Thiol-dependent passive K+Cl− transport in sheep red blood cells: VI. Functional heterogeneity and immunologic identity with volume-stimulated K+(Rb+) fluxes

Summary Ouabain-resistant (OR), volume-or N-ethylmaleimide (NEM)-stimulated K⁺(Rb⁺)Cl^– fluxes were measured in low-K⁺ sheep red cells and found to be functionally separate but immunologically similar. In anisosmotic solutions both K⁺ effluxes and Rb⁺ influxes of NEM-treated and control cells were additive. In contrast to the NEM-stimulated K⁺Cl^– flux, metabolic depletion did not reduce K⁺Cl^– flux of normal or swollen cells. The anion preference of OR K⁺ efflux in NEM-treated cells was Br^–>Cl^–>HCO ₃ ^– =F^–I^–=NO ₃ ^– =CNS^–, and hence consistent with a reported Br^–>Cl^–>NO ₃ ^– sequence of the volume-dependent K⁺Cl^– transport. Alloimmune anti-L_l antibodies known to decrease passive K⁺ fluxes in low K⁺ cells reduced by 51% both volume-and NEM-stimulated, furosemidesensitive Rb⁺Cl^– fluxes suggesting their immunologic identity, a conclusion also supported by anti-L₁ absorption studies. Since pretreatment with anti-L₁ prevented the activation of Rb⁺ influx by NEM, and the impermeant glutathionmaleimide-I did not stimulate Rb⁺Cl^– influx, the NEM reactive SH groups must be located apart from the L₁ antigen either within the membrane or on its cytoplasmic face. A model is proposed consisting of a K⁺Cl^– transport path(s) regulated by a protein with two functional subunits or domains; a chemically (C _s) and a volume (V _s)-stimulated domain, both interfacing with the L₁ surface antigen. Attachment of alloanti-L₁ from the outside reduces K⁺Cl^– transport stimulated throughC _s by NEM orV _s by cell swelling.     

Infliximab in ankylosing spondylitis: alone or in combination with methotrexate? A pharmacokinetic comparative study

Introduction  

Methotrexate (MTX) has been shown to modify infliximab pharmacokinetics in rheumatoid arthritis. However, its combination with infliximab in the treatment of ankylosing spondylitis (AS) is not recommended. The objective of this study was to examine the influence of MTX on infliximab exposure in patients with AS.     

Antibodies toward infliximab are associated with low infliximab concentration at treatment initiation and poor infliximab maintenance in rheumatic diseases

Introduction  

A proportion of patients receiving infliximab have antibodies toward infliximab (ATI), which are associated with increased risk of infusion reaction and reduced response to treatment. We studied the association of infliximab concentration at treatment initiation and development of ATI as well as the association of the presence of ATI and maintenance of infliximab.     

GSH depletion, K-Cl cotransport, and regulatory volume decrease in high-K/high-GSH dog red blood cells

Thiol reagents activateK-Cl cotransport (K-Cl COT), the Cl-dependent and Na-independentouabain-resistant K flux, in red blood cells (RBCs) of several species,upon depletion of cellular glutathione (GSH). K-Cl COT isphysiologically active in high potassium (HK), high GSH (HG) dog RBCs.In this unique model, we studied whether the same inverse relationshipexists between GSH levels and K-Cl COT activity found in other species.The effects of GSH depletion by three different chemical reactions[nitrite (NO₂)-mediated oxidation, diazene dicarboxylicacid bis-N,N-dimethylamide (diamide)-induceddithiol formation, and glutathione S-transferase (GST)-catalyzed conjugation of GSH with 1-chloro-2,4-dinitrobenzene (CDNB)] were tested on K-Cl COT and regulatory volume decrease (RVD).After 85% GSH depletion, all three interventions stimulated K-Cl COThalf-maximally with the following order of potency: diamide > NO₂ > CDNB. Repletion of GSH reversed K-Cl COTstimulation by 50%. Cl-dependent RVD accompanied K-Cl COT activationby NO₂ and diamide. K-Cl COT activation at concentrationratios of oxidant/GSH greater than unity was irreversible, suggestingeither nitrosothiolation, heterodithiol formation, or GST-mediateddinitrophenylation of protein thiols. The data support the hypothesisthat an intact redox system, rather than the absolute GSH levels,protects K-Cl COT activity and cell volume regulation from thiol modification.

     

NONOates regulate KCl cotransporter-1 and -3 mRNA expression in vascular smooth muscle cells

Nitric oxide (NO) donors regulate KCl cotransport (KCC) activity and cotransporter-1 and -3 (KCC1 and KCC3) mRNA expression in sheep erythrocytes and in primary cultures of rat vascular smooth muscle cells (VSMCs), respectively. In this study, we used NONOates as rapid and slow NO releasers to provide direct evidence implicating NO as a regulator of KCC3 gene expression at the mRNA level. In addition, we used the expression of KCC3 mRNA to further investigate the mechanism of action of these NO donors at the cellular level. Treatment of VSMCs with rapid NO releasers, like NOC-5 and NOC-9, as well as with the direct NO-independent soluble guanylyl cyclase (sGC) stimulator YC-1, acutely increased KCC3 mRNA expression in a concentration- and time-dependent manner. The slow NO releaser NOC-18 had no effect on KCC3 gene expression. A specific NO scavenger completely prevented the NONOate-induced KCC3 mRNA expression. Inhibition of sGC with LY-83583 blocked the NONOate- and YC-1-induced KCC3 mRNA expression. This study shows that in primary cultures of rat VSMCs, the fast NO releasers NOC-9 and NOC-5, but not the slow NO releaser NOC-18, acutely upregulate KCC3 mRNA expression in a NO/sGC-dependent manner.