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Purpose: To compare ocular pharmacokinetics (PK), systemic absorption, and injectability of triamcinolone acetonide (TA) suprachoroidal (SC) and intravitreal (IVT) suspensions. Methods: New Zealand White (NZW) rabbits received a bilateral injection of 4 mg TA injectable suspension, TRI (TRIESENCE ®; Alcon) through either microneedle-based SC or standard IVT injection. Another group of NZW rabbits received a bilateral SC injection (4 mg) of either TRI or a proprietary TA suspension for SC use, CLS-TA (Clearside Biomedical). Blood and ocular tissues were analyzed for TA over 3 months. Separately, injectability of TRI and CLS-TA through a proprietary SC delivery system (SCS Microinjector®; Clearside Biomedical) was compared using microinjector syringe-plunger glide force measurement. Results: Suprachoroidal delivery of TRI, compared with IVT-TRI, resulted in ∼12-fold higher exposure in the retinal pigment epithelium-choroid-sclera, and comparable exposure in the retina. Conversely, SC-TRI, compared to IVT-TRI, resulted in 460-, 34-, and 22-fold lower exposure in the lens, iris-ciliary body, and vitreous humor, and negligible exposure in the aqueous humor. SC injection of either CLS-TA or TRI resulted in comparable and sustained ocular TA levels. Plasma TA levels were generally undetectable in both studies. A significantly greater and more variable glide force was measured for TRI vs. CLS-TA. Conclusions: Suprachoroidal delivery of TA enabled high and durable TA levels targeted to the chorioretina with limited anterior segment exposure. SC delivery of either CLS-TA or TRI resulted in comparable ocular PK profiles with low systemic exposure; however, CLS-TA required lower and less variable glide force than TRI, potentiating more consistent, controlled administration. [ABSTRACT FROM AUTHOR]

Cavet, Megan E., Shafinaz Akhter, Rakhilya Murtazina, Fermin Sanchez de Medina, Chung-Ming Tse, and Mark Donowitz. Half-lives of plasma membrane [Na.sup.+]/[H.sup.+] exchangers NHE1-3: plasma membrane NHE2 has a rapid rate of degradation. Am J Physiol Cell Physiol 281: C2039--C2048, 2001.--The [Na.sup.+]/[H.sup.+] exchangers NHE2 and NHE3 are involved in epithelial [Na.sup.+] and HC[O.sup.-.sub.3] absorption. To increase insights into the functions of NHE2 vs. NHE3, we compared their cellular processing with each other and with the housekeeping isoform NHE1. Using biotinylated exchanger, we determined that the half-life of plasma membrane NHE2 was short (3 h) compared with that of NHE1 (24 h) and NHE3 (14 h) in both PS120 fibroblasts and Caco-2 cells. NHE2 transport and plasma membrane levels were reduced by 3 h of Brefeldin A treatment, whereas NHE 1 was unaffected. NHE2 was degraded by the lysosomes but not proteosomes, as demonstrated by increasing levels of endocytosed NHE2 protein after inhibition of the lysosomes, but not with proteosome inhibition. Unlike that of NHE3, basal NHE2 transport activity was not affected by phosphatidylinositol 3-kinase inhibition and did not appear to be localized in the juxtanuclear recycling endosome. Therefore, for NHE2, protein degradation and/or protein synthesis probably play important roles in its basal and regulated states. These results suggest fundamental differences in the cellular processing and trafficking of NHE2 and NHE3. These differences may underlie the specialized roles that these exchangers play in epithelial cells. sodium absorption; Caco-2 cells; lysosomes; trafficking Received 18 December 2000; accepted in final form 20 August 2001

Results demonstrate that six of the 11 membrane spanning domains of the sodium-potassium exchanger NHE3 act as signal anchors and stop transfer seuences and the remaining five exhibit only the former function. Membrane orientation of these domains are determined by their location with respect to end terminals of NHE3.

Research has been conducted on two exchanger isoforms Na (super)+ /H (super)+, NHE1 and NHE3. The identification of the domains that are involved in the NHE3 targeting has been investigated.

