Neurotypic cell attachment and growth on III-nitride lateral polarity structures.

III-nitride materials have recently received increasing levels of attention for their potential to successfully interface with, and sense biochemical interactions in biological systems. Expanding on available sensing schemes (including transistor-based devices,) a III-N lateral polarity structure capable of introducing quasi-phase matching through a periodic polarity grating presents a novel platform for second harmonic generation. This platform constitutes a non-linear optical phenomenon with exquisite sensitivity to the chemical state of a surface or interface. To characterize the response of a biological system to the nanostructured lateral polarity structures, we cultured neurotypic PC12 cells on AlGaN with varying ratios of Al:Ga - 0, 0.4, 0.6, and 1 - and on surfaces of varying pitch to the III-polar vs. N-polar grating - 5, 10, 20 and 50 µm. While some toxicity associated with increasing Al is observed, we documented and quantified trends in cell responses to the local material polarity and nanoscale roughness. The nitrogen-polar material has a significantly higher nanoscale roughness than III-polar regions, and a 80-200 nm step height difference between the III-polar and N-polar materials in the lateral polarity configuration generates adequate changes in topography to influence cell growth, improves cell adhesion and promotes cell migration along the direction of the features. As the designed material configuration is further explored for biochemical sensing, the lateral polarity scheme may provide a route in assessing the non-specific protein adsorption to this varying nano-topography that drives the subsequent cell response.

Physisorption of functionalized gold nanoparticles on AlGaN/GaN high electron mobility transistors for sensing applications.

AlGaN/GaN high electron mobility transistors (HEMTs) were used to measure electrical characteristics of physisorbed gold nanoparticles (Au NPs) functionalized with alkanethiols with a terminal methyl, amine, or carboxyl functional group. Additional alkanethiol was physisorbed onto the NP treated devices to distinguish between the effects of the Au NPs and alkanethiols on HEMT operation. Scanning Kelvin probe microscopy and electrical measurements were used to characterize the treatment effects. The HEMTs were operated near threshold voltage due to the greatest sensitivity in this region. The Au NP/HEMT system electrically detected functional group differences on adsorbed NPs which is pertinent to biosensor applications.

Low-density lipoprotein (LDL)-induced monocyte-endothelial cell adhesion, soluble cell adhesion molecules, and autoantibodies to oxidized-LDL in chronic renal failure patients on dialysis therapy.

Premature atherosclerosis is a major cause of morbidity and mortality in chronic renal failure patients undergoing dialysis. In this study, we compared autoantibodies to oxidized low-density lipoprotein (OX-LDL), soluble cell adhesion molecules (CAMs), and the effect of both LDL and OX-LDL on monocyte endothelial cell adhesion in chronic renal failure patients on hemodialysis (HD, n = 16) and peritoneal dialysis (PD, n = 17) compared with matched healthy control subjects (C, n = 17). In addition, we studied the effect of supplementation with RRR-alpha-tocopherol (AT) 800 IU/d for 12 weeks on the above measures. LDL and OX-LDL induced adhesion of U937 cells to cultured endothelium, soluble vascular cell adhesion molecule-1 (sVCAM-1), soluble intracellular adhesion molecule-1 (sICAM-1), and soluble E-selectin (sE-selectin); autoantibodies to OX-LDL and markers of lipid peroxidation were determined before and after AT supplementation. Native LDL from PD patients induced greater monocyte-endothelial cell adhesion than LDL from C subjects (43.8% +/- 17.0% v25.3% +/- 17.7%, respectively, P = .0028). OX-LDL from chronic renal failure patients on both PD and HD stimulated greater adhesion than OX-LDL from the C subjects (68.0% +/- 18.5% and 57.6% +/- 15.1% v 40.9% +/- 17.3%, respectively, P < .01); OX-LDL from PD patients induced greater adhesion than that from HD patients (P < .01). Plasma methylglyoxal levels were significantly increased in both HD and PD groups, with higher levels in the HD group. Chronic renal failure patients on HD and PD also had higher levels of plasma sVCAM-1 and sE-selectin than C subjects (P < .01), indicating endothelial activation. Titers of autoantibodies to OX-LDL were not elevated in renal failure patients. Supplementation with AT 800 IU/d for 12 weeks, while resulting in significant enrichment with AT in LDL, did not have a significant effect on any of the parameters studied. This study makes the novel observation that the LDL of chronic renal failure patients on HD and PD appears to be potentially more atherogenic, since it induces greater monocyte-endothelial cell adhesion.

Characterization of acute inhibition of Na/H exchanger NHE-3 by dopamine in opossum kidney cells.

