The product of phospholipase D (PLD) enzymatic action in cell membranes, phosphatidic acid (PA), regulates kinases implicated NADPH oxidase activation, as well the mammalian target rapamycin (mTOR) kinase. However, other protein targets for this lipid second messenger must exist order to explain key PA-mediated cellular functions. In study, PA was found specifically and saturably bind activate recombinant immunoprecipitated endogenous ribosomal S6 kinase (S6K) with a stoichiometry 94:1...

GABA depolarizes and excites central neurons during early development, becoming inhibitory hyperpolarizing with maturation. This "developmental shift" occurs abruptly, reflecting a decrease in intracellular Cl(-) concentration ([Cl(-)](i)) shift equilibrium potential due to upregulation of the K(+)-Cl(-) cotransporter KCC2b, neuron-specific extruder. In contrast, primary afferent (PANs) are depolarized by throughout adulthood because expression NKCC1, Na(+)-K(+)-2Cl(-) that accumulates above...

The feeding pattern and control of energy intake in mice housed groups are poorly understood. Here, we determined quantified the normal microstructure social male female C57BL/6J genetic background fed a chow diet. Mice at 10w, 20w 30w age showed expected increase lean fat mass, being latter more pronounced variable males than females. Under ad libitum conditions, old females significantly increased daily when adjusted to body weight relative age-matched males. This was combined result small...

Abstract Intracellular chloride concentration ([Cl − ] i ) in pancreatic β-cells is kept above electrochemical equilibrium due to the predominant functional presence of Cl loaders such as Na + K 2Cl co-transporter 1 ( Slc12a2 over extruders unidentified nature. Using molecular cloning, RT-PCR, Western blotting, immunolocalization and vitro assays, we establish that “neuron-specific” 2 (KCC2, Slc12a5 expressed several endocrine cells islet, including glucagon secreting α-cells, but...

Thiazide, thiazide-like, and loop diuretics are primarily known for inhibiting members of the SLC12A family Cl– transporters, which include Na+Cl– cotransporter (NCC), Na+K+2Cl– cotransporters (NKCC1 NKCC2) K+Cl– symporters (KCC1-4). While main pharmacological effect these is diuresis, achieved by promoting excretion excess water salt through kidneys, they have intriguing effects beyond their traditional ones cannot be solely attributed to on renal transport. Of particular interest role in...

Characterizing mechanisms regulating mammary cell growth and differentiation is vital, as they may contribute to breast carcinogenesis. Here, we examine a cross talk mechanism(s) downstream of prolactin (PRL), primary hormone, epidermal factor (EGF), an important proliferative factor, in epithelial differentiation. Our data indicate that EGF exerts inhibitory effects on PRL-induced cellular by interfering with Stat5a-mediated gene expression independent the PRL-proximal signaling cascade....

K-Cl cotransport (KCC) is activated by nitric oxide donors and appears to be regulated the cGMP signaling pathway. Expression of KCC mRNAs (KCC1–KCC4) in rat vascular smooth muscle cells (VSMCs) unknown. We have reported presence KCC1 KCC3 primary cultures VSMCs specific reverse transcription-polymerase chain reaction. KCC2 mRNA appeared at extremely low levels. KCC4 was undetectable. Semiquantitative reaction revealed a 2:1 KCC1/KCC3 ratio VSMCs. Depletion protein kinase G (PKG)-1 from did...

The solute carrier protein family 12 group A, member one (Slc12a1) and two (Slc12a2) encode several splice variants of the kidney-specific ubiquitous isoforms, respectively, bumetanide (BTD)-sensitive Na-dependent K2Cl co-transporter. Slc12a2 co-transporter is involved in maintenance a high intracellular chloride concentration [Cl–]i β-cells its inhibition with BTD blocks glucose-induced insulin secretion. In β-cells, plays an important role depolarization Glucose promotes electrogenic...