Cavet, Megan E., Shafinaz Akhter, Fermin Sanchez De Medina, Mark Donowitz, and Chung-Ming Tse. [Na.sup.+]/[H.sup.+] exchangers (NHE1-3) have similar turnover numbers but different percentages on the cell surface. Am. J. Physiol. 277 (Cell Physiol. 46): C1111-C1121, 1999.--NHE1, NHE2, and NHE3 are well-characterized cloned members of the mammalian [Na.sup.+]/[H.sup.+] exchanger (NHE) gene family. Given the specialized function and regulation of NHE1, NHE2, and NHE3, we compared basal turnover numbers of NHE1, NHE2, and NHE3 measured in the same cell system: PS120 fibroblasts lacking endogenous NHEs. NHE1, NHE2, and NHE3 were epitope tagged with vesicular stomatitis virus glycoprotein (VSVG). The following characteristics were determined on the same passage of cells transfected with NHEIV, NHE2V, or NHE3V: 1) maximal reaction velocity ([V.sub.max]) by [sup.22][Na.sub.+] uptake and fluorometery, 2) total amount of NHE protein by quantitative Western analysis with internal standards of VSVG-tagged maltose-binding protein, and 3) cell surface expression by cell surface biotinylation. Cell surface expression (percentage of total NHE) was 88.8 [+ or -] 3.5, 64.6 [+ or -] 3.3, 20.0 [+ or -] 2.6, and 14.0 [+ or -] 1.3 for NHEIV, 85- and 75-kDa NHE2V, and NHE3V, respectively. Despite these divergent cell surface expression levels, turnover numbers for NHE1, NHE2, and NHE3 were similar (80.3 [+ or -] 9.6, 92.1 [+ or -] 8.6, and 99.2 [+ or -] 9.1 [s.sup.-1], when [V.sub.max] was determined using [sup.22]Na uptake at 22[degrees]C and 742 [+ or -] 47, 459 [+ or -] 16, and 609 [+ or -] 39 [s.sup.-1] when [V.sub.max] was determined using fluorometry at 37[degrees]C). These data indicate that, in the same cell system, intrinsic properties that determine turnover number are conserved among NHE1, NHE2, and NHE3. sodium/hydrogen antiporter; quantitative Western analysis; cell surface biotinylation; PS120 fibroblasts

In vascular smooth muscle cells, Axl is a key receptor tyrosine kinase, because it is upregulated in injury, increases migration and neointima formation, and is activated by reactive oxygen species. Reaction of glutathione with cysteine residues (termed "glutathiolation") is an important posttranslational redox modification that may alter protein activity and protein-protein interactions. To investigate the mechanisms by which reactive oxygen species increase Axl-dependent vascular smooth muscle cell function we assayed for glutathiolated proteins that associated with Axl in a redox-dependent manner. We identified glutathiolated nonmuscle myosin heavy chain (MHC)-IIB as a novel Axl interacting protein. This interaction was specific in that other myosins did not interact with Axl. The endogenous ligand for Axl, Gas6, increased production of reactive oxygen species in vascular smooth muscle cells and also increased the association of Axl with MHC-IIB. Antioxidants ebselen and N-acetylcysteine decreased the association of Axl with MHC-IIB in response to both Gas6 and reactive oxygen species. Blocking the Axl-MHC-IIB interaction with the specific myosin II inhibitor blebbistatin decreased phosphorylation of Axl and activation of extracellular signal-regulated kinase 1/2 and Akt. Association of MHC-IIB with Axl was increased in balloon-injured rat carotid vessels. Finally, expression of MHC-IIB was upregulated in the neointima of the carotid artery after balloon injury similar to upregulation of Axl protein expression, as shown in our previous studies. These results demonstrate a novel interaction between Axl and MHC-IIB in response to reactive oxygen species. This interaction provides a direct link between Axl and molecular motors crucial for directed cell migration, which may mediate increased migration in vascular dysfunction. [ABSTRACT FROM AUTHOR]