Effect of hypertensive rat plasma on ion transport of cultured vascular smooth muscle

Abstract

We layered fresh, unprocessed plasma from healthy rats with early (less than or equal to 7 days) or benign, chronic (greater than 3 wk) one-kidney, one-clip hypertension and from paired one-kidney normotensive control rats over confluent primary-cultured rat aortic smooth muscle cells. Plasma from all rats increased cellular ouabain-sensitive 86Rb+ uptake and sodium content and decreased ouabain-insensitive 86Rb+ uptake compared with uptakes and content in the presence of balanced salt solution (P less than 0.01). Cells incubated in the presence of plasma from rats with early (P less than 0.02) or chronic hypertension (P less than 0.01) had significantly reduced ouabain-sensitive 86Rb+ uptake when compared with cells incubated in normotensive plasma, but their intracellular Na+ contents were not lower. We no longer detected this uptake difference when chronic hypertensives drank 0.9% NaCl instead of water. Plasma from hypertensive rats also altered ouabain-insensitive 86Rb+ uptake by the cultured cells. These findings of this new, reproducible, and specific assay system support the hypothesis that plasma factors inhibit the membrane sodium-potassium pump in vascular smooth muscle cells in this form of hypertension. The abnormality occurs in both early and chronic stages, but may not be related to sodium intake. The data also provide evidence for plasma factors in hypertension altering membrane K+ permeability.

Long-term captopril treatment. Angiotensin II receptors and responses.

“…The aortic cell cultures were prepared as described previously. 22 Approximately 10* cells were obtained per aorta, and confluency was reached within 13 days. Analysis of Ang II receptor binding was performed at this time.…”

Hypertensive dog plasma inhibits the Na+-K+ pump of cultured vascular smooth muscle.

“…Isolated medial tissue was minced with scissors into 2-mm 2 pieces and digested with collagenase/elastase as described for rat aorta. 1 Isolated cells were plated at 1.5 x lOVcm 2 in DMEM and 5% fetal calf serum. Plating efficiency was 50 to 70%.…”

“…The concentration of citrate was carefully controlled because of known effects on cell ^Rb* uptake. 1 In some cases protease inhibitors (aprotinin, 1 U/ml blood; bacitracin, 0.4 mM final concentration; and diisopropyl fluorophosphate, 0.1 mM final concentration) were also added to the paired blood samples. These levels of protease inhibitors in plasma had no effect on 86 Rb + uptake by cultured dog or rat vascular smooth muscle cells.…”

“…As in our previous study, 1 we used a modification of the procedure of Brock et al, 9 except that the preincubation time in plasma before adding w RbCl was increased to 15 minutes and ouabain-insensitive uptake was measured in the presence of 0.5 mM ouabain. Plasma was added at a concentration of 80% to media bathing the cells, as previously, and incubation with ^RbCl was 15 minutes at 37 °C (uptake linear for at least 30 minutes).…”

“…We recently reported that fresh, unprocessed plasma from rats with benign one-kidney, one clip hypertension reduces ouabain-sensitive 86 Rb + uptake of primary cultured rat aortic smooth muscle cells when compared with cells incubated in normotensive plasma. 1 This abnormality occurs in the very early (<1 week) and also chronic (>3 weeks) stages of the hypertension. The assay system we used has many advantages over previous methods: The assay involves vascular muscle cells, a postulated effector cell for the putative humoral inhibitor in hypertension; intracellular ion concentrations are easily measured, facilitating data interpretation; and use of unprocessed plasma avoids creation, modification, release, or destruction of inhibitory or stimulatory factors by acidification, boiling, or freezing.…”