Exercise effects on muscle beta-adrenergic signaling for MAPK-dependent NKCC activity are rapid and persistent.
(9/205)
This study investigated exercise adaptation of signaling mechanisms that control Na(+)-K(+)-2Cl(-) cotransporter (NKCC) activity in rat skeletal muscle. An acute bout of exercise increased total and NKCC-mediated (86)Rb influx. Inhibition of extracellular signal-regulated kinase (ERK) activation abolished the exercise-induced NKCC upregulation. Treadmill training (20 m/min, 20% grade, 30 min/day, 5 days/wk) stimulated total (86)Rb influx and increased NKCC activity in the soleus muscle after 2 wk and in the plantaris muscle after 4 wk. Exercise-induced NKCC activity was associated with a 1.4- to 2-fold increase in ERK phosphorylation. Isoproterenol, which activates ERK and NKCC in sedentary muscle, caused a remarkable inhibition of the exercise-induced NKCC activity. Furthermore, isoproterenol inhibition of exercise-induced NKCC activity was accompanied with decreased ERK phosphorylation in the plantaris muscle. Akt (protein kinase B) phosphorylation on both Thr(308) and Ser(473), which activates Akt and inhibits NKCC activity in sedentary muscle, was stimulated by acute and chronic exercise. This Akt activation was unaffected by isoproterenol. These results indicate an immediate and persistent exercise adaptation of the signal pathways that participate in the control of potassium transport. (+info)
Allopregnanolone enhancement of GABAergic transmission in rat medial preoptic area neurons.
(10/205)
Gamma-aminobutyric acid (GABA)-mediated transmission in the medial preoptic area (MPOA) of the hypothalamus plays an important role in functions such as sex steroid hormone dynamics and control of body temperature. The action of allopregnanolone, the primary metabolite of progesterone, on GABAergic transmission was investigated by employing patch clamp whole cell recording on acutely dissociated rat MPOA neurons with the functional connection of presynaptic terminals. Allopregnanolone enhanced spontaneous GABA release on the MPOA neurons and induced prolonged decay of miniature GABAergic-inhibitory postsynaptic currents (mIPSCs). The facilitation of GABA release from the presynaptic terminals by allopregnanolone disappeared in Ca2+-free extracellular solution. The presynaptic action of this neurosteroid was also blocked by bumetanide, a blocker of cation-Cl- cotransporters, and by removal of extracellular Na+. The results suggest that allopregnanolone enhances GABAergic transmission at the MPOA neurons by pre- and postsynaptic mechanisms. The enhancement of GABA release by allopregnanolone might require a high Cl- concentration in the presynaptic terminal maintained by Na+-dependent, bumetanide-sensitive mechanisms (e.g., Na+-K+-Cl- cotransporter) and might be mediated by Ca2+ influx into presynaptic terminal. (+info)
Hyposmotic shock: effects on rubidium/potassium efflux in normal and ischemic rat hearts, assessed by 87Rb and 31P NMR.
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The study evaluated effects of hyposmotic shock on the rate of Rb(+)/K(+) efflux, intracellular pH and energetics in Langendorff-perfused rat hearts with the help of 87Rb- and 31P-NMR. Two models of hyposmotic shock were compared: (1) normosmotic hearts perfused with low [NaCl] (70 mM) buffer, (2) hyperosmotic hearts equilibrated with additional methyl alpha-D-glucopyranoside (Me-GPD, 90 or 33 mM) or urea (90 mM) perfused with normosmotic buffer. Four minutes after hyposmotic shock, Rb(+) efflux rate constant transiently increased approximately two-fold, while pH transiently decreased by 0.08 and 0.06 units, in the first and the second models, respectively, without significant changes in phosphocreatine and ATP. Hyposmotic shock (second model) did not change the rate of Rb(+)/K(+) uptake, indicating that the activity of Na(+)/K(+) ATPase was not affected. Dimethylamiloride (DMA) (10 microM) abolished activation of the Rb(+)/K(+) efflux in the second model; however, Na(+)/H(+) exchanger was not involved, because intracellular acidosis induced by the hyposmotic shock was not enhanced by DMA treatment. After 12 or 20 min of global ischemia, the rate of Rb(+)/K(+) efflux increased by 120%. Inhibitor of the ATP-sensitive potassium channels, glibenclamide (5 microM), partially (40%) decreased the rate constant; however, reperfusion with hyperosmolar buffer (90 mM Me-GPD) did not. We concluded that the shock-induced stimulation of Rb(+)/K(+) efflux occurred, at least partially, through the DMA-sensitive cation/H(+) exchanger and swelling-induced mechanisms did not considerably contribute to the ischemia-reperfusion-induced activation of Rb(+)/K(+) efflux. (+info)
NO inhibits Na+-K+-2Cl- cotransport via a cytochrome P-450-dependent pathway in renal epithelial cells (MMDD1).
