AB3560P Sigma-AldrichAnti-Sodium Antibody, Potassium, Chloride Cotransporter 1

Ischemia in the immature brain is an important cause of neonatal seizures. Temporal evolution of acquired neonatal seizures and their response to anticonvulsants are of great interest, given the unreliability of the clinical correlates and poor efficacy of first-line anti-seizure drugs. The expression and function of the electroneutral chloride co-transporters KCC2 and NKCC1 influence the anti-seizure efficacy of GABAA-agonists. To investigate ischemia-induced seizure susceptibility and efficacy of the GABAA-agonist phenobarbital (PB), with NKCC1 antagonist bumetanide (BTN) as an adjunct treatment, we utilized permanent unilateral carotid-ligation to produce acute ischemic-seizures in post-natal day 7, 10, and 12 CD1 mice. Immediate post-ligation video-electroencephalograms (EEGs) quantitatively evaluated baseline and post-treatment seizure burdens. Brains were examined for stroke-injury and western blot analyses to evaluate the expression of KCC2 and NKCC1. Severity of acute ischemic seizures post-ligation was highest at P7. PB was an efficacious anti-seizure agent at P10 and P12, but not at P7. BTN failed as an adjunct, at all ages tested and significantly blunted PB-efficacy at P10. Significant acute post-ischemic downregulation of KCC2 was detected at all ages. At P7, males displayed higher age-dependent seizure susceptibility, associated with a significant developmental lag in their KCC2 expression. This study established a novel neonatal mouse model of PB-resistant seizures that demonstrates age/sex-dependent susceptibility. The age-dependent profile of KCC2 expression and its post-insult downregulation may underlie the PB-resistance reported in this model. Blocking NKCC1 with low-dose BTN following PB treatment failed to improve PB-efficacy.

Slc26a4 (Δ/Δ) mice are deaf, develop an enlarged membranous labyrinth, and thereby largely resemble the human phenotype where mutations of SLC26A4 cause an enlarged vestibular aqueduct and sensorineural hearing loss. The enlargement is likely caused by abnormal ion and fluid transport during the time of embryonic development, however, neither the mechanisms of ion transport nor the ionic composition of the luminal fluid during this time of development are known. Here we determine the ionic composition of inner ear fluids at the time at which the enlargement develops and the onset of expression of selected ion transporters. Concentrations of Na(+) and K(+) were measured with double-barreled ion-selective electrodes in the cochlea and the endolymphatic sac of Slc26a4 (Δ/+), which develop normal hearing, and of Slc26a4 (Δ/Δ) mice, which fail to develop hearing. The expression of specific ion transporters was examined by quantitative RT-PCR and immunohistochemistry. High Na(+) (∼141 mM) and low K(+) concentrations (∼11 mM) were found at embryonic day (E) 16.5 in cochlear endolymph of Slc26a4 (Δ/+) and Slc26a4 (Δ/Δ) mice. Shortly before birth the K(+) concentration began to rise. Immediately after birth (postnatal day 0), the Na(+) and K(+) concentrations in cochlear endolymph were each ∼80 mM. In Slc26a4 (Δ/Δ) mice, the rise in the K(+) concentration occurred with a ∼3 day delay. K(+) concentrations were also found to be low (∼15 mM) in the embryonic endolymphatic sac. The onset of expression of the K(+) channel KCNQ1 and the Na(+)/2Cl(-)/K(+) cotransporter SLC12A2 occurred in the cochlea at E19.5 in Slc26a4 (Δ/+) and Slc26a4 (Δ/Δ) mice. These data demonstrate that endolymph, at the time at which the enlargement develops, is a Na(+)-rich fluid, which transitions into a K(+)-rich fluid before birth. The data suggest that the endolymphatic enlargement caused by a loss of Slc26a4 is a consequence of disrupted Na(+) transport.

The Na(+)-K(+)-2Cl(-) cotransporter-2 (NKCC2) has long been recognized as a kidney-specific transporter and is important in salt reabsorption. NKCC2 has been found in the gastric mucosa; however, its cellular distribution and function remain obscure. The present study characterized the distribution pattern of NKCC2 in mammalian gastric mucosa and investigated its response to osmotic challenge. Reverse transcription with the polymerase chain reaction, Western blot and immunofluorescence were used to determine NKCC2 expression and localization. The effect of osmotic shock on NKCC2 expression was studied in isolated gastric mucosa with variable osmolarity treatment. Results from all of the above studies were compared with those of NKCC1. Our data indicated that NKCC1 and NKCC2 were expressed in the gastric mucosa of rat, mouse and human. The mRNA transcripts and proteins for NKCC1 and NKCC2 were broadly expressed in the rat gastric mucosa. In rat and mouse, NKCC1 was largely confined to the lower part of the oxyntic and pyloric gland areas, whereas NKCC2 extended throughout the gastric glands. NKCC1 immunoreactivity was strongly expressed in the parietal and chief cells but was weaker in the mucous cells. NKCC2 was abundantly located in the parietal and mucous cells but faintly distributed in the chief cells. Hypertonic treatment increased the protein level of NKCC1 and caused evident membrane translocation. In contrast, NKCC2 was significantly downregulated and no obvious membrane translocation was observed. Thus, NKCC2 displayed a more ubiquitous distribution in the gastric mucosa and might work coordinately with NKCC1 to maintain cell volume homeostasis under hypertonic conditions.

