Gamba, G. et al. Molecular cloning, primary structure, and characterization of two members of the mammalian electroneutral sodium-(potassium)-chloride cotransporter family expressed in kidney. J. Biol. Chem. 269, 17713−17722
Electrically silent Na(+)-(K+)-Cl- transporter systems are present in a wide variety of cells and serve diverse physiological functions. In chloride secretory and absorbing epithelia, these cotransporters provide the chloride entry mechanism crucial for transcellular chloride transport. We have isolated cDNAs encoding the two major electroneutral sodium-chloride transporters present in the mammalian kidney, the bumetanide-sensitive Na(+)-K(+)-Cl- symporter and thiazide-sensitive Na(+)-Cl- cotransporter, and have characterized their functional activity in Xenopus laevis oocytes. Despite their differing sensitivities to bumetanide and thiazides and their different requirements for potassium, these approximately 115-kDa proteins share significant sequence similarity (approximately 60%) and exhibit a topology featuring 12 potential membrane-spanning helices flanked by long non-hydrophobic domains at the NH2 and COOH termini. Northern blot analysis and in situ hybridization indicate that these transporters are expressed predominantly in kidney with an intrarenal distribution consistent with their recognized functional localization. These proteins establish a new family of Na(+)-(K+)-Cl- cotransporters.
"NKCC2 is present in the medullary and cortical thick ascending limbs (MAL and CAL, respectively) and is sensitive to furosemide, a most potent diuretic   . Three types of isoforms of NKCC2 have been reported; a, b, and f types      . The F type has been reported in the outer medullary collecting ducts (OMCD), which was not confirmed by immunohistochemistry. Water reabsorption through AQP2 is sensitive to vasopressin. "
[Show abstract][Hide abstract] ABSTRACT: Sodium reabsorption via Na-K-2Cl cotransporter 2 (NKCC2) in the thick ascending limbs has a major role for medullary osmotic gradient and subsequent water reabsorption in the collecting ducts. We investigated intrarenal localization of three isoforms of NKCC2 mRNA expressions and the effects of dehydration on them in rats. To further examine the mechanisms of dehydration, the effects of hyperosmolality on NKCC2 mRNA expression in microdissected renal tubules was studied. RT-PCR and RT-competitive PCR were employed. The expressions of NKCC2a and b mRNA were observed in the cortical thick ascending limbs (CAL) and the distal convoluted tubules (DCT) but not in the medullary thick ascending limbs (MAL), whereas NKCC2f mRNA expression was seen in MAL and CAL. Two-day dehydration did not affect these mRNA expressions. In contrast, hyperosmolality increased NKCC2 mRNA expression in MAL in vitro. Bradykinin dose-dependently decreased NKCC2 mRNA expression in MAL. However, dehydration did not change NKCC2 protein expression in membrane fraction from cortex and outer medulla and in microdissected MAL. These data show that NKCC2a/b and f type are mainly present in CAL and MAL, respectively. Although NKCC2 mRNA expression was stimulated by hyperosmolality in vitro, NKCC2 mRNA and protein expressions were not stimulated by dehydration in vivo. These data suggest the presence of the inhibitory factors for NKCC2 expression in dehydration. Considering the role of NKCC2 for the countercurrent multiplier system, NKCC2f expressed in MAL might be more important than NKCC2a/b.
Biochemical and Biophysical Research Communications 09/2014; 453(3). DOI:10.1016/j.bbrc.2014.09.089 · 2.30 Impact Factor
"A more modern approach to analyse the NKCC and KCC activity consists in measurement of the 86 Rb + flux through the membrane. This approach was used successfully to clone and characterize NKCC1 (Gamba et al., 1994; Xu et al., 1994), KCC1 (Gillen et al., 1996) and KCC2 (Payne, 1997), and continues to be extensively utilized for characterization of different mutants and variants of CCC. Although the above approaches strongly contributed to study the functional properties of CCC in homogenous cell preparations (erythrocytes, oocytes, different heterologous cell lines), they have limited applications for studying tissues composed of different cell types (i.e., brain slices, neuronal cultures). "
[Show abstract][Hide abstract] ABSTRACT: In the mammalian central nervous system (CNS), the inhibitory strength of chloride (Cl(-))-permeable GABAA and glycine receptors (GABAAR and GlyR) depends on the intracellular Cl(-) concentration ([Cl(-)]i). Lowering [Cl(-)]i enhances inhibition, whereas raising [Cl(-)]i facilitates neuronal activity. A neuron's basal level of [Cl(-)]i, as well as its Cl(-) extrusion capacity, is critically dependent on the activity of the electroneutral K(+)-Cl(-) cotransporter KCC2, a member of the SLC12 cation-Cl(-) cotransporter (CCC) family. KCC2 deficiency compromises neuronal migration, formation and the maturation of GABAergic and glutamatergic synaptic connections, and results in network hyperexcitability and seizure activity. Several neurological disorders including multiple epilepsy subtypes, neuropathic pain, and schizophrenia, as well as various insults such as trauma and ischemia, are associated with significant decreases in the Cl(-) extrusion capacity of KCC2 that result in increases of [Cl(-)]i and the subsequent hyperexcitability of neuronal networks. Accordingly, identifying the key upstream molecular mediators governing the functional regulation of KCC2, and modifying these signaling pathways with small molecules, might constitute a novel neurotherapeutic strategy for multiple diseases. Here, we discuss recent advances in the understanding of the mechanisms regulating KCC2 activity, and of the role these mechanisms play in neuronal Cl(-) homeostasis and GABAergic neurotransmission. As KCC2 mediates electroneutral transport, the experimental recording of its activity constitutes an important research challenge; we therefore also, provide an overview of the different methodological approaches utilized to monitor function of KCC2 in both physiological and pathological conditions.
"Potassium demonstrated a slightly increasing growth profile throughout gestation, confirming observations reported by Benzie et al. (1973). These results are in accordance with the maturation of distal and collecting tubules that are responsible for potassium handling by the fetal kidneys (Gamba et al. 1994). "
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to determine the values of some metabolites, ions, and enzymes in maternal blood serum and fetal
fluids in relation to gestation day in cattle. Gravid uteri of cattle were collected after slaughter. The allantoic and amniotic
fluids as well as maternal blood samples were collected. Fetal ages were determined according to crown–rump length by applying
the age estimation formula that previously was presented for cattle. The pregnancies were divided according to gestation days
into four groups: 0–50, 51–100, 101–150, and 151–200days. With the progress of pregnancy, the biochemical levels of fetal
fluids and maternal serum changed as follows: there was a rise of total protein, urea, creatinine, and alkaline phosphatase
values in fetal fluids and serum; the levels of glucose in maternal serum and potassium in fetal fluids increased; cholesterol,
triglyceride, phosphorus, and sodium contents of fetal fluids and serum decreased; the concentrations of glucose and calcium
in fetal fluids, lactate dehydrogenase (LDH) in serum and allantoic fluid, potassium in serum, and aspartate aminotransferase
(ASAT) in amniotic fluid and serum dropped. The values of LDH in amniotic fluid, ASAT in allantoic fluid, alanine aminotransferase
in amniotic and allantoic fluids, and calcium in maternal serum remained unchanged.
KeywordsBiochemical composition–Gestation–Fetal fluids–Maternal blood–Cattle
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