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Nitric oxide (NO) exerts direct effects on nephron transport. We determined the effect of NO on Na(+)-K(+)-2Cl(-) cotransport in a cell line (MMDD1) with properties of macula densa. Na(+)-K(+)-2Cl(-) cotransport was measured as bumetanide-sensitive (86)Rb(+) uptake in the presence of ouabain. MMDD1 cells expressed mRNA for the neuronal isoform of nitric oxide synthase, as well as NKCC1 and NKCC2(B) isoforms of the Na(+)-K(+)-2Cl(-) cotransporter. Preincubation of cells with the NO donors sodium nitroprusside (SNP) or S-nitroso-N-acetylpenicillamine (SNAP) caused concentration-dependent inhibition of Na(+)-K(+)-2Cl(-) cotransport. Both apical and basolateral Na(+)-K(+)-2Cl(-) cotransport was inhibited by NO donors. SNP or SNAP had no significant effect on cellular levels of cGMP, cAMP, cytosolic calcium, or phosphorylation of ERK1 and ERK2. In contrast, the inhibitors of cytochrome P-450, 1-aminobenzotriazole (ABT; 10(-3) M) or ketoconazole (1.5 x 10(-5) M), completely reversed the inhibitory effect of SNAP on apical or basolateral Na(+)-K(+)-2Cl(-) cotransport [apical: control 1.18 +/- 0.15 vs. SNAP (10(-4) M) 0.41 +/- 0.05 pmol x mg(-1) x 5 min(-1); P < 0.001; SNAP (10(-4) M) + ABT 1.32 +/- 0.10 pmol x mg(-1) x 5 min(-1); P = not significant vs. control; n = 5]. The cytochrome P-450 epoxyeicosatrienoic acid (EET) metabolite 14,15-EET (5 x 10(-7) M) inhibited both apical and basolateral cotransport, whereas 8,9-EET and 11,12-EET had no significant effect. Although 20-hydroxyeicosatetraenoic acid inhibited apical cotransport, the inhibitor of omega-hydroxylase activity HET0016 did not reverse SNAP-mediated inhibition of apical cotransport. These data indicate that NO inhibits apical and basolateral Na(+)-K(+)-2Cl(-) cotransport in MMDD1 cells. The results suggest that the inhibitory pathway is independent of cGMP and might involve stimulation of a cytochrome P-450-dependent pathway. (+info)
Na-K-Cl cotransporter contributes to glutamate-mediated excitotoxicity.
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We hypothesized that cation-dependent Cl- transport protein Na-K-Cl cotransporter isoform 1 (NKCC1) plays a role in the disruption of ion homeostasis in cerebral ischemia. In the current study, a role for NKCC1 in neuronal death was elucidated in neurotoxicity induced by glutamate and oxygen and glucose deprivation (OGD). Incubation of cortical neurons cultured for 14-15 d in vitro (DIV) with 100 microm glutamate for 24 hr resulted in 50% cell death. Three hours of OGD followed by 21 hr of reoxygenation led to 70% cell death. Inhibition of NMDA receptors with dizocilpine hydrogen maleate (1 microm) prevented both OGD- and glutamate-mediated cell death. Moreover, blocking of NKCC1 activity with bumetanide (5-10 microm) abolished glutamate- or OGD-induced neurotoxicity. Bumetanide was ineffective if added after 10-120 min of glutamate incubation or 3-6 hr of OGD treatment. Accumulation of intracellular Na+ and 36Cl content after NMDA receptor activation was inhibited by bumetanide. Blockage of NKCC1 significantly attenuated cell swelling after OGD or NMDA receptor activation. This neuroprotection was age dependent. Inhibition of NKCC1 did not protect DIV 7-8 neurons against OGD-mediated cell death. In contrast, cell death in DIV 7-8 neurons was prevented by the protein-synthesis inhibitor, cycloheximide. Taken together, the results suggest that NKCC1 activity is involved in the acute excitotoxicity as a result of excessive Na+ and Cl- entry and disruption of ion homeostasis. (+info)
Deafness disrupts chloride transporter function and inhibitory synaptic transmission.