Persistent inflammation is associated with a shift in spinal GABA(A) signaling from inhibition to excitation such that GABA(A)-receptor activation contributes to inflammatory hyperalgesia. We tested the hypothesis that the primary afferent is the site of the persistent inflammation-induced shift in GABA(A) signaling which is due to a Na(+)-K(+)-Cl(-)-co-transporter (NKCC1)-dependent depolarization of the GABA(A) current equilibrium potential (E(GABA)). Acutely dissociated retrogradely labeled cutaneous dorsal root ganglion (DRG) neurons from naïve and inflamed (3 days after a subcutaneous injection of complete Freund's adjuvant) adult male rats were studied with Ca(2+) imaging, western blot and gramicidin-perforated patch recording. GABA evoked a Ca(2+) transient in a subpopulation of small- to medium-diameter capsaicin-sensitive cutaneous neurons. Inflammation was associated with a significant increase in the magnitude of GABA-induced depolarization as well as the percentage of neurons in which GABA evoked a Ca(2+) transient. There was no detectable change in NKCC1 protein or phosphoprotein at the whole ganglia level. Furthermore, the increase in excitatory response was comparable in both HEPES- and HCO(3)(-)-buffered solutions, but was only associated with a depolarization of E(GABA) in HCO(3)(-)-based solution. In contrast, under both recording conditions, the excitatory response was associated with an increase in GABA(A) current density, a decrease in low threshold K(+) current density, and resting membrane potential depolarization. Our results suggest that increasing K(+) conductance in afferents innervating a site of persistent inflammation may have greater efficacy in the inhibition of inflammatory hyperalgesia than attempting to drive a hyperpolarizing shift in E(GABA).

The kidney is an important target for the actions of the renin-angiotensin system (RAS) and this tissue contains a complete local RAS that expresses the bioactive peptides angiotensin II (ANG II) and Ang-(1-7). We find both angiotensin type 1 (AT(1)R) and type 2 (AT(2)R) receptors expressed on renal nuclei that stimulate reactive oxygen species and nitric oxide (NO), respectively. Since Ang-(1-7) also exhibits actions within the kidney and the Ang-(1-7)/Mas receptor protein contains a nuclear localization sequence, we determined the expression of Ang-(1-7) receptors in nuclei isolated from the kidneys of young adult sheep. Binding studies with (125)I-[Sar(1)Thr(8)]-ANG II revealed sites sensitive to the Ang-(1-7) antagonist [d-Ala(7)]-Ang-(1-7) (DALA, A779), as well as to AT(2) and AT(1) antagonists. Incubation of Ang-(1-7) [10(-15) to 10(-9) M] with isolated cortical nuclei elicited a dose-dependent increase in the fluorescence of the NO indicator [4-amino-5-methylamino-2',7']-difluorofluorescein diacetate. The NO response to Ang-(1-7) was abolished by the NO inhibitor N-nitro-l-arginine methyl ester and DALA, but not the AT(1) antagonist losartan or the AT(2) blocker PD123319. Immunofluorescent studies utilizing the Ang-(1-7)/Mas receptor antibody revealed immunolabeling of the proximal tubules but not staining within the glomerulus in cortical sections of the sheep kidney. In the nuclear fraction of isolated proximal tubules, immunoblots revealed the precursor angiotensinogen and renin, as well as functional activity for ACE, ACE2, and neprilysin. We conclude that renal nuclei express Ang-(1-7)/Mas receptors that are functionally linked to NO formation. The marked sensitivity of the intracellular NO response to Ang-(1-7) implicates a functional role of the Ang-(1-7) axis within the nucleus. Moreover, evidence for the precursor and enzymatic components of the RAS within the nuclear compartment of the proximal tubules provides a potential pathway for the intracellular generation of Ang-(1-7).

Muscle activity is associated with potassium displacements, which may cause fatigue. It was reported previously that the density of the large-conductance Ca2+-dependent K+ (BK(Ca)) channel is higher in the T tubule membrane than in the sarcolemmal membrane and that the opposite is the case for the ATP-sensitive K+ (K(ATP)) channel. In the present experiments, we investigated the subcellular localizations of the strong inward rectifier 2.1 K+ (Kir2.1) channel and the Na+-K+-2Cl- (NKCC)1 cotransporter with Western blot analysis of different muscle fractions. Furthermore, muscle function was studied while trying to manipulate the opening probability or transport capacity of these proteins during electrical stimulation of isolated soleus muscles. All experiments were made with excised muscle from male Wistar rats. Kir2.1 channels were almost undetectable in the sarcolemmal membrane but present in the T tubule membrane, whereas NKCC1 cotransporters were present in the sarcolemmal membrane. For muscles incubated in a buffer containing pinacidil, NS1619, Ba2+, or bumetanide, there was a faster reduction in peak force (P less than 0.05). Furthermore, bumetanide incubation reduced the peak force at the onset of electrical stimulation (P less than 0.05). Thus the effects on muscle force indicate that these drugs can affect K+-transporting proteins and thereby influence K+ accumulation, especially in the T tubules, suggesting that K(ATP) and BK(Ca) channels are responsible for K+ release and decrease in force during repeated muscle contractions, whereas Kir2.1 and NKCC1 may have a role in K+ reuptake.