Angiotensin increases Na+ entry and Na+/K+ pump activity in cultures of smooth muscle from rat aorta.

Abstract

Angiotensin markedly altered the Na' permeability of smooth muscle cells cultured from explants of rat aorta. The rate ofnet Na' uptake was followed in the presence ofouabain in order to block Na' efflux via the Na+/K' pump. Angiotensin H (All) or angiotensin m (AHI) increased net Na' uptake by about 3-fold. Maximal stimulation of Na' uptake was produced by about 10 nM AUl. Bradykinin and the angiotensin antagonist [Sari, Ileu5, Ala8JAII had no significant effect on net Na+ uptake. Angiotensin also enhanced the activity of the Na+/K+ pump, which was assayed by following the rate of ouabain-sensitive wRb+ uptake by the cells. AII and AI nearly doubled ouabain-sensitive 86Rb+ uptake, but bradykinin, norepinephrine, and [SariIleu5,Ala8]AII had no effect. In the presence of ouabain, 86Rb+ uptake was not significantly affected by AUl or AM, indicating that angiotensin did not alter passive permeability to Rb+. Loading the cells with Na+, either by incubation in K+-free medium or exposure to the Na+-selective ionophore monensin, markedly increased ouabainsensitive 86Rb+ uptake. This result indicates that the activity ofthe Na+/K+ pump is limited by the low level of Na+ that is normally in the cells. All had no effect on the activity of the Na+/K+ pump in Na+-loaded cells. These results suggest that All or AIM stimulates the Na+/K+ pump in cultured aortic muscle cells by increasing its Na+ supply.The octapeptide hormone angiotensin II (All) is a highly potent vasoconstrictor (1-3). Specific receptors on the surface of smooth muscle cells (4) mediate the contractile response to All, which occurs when the level of free Ca2" in the cytoplasm increases (5, 6). It is unclear what transmembrane events are elicited when All binds to its receptor on the cell surface.All is known to depolarize the smooth muscle membrane in various tissues (7)(8)(9). Recently, Hamon and Worcel (9) reported evidence suggesting that All depolarizes uterine smooth muscle by increasing the conductance of the membrane to Na+. Alterations in Na+ permeability also appear to be involved in vasoconstriction by angiotensin. Increasing the Na+ concentration in the medium bathing segments of rat aorta potentiates the contractile response to All (10), and perfusing rat tail arteries with low Na+ decreases the constriction elicted by All (11). However, the reported influences of angiotensin on the Na+ movements in isolated vascular tissue are ambiguous. Although Friedman and Allardyce (12) reported that angiotensin decreased the Na' activity in the medium bathing an artery, Guignard and Friedman (13) found that pressor doses ofAll had no such effect.All was recently shown to stimulate phosphorylation of the 20,000-dalton light chain of myosin in primary cultures of vascular smooth muscle (14). Therefore, it is feasible to study vasoactive mechanisms in cultured cells. In an attempt to elucidate further the effects of angiotensin on cation transport, we have examined the influence of vasoactive agents on Na' and K+ movements in cultures of rat aor...

Phorbol Diacetate Potentiates Na + -K + ATPase Inhibition by a Putative Endogenous Ligand, Marinobufagenin

“…[1][2][3] Protein kinases phosphorylate the sodium pump in a tissue-and isoform-specific fashion. 4,5 Protein kinase C (PKC) directly phosphorylates the ␣ 1 -NKA isoform.…”

Modulation of aortic smooth muscle cell membrane potential by extracellular calcium.

“…RASM cells were prepared by a method modified from Brock et al 18 The thoracic aortas of 10-week-old Wistar rats were removed, trimmed of fat, and rinsed with a physiological salt solution (PSS 1) of the following composition (mM): NaCl 110, KC1 5.4, MgSO 4 0.4, Na 2 HPO 4 1.04, NaHCO 3 22, CaCl 2 1.0, Glucose 5, iV-(2-hydroxyethyl)piperazine-7V'-2-ethanesulfonic acid (HEPES) 25, and gassed with 95% O 2 and 5% CO 2 ; pH 7.4 at 37° C. After a 30-minute treatment with 1.0 mg/ml collagenase type II (Sigma Chemical Co., St. Louis, Missouri) and 0.5 mg/ml elastase (Calbiochem-Behring, San Diego, California), the tunica adventitia was removed, and the remaining vessel core was reincubated for 2 hours in PSS 1 containing the same enzyme mixture. The solution was then filtered to remove vessel fragments, and the cell suspension was centrifuged and washed three times with PSS 1.…”

Direct measurement of sodium influx in vascular smooth muscle. Stimulation by aldosterone.

“…Brock et al 13 used cultured smooth muscle from aorta and measured the increase in the intracellular sodium content caused by ouabain. They washed out the extracellular sodium by rinsing with cold MgCl 2 solution, but it is difficult to judge from their description of this method how effective it was.…”

“…The method originally was developed for rat tail artery, 6 but it has been used successfully in other preparations, including aorta 13 and portal vein. 14 In rabbit portal vein, the decline in the intracellular sodium content during 30 to 120 minutes in cold, lithium-substituted, sodium-free solution, as measured by the electron microprobe, is similar to that seen in rat tail artery under similar conditions.…”