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Loss of sensory function leads to atrophy or death within the developing CNS, yet little is known about the physiology of remaining synapses. After bilateral deafening, gramicidin-perforated-patch recordings were obtained from gerbil inferior colliculus neurons in a brain slice preparation. Afferent-evoked IPSPs had a diminished ability to block current-evoked action potentials in deafened neurons. This change could be attributed, in part, to a loss of potassium-dependent chloride transport function, with little change in K-Cl cotransporter expression. Treatments that suppressed chloride cotransport (bumetanide, cesium, and genistein) had little or no effect on neurons from deafened animals. These same treatments depolarized the E(IPSC) of control neurons. Semiquantitative RT-PCR and immunohistochemical staining indicated no change in the expression of chloride cotransporter mRNA or protein after deafness. Therefore, profound hearing loss leads rapidly to the disruption of chloride homeostasis, which is likely attributable to the dysfunction of the potassium-dependent chloride cotransport mechanism, rather than a downregulation of its expression. This results in inhibitory synapses that are less able to block excitatory events. (+info)
Bumetanide, the specific inhibitor of Na+-K+-2Cl- cotransport, inhibits 1alpha,25-dihydroxyvitamin D3-induced osteoclastogenesis in a mouse co-culture system.
(15/205)
The Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) is responsible for ion transport across the secretory and absorptive epithelia, the regulation of cell volume, and possibly the modulation of cell growth and development. It has been reported that a variety of cells, including osteoblasts, contain this cotransporter. In this study, the physiological role of NKCC1 in osteoclastogenesis was exploited in a co-culture system. Bumetanide, a specific inhibitor of NKCC1, reduced the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells. In order to investigate the mechanism by which bumetanide inhibits osteoclastogenesis, the mRNA expressions of the receptor activator of nuclear factor (NF)-kappaB ligand (RANKL) and osteoprotegerin (OPG) were analysed by RT-PCR. Exposure of osteoblastic cells to a medium containing 1 micro M bumetanide reduced RANKL mRNA expression induced by 10 nM 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3, in a dose-dependent manner. In addition, RANKL expression was also analysed with enzyme-linked immunosorbant assay (ELISA) using anti-RANKL antibody. The expression of RANKL was decreased with the increase of bumetanide concentration. In contrast, the expression of OPG mRNA, a novel tumour necrosis factor (TNF) receptor family member was increased in the presence of bumetanide. These results imply that bumetanide inhibits osteoclast differentiation by reducing the RANKL/OPG ratio in osteoblastic cells. However, no significant difference in M-CSF mRNA expression was observed when bumetanide was added. Also, we found that the phosphorylation of c-Jun NH2-terminal kinase (JNK), which regulates the activity of various transcriptional factors, was reduced by bumetanide treatment. Conclusively, these findings suggest that NKCC1 in osteoblasts has a pivotal role in 1alpha,25(OH)2D3-induced osteoclastogenesis partly via the phosphorylation of JNK. (+info)
Chloride cotransport in the membrane of earthworm body wall muscles.
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The resting membrane potential (V(m)) of isolated somatic longitudinal muscles of the earthworm Lumbricus terrestris was studied by glass microelectrodes. The inhibition of chloride permeability by low pH did not affect V(m) of the muscle fibers in isolated somatic longitudinal muscles of the earthworm Lumbricus terrestris which was -48.7 mV (inside negative) at pH 7.3 and -49.1 at pH 5.6. On the other hand, bathing the muscles in Cl(-) and Na(+)-free solutions, or application of the chloride transporter inhibitor furosemide and Na(+)-K(+)-ATPase inhibitor ouabain depolarized the V(m) by 3-5 mV. The effects of a Cl(-) -free solution and ouabain were not additive. This demonstrates relatively small contribution of equilibrium potential for Cl(-) to the resting membrane potential and electrogenic effect of Na(+)K(+)-ATPase which is dependent on the supply of Na(+)(i) ions by furosemide-sensitive and Cl(-)(e)- and Na(+)(e)-dependent electroneutral transport (most probably Na(+)K(+)Cl(-) cotransport). (+info)