Pharmacological evidence for a KATP channel in renin-secreting cells from rat kidney

Abstract

1. Openers of the ATP-sensitive potassium channel (KATP channel) increase and blockers decrease renin secretion. Here we report the effects of levcromakalim (LCRK, a channel opener) and glibenclamide (GBC, a blocker) on membrane potential, whole-cell current and the cytoplasmic Ca2+ concentration of renin-secreting cells (RSC). Studies were performed on afferent arterioles from the kidney of Na+-depleted rats. 2. As monitored with the fluorescent oxonol dye DiBAC4(3), LCRK (0.3 and 1 microM) induced a hyperpolarization of approximately 15 mV which was abolished by GBC (1 microM). 3. Whole-cell current-clamp experiments showed that RSC had a membrane potential of -61 +/- 1 mV (n = 16). LCRK (1 microM) induced a hyperpolarization of 9.9 +/- 0.2 mV (n = 16) which, in the majority of cells, decreased slowly with time. 4. Capacitance measurements showed a strong electrical coupling of the cells in the preparation. 5. At -60 mV, LCRK induced a hyperpolarizing current in a concentration-dependent manner with an EC50 of 152 +/- 31 nM and a maximum current of about 200 pA. 6. Application of GBC (1 microM) produced no effect; however, when applied after LCRK (300 nM), GBC inhibited the opener-induced hyperpolarizing current with an IC50 of 103 +/- 36 nM. 7. LCRK (0.3 and 1 microM) did not significantly affect the cytoplasmic Ca2+ concentration either at rest or after stimulation by angiotensin II. 8. The data show that LCRK induces a GBC-sensitive hyperpolarizing current in rat RSC. This current presumably originates from the activation of KATP channels which pharmacologically resemble those in vascular smooth muscle cells. The stimulatory effect of KATP channel opening on renin secretion is not mediated by a decrease in intracellular Ca2+ concentration.

Figure 1. Glomerular preparation from the kidney of a Na⁺-depleted rat

The preparation was exposed to collagenase to allow seal formation for patch-clamp experiments. This treatment completely removed Bowman's capsule around the glomeruli. RSC in the afferent arteriole appear like a bunch of grapes which is typical for salt-depleted rats. Only vessels with these bulgy roundish cells were used for experiments. G denotes a glomerulus and AA the afferent arteriole. The scale bar represents 100 μm.

Figure 4. LCRK-induced current (I_K(LCRK)) in RSC

A, whole-cell recording at physiological K⁺ concentrations and a temperature of 37 °C; holding potential was −60 mV. Application of 0.1 and 10 μm LCRK induced concentration-dependent outward currents (I_K(LCRK)). Note the rapid fading of the current in the continued presence of 10 μm LCRK. B, concentration dependence of I_K(LCRK). Currents were normalized with respect to the maximal current (I_max) with 10 μm LCRK; I_max was 189 pA (median with 95 % confidence intervals of 161 and 368 pA; n = 20). The normalized values determined in individual experiments (small circles) and the means (larger circles) and s.e.m. are shown; for clarity, symbols are shifted to the left and right, respectively. The curve shows the Hill fit to the individual values giving a mid-point (EC₅₀) of 152 ± 31 nm and a Hill coefficient of 1.32 ± 0.25.

Figure 5. Inhibition of I_K(LCRK) by GBC

A, original trace showing the reduction of the current induced by 0.3 μm LCRK by GBC (0.1 μm). Within the time course of the experiment, the effect of GBC could be washed out only partially. Data were recorded in the whole-cell configuration under a physiological K⁺ gradient; holding potential was −60 mV and temperature was 37 °C. B, concentration-dependent inhibition of I_K(LCRK) by GBC. The degree of inhibition was normalized with respect to the current induced by 0.3 μm LCRK prior to the addition of GBC. Normalized individual values and means ±s.e.m. are shown; for clarity, symbols are shifted to the left and right, respectively. The curve shows the Hill fit to the individual values giving an IC₅₀ value of 103 ± 36 nm and a Hill coefficient of 1.38 ± 0.58.

Figure 2. Changes in DiBAC₄(3) fluorescence induced by levcromakalim

Glomeruli were incubated with the membrane potential-sensitive oxonol dye DiBAC₄(3) and the epifluorescence from the afferent arteriole near the entrance into the glomerulus was monitored. A, levcromakalim (LCRK, 0.3 μm) induced a sustained decrease in fluorescence by 4.5 % corresponding to a hyperpolarization of ≈18 mV. B, reversal of the LCRK (0.3 μm)-induced decrease in fluorescence (2 %, corresponding to a hyperpolarization of ≈8 mV) by glibenclamide (GBC, 1 μm). The slight increase in fluorescence during the time period shown probably reflects continued dye uptake.

Figure 3. Effects of LCRK and GBC on membrane potential in RSC

Membrane potential was measured with the whole-cell patch-clamp technique in the current-clamp mode. A, trace showing the effect of LCRK (1 μm) on membrane potential. Note the fading of the effect during prolonged application of the agonist (−50 % within 5 min after reaching the maximum) and the small response to a second challenge with LCRK (1 μm) 10 min after washout. B and C, superfusion of solvent (S, 0.1 per thousand ethanol + 0.1 per thousand DMSO), GBC (1 μm) and LCRK (1 μm). Note the spiking activity in these traces; see text for details.

Figure 6. [Ca²⁺]_i in RSC loaded with fura-2

A, removal of Ca²⁺ from the bath solution by EGTA induced a decrease in [Ca²⁺]_i by 75 nm, which was reversible upon washout of EGTA. B, an increase of [Ca²⁺] in the bath from 1 to 20 mM increased [Ca²⁺]_i by 137 nm; LCRK (0.3 μm) was without affect on [Ca²⁺]_i. C, double stimulation of the preparation with angiotensin II (ANG II) increased [Ca²⁺]_i by 64 and 69 nm. Application of LCRK (0.3 μm) prior to and during the second challenge did not modify the response. D, superfusion of 60 mM K⁺ increased [Ca²⁺]_i by 43 and 40 nm.