysiological mechanism is unclear. Our objective was to determine the effect of the GH excess on renal and extrarenal epithelial sodium channel (ENaC) activity. We conducted a prospective randomized open-label blinded-endpoint (PROBE) crossover study (www.ClinicalTrials.gov Identifier: NCT00531908) at a tertiary referral medical center and clinical investigation center. Sixteen patients (five females, 11 males) with acromegaly were randomly assigned to receive 20 mg amiloride (an ENaC blocker) and 25 mg furosemide (a Na-K-2Cl cotransporter blocker) under a high-sodium diet to suppress endogenous renin and aldosterone. Diuretic-induced changes in the urinary Na/K ratio (reflecting coupling between ENaC-mediated Na reabsorption and distal K secretion) and the intranasal amiloride-sensitive potential (reflecting extrarenal ENaC activity) were measured before and 6 months after (range, 1-12 months) treatment of acromegaly. Serum IGF-I concentrations normalized in all the patients after treatment of acromegaly. Baseline plasma renin and aldosterone concentrations remained unchanged after treatment. Active acromegaly, compared with controlled disease, was associated with an enhanced response [median (interquartile range)] to amiloride [urinary Na/K, 13.9 (9.8-19.5) vs. 6.3 (4.3-8.4) mmol/mmol, P = 0.0003], a reduced response to furosemide [urinary Na/K, 5.2 (4.6-7.2) vs. 7.1 (5.4-8.8) mmol/mmol, P =0.0151], and an increased intranasal amiloride-sensitive potential [5.8 (11.9-3.8) vs. 4.2 (6.4-2.1) mV, P = 0.031], respectively. GH/IGF-I excess in humans is associated with enhanced renal and extrarenal ENaC activity that may contribute to soft-tissue swelling and volume expansion in acromegaly.

Implications for glycine receptors and astrocytes in ethanol-induced elevation of dopamine levels in the nucleus accumbens

Elevated dopamine levels are believed to contribute to the rewarding sensation of ethanol (EtOH), and previous research has shown that strychnine-sensitive glycine receptors in the nucleus accumbens (nAc) are involved in regulating dopamine release and in mediating the reinforcing effects of EtOH. Furthermore, the osmoregulator taurine, which is released from astrocytes treated with EtOH, can act as an endogenous ligand for the glycine receptor, and increase extracellular dopamine levels. The aim of this study was to address if EtOH-induced swelling of astrocytes could contribute to elevated dopamine levels by increasing the extracellular concentration of taurine. Cell swelling was estimated by optical sectioning of fluorescently labeled astrocytes in primary cultures from rat, and showed that EtOH (25-150 mM) increased astrocyte cell volumes in a concentration- and ion-dependent manner. The EtOH-induced cell swelling was inhibited in cultures treated with the Na(+) /K(+) /2Cl⁻ cotransporter blocker furosemide (1 mM), Na(+) /K(+) -ATPase inhibitor ouabain (0.1 mM), potassium channel inhibitor BaCl₂ (50 µM) and in cultures containing low extracellular sodium concentration (3 mM). In vivo microdialysis performed in the nAc of awake and freely moving rats showed that local treatment with EtOH enhanced the concentrations of dopamine and taurine in the microdialysate, while glycine and β-alanine levels were not significantly modulated. EtOH-induced dopamine release was antagonized by local treatment with the glycine receptor antagonist strychnine (20 µM) or furosemide (100 µM or 1 mM). Furosemide also prevented EtOH-induced taurine release in the nAc. In conclusion, our data suggest that extracellular concentrations of dopamine and taurine are interconnected and that swelling of astrocytes contributes to the acute rewarding sensation of EtOH.

Progressive NKCC1-Dependent Neuronal Chloride Accumulation during Neonatal Seizures

Seizures induce excitatory shifts in the reversal potential for GABA(A)-receptor-mediated responses, which may contribute to the intractability of electro-encephalographic seizures and preclude the efficacy of widely used GABAergic anticonvulsants such as phenobarbital. We now report that, in intact hippocampi prepared from neonatal rats and transgenic mice expressing Clomeleon, recurrent seizures progressively increase the intracellular chloride concentration ([Cl(-)](i)) assayed by Clomeleon imaging and invert the net effect of GABA(A) receptor activation from inhibition to excitation assayed by the frequency of action potentials and intracellular Ca(2+) transients. These changes correlate with increasing frequency of seizure-like events and reduction in phenobarbital efficacy. The Na(+)-K(+)-2Cl(-) (NKCC1) cotransporter blocker bumetanide inhibited seizure-induced neuronal Cl(-) accumulation and the consequent facilitation of recurrent seizures. Our results demonstrate a novel mechanism by which seizure activity leads to [Cl(-)](i) accumulation, thereby increasing the probability of subsequent seizures. This provides a potential mechanism for the early crescendo phase of neonatal seizures.