BACKGROUND: Dopamine (DA) is a principal natriuretic hormone that defends extracellular fluid volume from a Na load. Natriuresis is effected partly through inhibiting the proximal tubule Na/H exchanger NHE-3. Changes in NHE-3 phosphorylation is one mechanism by which NHE-3 activity is regulated. METHODS: We used opossum kidney (OK) cells to characterize the differential and synergistic effects of DA receptor subtype-1 (DA1) and -2 (DA2) agonists and the effect of blockade of protein kinase A (PKA) or protein kinase C (PKC) on NHE-3 activity and phosphorylation. RESULTS: DA and DA1 agonists inhibited NHE-3 activity, and DA1 antagonist blocked the effect of either DA or DA1 agonist. DA2 agonist alone had no effect, but DA2 antagonist reduced the DA effect on NHE-3 activity. DA1 and DA2 agonists together were more potent than DA1 alone. PKA inhibition eliminated the effect of DA1 agonist and partially blocked the effect of DA on NHE-3 activity. PKC inhibition did not block the DA effect. DA1 agonist and PKA activation phosphorylated NHE-3 on identical sites. Despite lack of effect on NHE-3 activity, DA2 agonists increased NHE-3 phosphorylation. DA-induced NHE-3 phosphorylation was distinct from DA1 and PKA but closely resembled DA2. CONCLUSION: We postulate the following: (1) DA modifies NHE-3 phosphorylation by activating PKA through DA1 and by other kinases/phosphatases via DA2. (2) DA1 is sufficient to inhibit NHE-3, while DA2 is insufficient but plays a synergistic role by altering NHE-3 phosphorylation.

Acute regulation of Na+/H+ exchanger NHE3 by parathyroid hormone via NHE3 phosphorylation and dynamin-dependent endocytosis.

Parathyroid hormone (PTH) is a potent inhibitor of mammalian renal proximal tubule Na(+) transport via its action on the apical membrane Na(+)/H(+) exchanger NHE3. In the opossum kidney cell line, inhibition of NHE3 activity was detected from 5 to 45 min after PTH addition. Increase in NHE3 phosphorylation on multiple serines was evident after 5 min of PTH, but decrease in surface NHE3 antigen was not detectable until after 30 min of PTH. The decrease in surface NHE3 antigen was due to increased NHE3 endocytosis. When endocytic trafficking was arrested with a dominant negative dynamin mutant (K44A), the early inhibition (5 min) of NHE3 activity by PTH was not affected, whereas the late inhibition (30 min) and decreased surface NHE3 antigen induced by PTH were abrogated. We conclude that PTH acutely inhibits NHE3 activity in a biphasic fashion by NHE3 phosphorylation followed by dynamin-dependent endocytosis.

Dual mechanisms of regulation of Na/H exchanger NHE-3 by parathyroid hormone in rat kidney.

Parathyroid hormone (PTH) is a potent inhibitor of mammalian renal proximal tubule sodium absorption via suppression of the apical membrane Na/H exchanger (NHE-3). We examined the mechanisms by which PTH inhibits NHE-3 activity by giving an acute intravenous PTH bolus to parathyroidectomized rats. Parathyroidectomy per se increased apical membrane NHE-3 activity and antigen. Acute infusion of PTH caused a time-dependent decrease in NHE-3 activity as early as 30 min. Decrease in NHE-3 activity at 30 and 60 min was accompanied by increased NHE-3 phosphorylation. In contrast to the rapid changes in NHE-3 activity and phosphorylation, decrease in apical membrane NHE-3 antigen was not detectable until 4-12 h after the PTH bolus. The decrease in apical membrane NHE-3 occurred in the absence of changes in total renal cortical NHE-3 antigen. Pretreatment of the animals with the microtubule-disrupting agent colchicine blocked the PTH-induced decrease in apical NHE-3 antigen. We propose that PTH acutely cause a decrease in NHE-3 intrinsic transport activity possibly via a phosphorylation-dependent mechanism followed by a decrease in apical membrane NHE-3 antigen via changes in protein trafficking.

Acute inhibition of Na/H exchanger NHE-3 by cAMP. Role of protein kinase a and NHE-3 phosphoserines 552 and 605.

Regulation of the renal Na/H exchanger NHE-3 by protein kinase A (PKA) is a key intermediate step in the hormonal regulation of acid-base and salt balance. We studied the role of NHE-3 phosphorylation in this process in NHE-deficient AP-1 cells transfected with NHE-3 and in OKP cells expressing native NHE-3. A dominant-negative PKA-regulatory subunit completely abolished the effect of cAMP on NHE-3 activity demonstrating a role of PKA in the functional regulation of NHE-3 by cAMP. NHE-3 isolated from cAMP-treated cells showed lower phosphorylation by purified PKA in vitro suggesting that NHE-3 is a PKA substrate in vivo. Although changes in NHE-3 whole protein phosphorylation is difficult to detect in response to cAMP addition, the tryptic phosphopeptide map of in vivo phosphorylated NHE-3 showed a complex pattern of constitutive and cAMP-induced phosphopeptides. To test the causal relationship between phosphorylation and activity, we mutated eight serines in the cytoplasmic domain to glycine or alanine. Single or multiple mutants harboring S552A or S605G showed no PKA activation or reduced regulation by PKA activation. Ser-552 and Ser-605 were phosphorylated in vivo. However, multiple mutations of serines other than Ser-552 or Ser-605 also reduced the functional PKA regulation. We conclude that regulation of NHE-3 by PKA in vivo involves complex mechanisms, which include phosphorylation of Ser-552 and Ser-605.