Ca²⁺ signaling and membrane potential in descending vasa recta pericytes and endothelia

Abstract

We devised a method for removal of pericytes from isolated descending vasa recta (DVR). After enzymatic digestion, aspiration of a descending vas rectum into a micropipette strips the pericytes from the abluminal surface. Pericytes and denuded endothelia can be recovered for separate study. Using fura 2-loaded preparations, we demonstrated that 10 nM angiotensin II (ANG II) elevates pericyte intracellular Ca(2+) concentration ([Ca(2+)](i)) and suppresses endothelial [Ca(2+)](i). The anion transport blocker probenecid helps retain fura 2 in the pericyte cytoplasm. DVR endothelia were accessed for membrane potential measurement by perforated-patch whole cell recording by using the pericyte-stripping technique and by turning nondigested vessels inside out with concentric micropipettes. By either method of access, 10 nM ANG II depolarized (n = 20) and 100 nM bradykinin hyperpolarized (n = 25) the endothelia. We conclude that isolated endothelia and pericytes remain functional for study of [Ca(2+)](i) responses and that ANG II and bradykinin receptors exist separately on each cell type.

Physiology of the renal medullary microcirculation

“…As expected, BK generates a peak-and-plateau Ca 2ϩ response, enhances NO generation (Fig. 8), and induces vasodilation (112,129,130). An unexpected finding is that the vasoconstrictor ANG II suppresses basal Ca 2ϩ and inhibits BK-, acetylcholine-, thapsigargin-, and cyclopiazonic acid-induced Ca 2ϩ responses in DVR endothelia (Fig.…”

“…As expected for signaling via the ANG II AT 1 receptor, a classic peak-and-plateau intracellular Ca 2ϩ response is elicited in fura 2-loaded pericytes ( Fig. 3) (130,176). Both whole cell electrophysiological recording and measurements with a potentially sensitive fluorescent probe showed that ANG II depolarizes the pericyte cell membrane (Fig.…”

“…An unexpected finding is that the vasoconstrictor ANG II suppresses basal Ca 2ϩ and inhibits BK-, acetylcholine-, thapsigargin-, and cyclopiazonic acid-induced Ca 2ϩ responses in DVR endothelia (Fig. 7, B and C) (114,130). This is surprising for several reasons.…”

“…Both whole cell electrophysiological recording and measurements with a potentially sensitive fluorescent probe showed that ANG II depolarizes the pericyte cell membrane (Fig. 4) (107,130,175,176). This seems to occur through activation of a Ca 2ϩ -sensitive Cl Ϫ conductance that shifts membrane potential away from the equilibrium potential of the K ϩ ion toward that of Cl Ϫ (107,175).…”

“…B: isolated collagenase-treated vessel has been drawn into a glass micropipette with a heat-polished opening of ϳ6 m diameter, stripping pericytes from the abluminal surface. This process can be continued to isolate a group of pericytes for loading with the Ca 2ϩ -sensitive fluorescent indicator fura 2 (130). Bar ϭ ϳ10 m. C: intracellular Ca 2ϩ concentration ([Ca 2ϩ ]i) response of fura 2-loaded DVR pericytes to ANG II (10 nM) is shown in the presence and absence of diltiazem, n ϭ 6 and 7, respectively.…”