Functional identification of an outwardly rectifying pH‐ and anesthetic‐sensitive leak K+ conductance in hippocampal astrocytes

Astrocytes function as spatial K(+) buffers by expressing a rich repertoire of K(+) channels. Earlier studies suggest that acid-sensitive tandem-pore K(+) channels, mainly TWIK-related acid-sensitive K(+) (TASK) channels, mediate part of the passive astroglial membrane conductance. Here, using a combination of electrophysiology and pharmacology, we investigated the presence of TASK-like conductance in hippocampal astrocytes of rat brain slices. Extracellular pH shifts to below 7.4 (or above 7.4) induced a prominent inward (or outward) current in astrocytes in the presence of tetrodotoxin, a Na(+) channel blocker, and 4,4'-diisothiocyanatostilbene-2,2'-disulfonate, a co-transporter blocker. The pH-sensitive current was insensitive to quinine, a potent blocker of tandem-pore K(+) channels including TWIK-1 and TREK-1 channels. Voltage-clamp analysis revealed that the pH-sensitive current exhibited weak outward rectification with a reversal potential of -112 mV, close to the Nernst equilibrium potential for K(+) . Furthermore, the current-voltage relationship was well fitted with the Goldman-Hodgkin-Katz current equation for the classical open-rectifier 'leak' K(+) channel. The pH-sensitive K(+) current was potentiated by TASK channel modulators such as the volatile anesthetic isoflurane but depressed by the local anesthetic bupivacaine. However, unlike TASK channels, the pH-sensitive current was insensitive to Ba(2+) and quinine. Thus, the molecular identity of the pH-sensitive leak K(+) channel is unlikely to be attributable to TASK channels. Taken together, our results suggest a novel yet unknown leak K(+) channel underlying the pH- and anesthetic-sensitive background conductance in hippocampal astrocytes.

Oxytocin is neuroprotective against oxygen–glucose deprivation and reoxygenation in immature hippocampal cultures

Oxytocin triggers an excitatory-to-inhibitory switch in GABA (γ-aminobutyric acid) actions in immature neurons and this was found to increase their resistance to anoxic episodes. In this study we examined the neuroprotective effect of oxytocin on immature hippocampal cultures subjected to oxygen–glucose deprivation (OGD) both immediately after the insult, as well as after 6 h of reoxygenation. We measured metabolic activity fluorometrically using resazurin and found that cellular viability was increased in the oxytocin treated group both immediately after OGD, as well as after 6 h of reoxygenation. While the oxytocin receptor antagonist atosiban blocked the effect of oxytocin, the Na +–K +–2Cl − cotransporter (NKCC1) blocker bumetanide protected neurons after reoxygenation. The effects of oxytocin are dose-related. Our results suggest that oxytocin exerts a prolonged neuroprotective action on fetal neurons. Perinatal pharmacologic manipulation of oxytocin receptors may have detrimental effects by increasing susceptibility of the fetal brain to hypoxic-ischemic insults.

The multi‐herbal drug STW 5 (Iberogast®) has prosecretory action in the human intestine

There is growing evidence that STW 5 (Iberogast), fixed combination of hydroethanolic herbal extracts), besides being effective in functional dyspepsia, also improves symptoms in irritable bowel syndrome (IBS). Clinical data indicate that modulation of mucosal secretion is a promising approach to treat intestinal disorders associated with IBS. We therefore explored the effect of STW 5 on secretion in the human intestine and the mechanisms by which it acts. The Ussing chamber technique was used to measure mucosal secretion in human intestinal mucosa/submucosa preparations and in human epithelial cell line T84. In addition, we recorded STW 5 effects on human enteric neurons with voltage sensitive dye imaging. In human tissue and T84 cells STW 5 induced a dose-dependent increase in ion secretion that was significantly reduced by the Na-K-Cl cotransporter blocker bumetanide, the adenylate cyclase inhibitor MDL-12 330, the non-specific and selective cystic fibrosis transmembrane conductance regulator (CFTR) inhibitors glibenclamide and CFTR(inh)-172, respectively, and the blocker of calcium dependent Cl(-) channels (ClCa) SITS (4-acetamido-4-isothiocyanatostilbene-2,2-disulphonic acid). It was unaffected by amiloride, a blocker of epithelial Na(+) channels. In human tissue, the nerve blocker tetrodotoxin significantly suppressed the STW 5 response. STW 5 evoked an increased spike discharge in 51% of human submucous neurons. Results suggest that STW 5 is a secretogogue in the human intestine by direct epithelial actions and through activation of enteric neurons. The prosecretory effect is due to increased epithelial Cl(-) fluxes via CFTR and Ca-dependent ClCa channels. STW 5 may be a novel option to treat secretory disorders associated with IBS and constipation.