The Na + K 2Cl − cotransporter-1 ( Slc12a2 , NKCC1) is widely distributed and involved in cell volume/ion regulation. Functional NKCC1 locates the plasma membrane of all cells studied, particularly basolateral most polarized cells. Although mechanisms sorting are poorly understood, it assumed that N-glycosylation necessary. Here, we characterize expression, N-glycosylation, distribution COS7 We show ~25% complex N-glycosylated whereas rest corresponds to core/high-mannose hybrid-type forms....

The renin angiotensin system (RAS) plays a vital role in the regulation of cardiovascular and renal functions. COS7 is robust easily transfectable cell line derived from kidney African green monkey, Cercopithecus aethiops. aims this study were to 1) demonstrate presence an endogenous functional RAS COS7, 2) investigate disintegrin metalloproteinase-17 (ADAM17) ectodomain shedding converting enzyme-2 (ACE2). Reverse transcription coupled gene-specific polymerase chain reaction demonstrated...

Cystic fibrosis (CF) is due to mutations in the CF-transmembrane conductance regulator ( CFTR ) and CF-related diabetes (CFRD) its most common co-morbidity, affecting ~50% of all CF patients, significantly influencing pulmonary function longevity. Yet, complex pathogenesis CFRD remains unclear. Two non-mutually exclusive underlying mechanisms have been proposed CFRD: i damage endocrine cells secondary severe exocrine pancreatic pathology ii intrinsic β-cell impairment secretory response...

The mammalian target of rapamycin (mTOR) and S6 kinase (S6K) pathway is essential for cell differentiation, growth, survival. Phospholipase D2 (PLD2) plays a key role in mTOR/S6K mitogenic signaling. However, the impact PLD on gene expression not known. Here we show that interleukin-8 (IL-8) increases mRNA levels PLD2, mTOR, S6K, with PLD2 preceding time. Silencing abrogated IL-8-induced expression, whereas silencing mTOR or S6K resulted large (>3-fold >5-fold, respectively) increased RNA,...

The effects of the tri-iodothyronine (T(3)) secreted by thyroid cells on growth thyrocyte are poorly known. In this study we analyzed T(3) proliferation bovine follicles in primary culture previously depleted endogenous T(3). Cellular deoxiribonucleic acid (DNA) synthesis, determined [(3)H]thymidine incorporation, was stimulated (0.1-5.0 nM) for 24 h a concentration-dependent fashion with maximal effect at 1.0 nM (P<0.01). This action time-dependent when assayed from 12 to 72 h. induction...

Abstract Human neutrophils exhibit a regulated phospholipase D (PLD) activity that can be measured biochemically in vitro. However, the precise expression pattern of PLD isoforms and their specific biological role(s) are not well understood. Neutrophil mRNA is intrinsically difficult to isolate as result extremely high content lytic enzymes cell's lysosomal granules. Reverse transcription coupled polymerase chain reaction indicated pure populations human had...

The fetal human lens epithelial cell (LEC) line (FHL124) possesses all four K(+)Cl(-) (KCC) cotransporter isoforms, KCC1-4, despite KCC2 being typically considered a neuronal isoform. Since at least two spliced variants, KCC2a and KCC2b, are co-expressed in cells of the central nervous system, this study sought to define expression profile FHL124 cells. KCC2a, but not KCC2b transcripts were detected by reverse transcriptase polymerase chain reaction (RT-PCR). Proteins molecular weights...

The products of the Slc12a1 and Slc12a2 genes, commonly known as Na + -dependent K 2Cl − co-transporters NKCC2 NKCC1, respectively, are targets for diuretic bumetanide. NKCCs implicated in regulation intracellular chloride concentration ([Cl ] i ) pancreatic β-cells, such, they may play a role glucose-stimulated plasma membrane depolarization insulin secretion. Unexpectedly, permanent elimination NKCC1 does not preclude secretion, an event potentially linked to homeostatic additional Cl...