GABAergic signaling induces divergent neuronal Ca2+ responses in the suprachiasmatic nucleus network

Intercellular communication between gamma-aminobutyric acid (GABA)ergic suprachiasmatic nucleus (SCN) neurons facilitates light-induced phase changes and synchronization of individual neural oscillators within the SCN network. We used ratiometric Ca(2+) imaging techniques to record changes in the intracellular calcium concentration ([Ca(2+)](i)) to study the role of GABA in interneuronal communication and the response of the SCN neuronal network to optic nerve stimulations that mimic entraining light signals. Stimulation of the retinohypothalamic tract (RHT) evoked divergent Ca(2+) responses in neurons that varied regionally within the SCN with a pattern that correlated with those evoked by pharmacological GABA applications. GABA(A) and GABA(B) receptor agonists and antagonists were used to evaluate components of the GABA-induced changes in [Ca(2+)](i). Application of the GABA(A) receptor antagonist gabazine induced changes in baseline [Ca(2+)](i) in a direction opposite to that evoked by GABA, and similarly altered the RHT stimulation-induced Ca(2+) response. GABA application induced Ca(2+) responses varied in time and region within the SCN network. The NKCC1 cotransporter blocker, bumetanide, and L-type calcium channel blocker, nimodipine, attenuated the GABA-induced rise of [Ca(2+)](i). These results suggest that physiological GABA induces opposing effects on [Ca(2+)](i) based on the chloride equilibrium potential, and may play an important role in neuronal Ca(2+) balance, synchronization and modulation of light input signaling in the SCN network.

Neuropeptides modulate compound postsynaptic potentials in basolateral amygdala

Previous behavioral studies have shown that neuropeptides intrinsic to the amygdala formation can alter fear and anxiety states. We have previously shown that the anxiogenic neuropeptide cholecystokinin (CCK) increases inhibitory neurotransmission in basolateral amygdala. We have since observed that CCK induces synchronized rhythmic activity composed of compound postsynaptic potentials (cPSPs). We have now further characterized these cPSPs by inducing cPSPs routinely in 5 mM extracellular K+. CCK facilitated cPSP occurrence in a dose dependent manner in brain slices from both young and mature rats. The cPSPs were attenuated by glutamate receptor antagonists (NBQX or DL-AP5) or low concentrations of GABAA receptor antagonists (bicuculline methiodide (BMI), SR95531, or picrotoxin), but not by the GABAB receptor antagonist, CGP52432. Low concentrations of tetrodotoxin (TTX, 10 nM) also attenuated the cPSPs. The Na–K–2Cl cotransporter blocker, bumetanide (1 or 10 μM) also blocked the cPSPs. The anxiogenic neuropeptide corticotropin-releasing factor (CRF) facilitated cPSPs while anxiolytic neuropeptides (neuropeptide Y (NPY) and somatostatin) attenuated cPSPs. The benzodiazepine agonist diazepam dose-dependently modulated cPSPs. Mefloquine facilitated cPSPs within 10 min of application. We hypothesize that cPSPs are generated by positive feedback between a subset of interneurons and a subset of glutamatergic projection neurons.

Presynaptic glycine receptors facilitate spontaneous glutamate release onto hilar neurons in the rat hippocampus

Although glycine receptors are found in most areas of the brain, including the hippocampus, their functional significance remains largely unknown. In the present study, we have investigated the role of presynaptic glycine receptors on excitatory nerve terminals in spontaneous glutamatergic transmission. Spontaneous EPSCs (sEPSCs) were recorded in mechanically dissociated rat dentate hilar neurons attached with native presynaptic nerve terminals using a conventional whole-cell patch recording technique under voltage-clamp conditions. Exogenously applied glycine or taurine significantly increased the frequency of sEPSCs in a concentration-dependent manner. This facilitatory effect of glycine was blocked by 1 microM strychnine, a specific glycine receptor antagonist, but was not affected by 30 microM picrotoxin. In addition, Zn(2+) (10 microM) potentiated the glycine action on sEPSC frequency. Pharmacological data suggested that the activation of presynaptic glycine receptors directly depolarizes glutamatergic terminals resulting in the facilitation of spontaneous glutamate release. Bumetanide (10 microM), a specific Na-K-2C co-transporter blocker, gradually attenuated the glycine-induced sEPSC facilitation, suggesting that the depolarizing action of presynaptic glycine receptors was due to a higher intraterminal Cl(-) concentration. The present results suggest that presynaptic glycine receptors on excitatory nerve terminals might play an important role in the excitability of the dentate gyrus-hilus-CA3 network in physiological and/or pathological conditions.

The Mechanisms of Acute Ischemic Injury in the Cell Processes of Developing White Matter Astrocytes

Astrocytes are fundamentally important to the maintenance and proper functioning of the central nervous system. During the period of development when myelination is occurring, white matter astrocytes are particularly sensitive to ischemic injury and their failure to regulate glutamate during ischemic conditions may be an important factor in excitotoxic injury. Here, we have identified key mechanisms of injury that operate on the processes of immature white matter astrocytes during oxygen-glucose deprivation (OGD) using GFAP-GFP mice. Oxygen-glucose deprivation produced a parallel loss of astrocyte processes and somata, assessed by both the retention of GFP fluorescence within these structures and by quantitative electron microscopy. Oxygen-glucose deprivation-induced process loss was Ca(2+) independent and had two distinct mechanisms. Substituting either extracellular Na(+) or Cl(-), or perfusion with the Na-K-Cl co-transport blocker bumetanide, provided protection up to 40 mins of OGD but not beyond that point. HCO(3)(-) substitution or perfusion with 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid provided complete protection of the processes up to 60 mins of OGD. Zero-Na(+)/zero-K(+) conditions provided complete protection from OGD-induced injury of processes and somata at all time points. We conclude that acute ischemic-type injury of immature astrocytes follows a cytotoxic ion influx mediated in part by Na-K-Cl co-transport and in part by Na(+)- and K(+)-dependent HCO(3)(-) transport, a mechanism that is common to both cell processes and somata. This work provides a basis on which preventative strategies may be developed to protect white matter astrocytes from ischemic injury in susceptible individuals.

Acute ischemic injury of astrocytes is mediated by Na-K-Cl cotransport and not Ca2+ influx at a key point in white matter development.

Cerebral palsy is a common birth disorder that frequently involves ischemic-type injury to developing white matter (WM). Dead glial cells are a common feature of this injury and here we describe a novel form of acute ischemic cell death in developing WM astrocytes. Ischemia, modeled by the withdrawal of oxygen and glucose, evoked [Ca2+]i increases and cell death in astrocytes in post-natal day 10 (P10) rat optic nerve (RON). Removing extracellular Ca2+ prevented increases in [Ca2+]i but increased the amount of cell death. Astrocytes showed rapid [Na+]i increases during ischemia and cell death was reduced to control levels by substitution of extracellular Na+ or Cl- or by perfusion with bumetanide, a selective Na-K-Cl cotransport (NKCC) blocker. Astrocytes showed marked swelling during ischemia in the absence of extracellular Ca2+, which was blocked by bumetanide. Raising the extracellular osmolarity to limit water uptake reduced ischemic astrocyte death to control levels. Ultrastructural examination showed that post-ischemic astrocytes had lost their processes and frequently were necrotic, effects partially prevented by bumetanide. At this point in development, therefore, NKCC activation in astrocytes during ischemia produces an osmo-regulatory challenge. Astrocytes can subsequently regulate their cell volume in a Ca2+-dependent fashion but this will require ATP hydrolysis and does not protect the cells against acute cell death.

Ca 2+ dependent k + efflux in hypertension: an 39k-nmr spectroscopic study

P-387 Key Words: Calcium, Potassium,

Cellular potassium ion deprivation enhances apoptosis induced by cisplatin.

The anticancer drug cisplatin induces cell death by apoptosis. Apoptosis is dependent on cellular loss of potassium ions (K+). We have recently shown that the antifungal drug amphotericin B (enhancing K+ efflux), combined with the Na+, K+, 2Cl(-)-cotransport blocker bumetanide (decreasing K+ influx), augmented cisplatin-induced apoptosis in vitro. We therefore quantified K+ fluxes with the K+ analogue rubidium (86Rb+) in cisplatin-induced apoptosis of mesothelioma cells treated with bumetanide and amphotericin B. Bumetanide combined with amphotericin B enhanced cisplatin-induced apoptosis by a pronounced initial reduction of K+ influx due (in addition to Na+, K+, 2Cl(-)-cotransport inhibition) also to Na+, K+, ATPase pump inhibition. As 86Rb+ efflux was initially preserved, combination of the drugs would lead to net K+ loss. Combinations of K+ flux modulators leading to cellular potassium ion deprivation thus augments cisplatin-induced apoptosis and could therefore possibly be used to enhance the antitumour efficacy of cisplatin treatment.

The 45-year story of the development of an anti-aldosterone more specific than spironolactone

At the early stage of its development in 1957, the daily dose of spironolactone necessary to improve various pathological conditions was not precisely determined and dose-dependent sexual side effects limited its long-term use. Prescription of high daily doses and absence of selectivity for the mineralocorticoid receptor explain these limitations. The 9–11α epoxy group added to mexrenone by the Ciba-Geigy chemists in 1984 and improved chemical synthesis at Searle, permitted the original international clinical development of a selective antagonist for high blood pressure and congestive heart failure treatment. This review deals with the main methodological issues of a 20-year biological and clinical development of eplerenone, the second antimineralocorticoid drug. The investigation of a large range of daily doses (25–400 mg) initially selected in normal volunteers by the 9α-fluorohydrocortisone test has led to the conclusion that 50–100 mg q.i.d. doses of eplerenone offer a favorable benefit/risk ratio in various patient populations by neutralization of the aldosterone effects on blood pressure and target organ damage. The absence of sexual side-effects has confirmed the clinical relevance of the initial biological hypothesis on the need for more selectivity at the androgen and progestogen receptor sites. Widening the distance between efficacy and adverse effects of an anti-mineralocorticoid drug will facilitate the long-term maintenance of a moderately negative sodium balance and a slightly positive potassium balance, while minimizing the direct effects of salt and aldosterone on the heart, vessels, brain, and kidneys. Wide use in unselected patients and additional controlled clinical trials are necessary to confirm the benefits expected from animal and clinical research given that a 45-year interval also characterizes the story of the Na–Cl cotransporter (NCC) blocker, chlorthalidone, from its initial clinical use to the demonstration of its beneficial effects on cardiovascular morbidity and mortality.

ATP release triggered by activation of the Ca2+-activated K+ channel in human airway Calu-3 cells.

Airway mucociliary clearance is subject to the autocrine/paracrine regulation of extracellular nucleotides released from the airway epithelial cells. The present study was performed in pursuit of effective modulators of ATP release under physiologic conditions in polarized human airway epithelial cells (Calu-3). Neither isoproterenol, forskolin, nor ionomycin augmented extracellular ATP release detected by luciferase assay. However, direct activation of the human intermediate conductance, Ca(2+)-activated K(+) channel (hIK-1) by 1-ethyl-2-benzimdazolinone (1-EBIO, 1 mM) and chlorzoxazone (CZ, 1 mM) increased ATP release predominantly in the apical compartment. Measurement of fluo-3 signals revealed that 1-EBIO- and CZ-stimulated cytosolic Ca(2+) mobilization was suppressed by the presence of MRS-2179, a specific P2Y(1) receptor antagonist. The hIK-1-mediated ATP release was inhibited by a hIK-1 blocker (charybdotoxin), and an Na(+)-K(+)-2Cl(-) cotransport blocker (bumetanide) without interruption by GdCl(3), an inhibitor of stretch-activated nonselective cation (SA) channels, or glybenclamide, a blocker of the cystic fibrosis transmembrane conductance regulator (CFTR). These results suggest that a cell volume decrease via the hIK-1-mediated KCl loss and the resultant induction of a regulatory volume increase via the Na(+)-K(+)-2Cl(-) transporter may trigger release of ATP, which causes P2Y(1)-mediated Ca(2+) mobilization, through mechanisms unrelated to the CFTR and SA channels.