The Calcium Paradoxon of Renin Release

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The Calcium Paradoxon of Renin Release by Christian Grünberger, Birgit Obermayer, Jürgen Klar, Armin Kurtz, and Frank Schweda Circulation Research Volume 99(11):1197-1206 November 24, 2006 Copyright © American Heart Association, Inc. All rights reserved.

Figure 1. Effects of angiotensin II, endothelin-1, or thapsigargin on the intracellular calcium concentration of isolated JG cells determined by fura-2/AM measurements. Figure 1. Effects of angiotensin II, endothelin-1, or thapsigargin on the intracellular calcium concentration of isolated JG cells determined by fura-2/AM measurements. A, Original traces of fura-2/AM ratio 340 nm:380 nm. B, Fura-2/AM 340:380 nm ratio: control, peak, and 5 minutes after start of superfusion with drugs (n=5). C, Effects of angiotensin II (angII), endothelin-1 (endo), or thapsigargin (thapsi) on the intracellular calcium concentration of JG cells in the presence of 250 μmol/L cAMP-AM (n=4). #P<0.05 vs control alone. Christian Grünberger et al. Circ Res. 2006;99:1197-1206 Copyright © American Heart Association, Inc. All rights reserved.

Figure 2. Effects of angiotensin II (angII) (1 μmol/L), endothelin-1 (endo) (10 nmol/L), or thapsigargin (thapsi) (1 μmol/L) on renin release (percentage of total renin content; black bars) and intracellular cAMP content (open bars) of primary cultured JG cells in the presence of isoproterenol (isoprot.) (100 nmol/L; n=5) (A), forskolin (5 μmol/L; n=5) (B), IBMX (100 μmol/L; n=4) (C), or cAMP-AM (250 μmol/L; n=5) (D). Figure 2. Effects of angiotensin II (angII) (1 μmol/L), endothelin-1 (endo) (10 nmol/L), or thapsigargin (thapsi) (1 μmol/L) on renin release (percentage of total renin content; black bars) and intracellular cAMP content (open bars) of primary cultured JG cells in the presence of isoproterenol (isoprot.) (100 nmol/L; n=5) (A), forskolin (5 μmol/L; n=5) (B), IBMX (100 μmol/L; n=4) (C), or cAMP-AM (250 μmol/L; n=5) (D). E, Role of angiotensin II AT1 and AT2 receptors on the effects of angiotensin II on renin release (black bars) and intracellular cAMP content (open bars) of JG cells. AT1 receptors were blocked by losartan (10 μmol/L); AT2 receptors by PD123319 (10 μmol/L) (n=4). *P<0.05 vs control, #P<0.01 vs control, +P<0.05 vs forskolin/IBMX/cAMP-AM, §P<0.01 vs isoproterenol/IBMX. Christian Grünberger et al. Circ Res. 2006;99:1197-1206 Copyright © American Heart Association, Inc. All rights reserved.

Figure 3. Relationship between cAMP levels and renin release Figure 3. Relationship between cAMP levels and renin release. cAMP levels and renin release were determined under control conditions and after stimulation by either isoproterenol (100 nmol/L), cAMP-AM (250 μmol/L), forskolin (5 μmol/L), IBMX (100 μmol/L), in the absence (normal calcium, white circles) or the presence (high calcium, black circles) of angiotensin II, endothelin-1, or thapsigargin. Figure 3. Relationship between cAMP levels and renin release. cAMP levels and renin release were determined under control conditions and after stimulation by either isoproterenol (100 nmol/L), cAMP-AM (250 μmol/L), forskolin (5 μmol/L), IBMX (100 μmol/L), in the absence (normal calcium, white circles) or the presence (high calcium, black circles) of angiotensin II, endothelin-1, or thapsigargin. Solid line indicates regression line for normal calcium values; dashed line, regression line for high-calcium values. Christian Grünberger et al. Circ Res. 2006;99:1197-1206 Copyright © American Heart Association, Inc. All rights reserved.

Figure 4. Semiquantification of mRNA expression of AC1 to AC9 in freshly isolated JG cells or denuded aorta of mice by real-time PCR. AC2 and AC7 mRNA was detected in neither aortas nor JG cells. Figure 4. Semiquantification of mRNA expression of AC1 to AC9 in freshly isolated JG cells or denuded aorta of mice by real-time PCR. AC2 and AC7 mRNA was detected in neither aortas nor JG cells. AC3 and AC8 mRNA was only detected in aortas. *P<0.05 vs aorta, #P<0.01 vs aorta. n.d. indicates not detectable. Data are mean+SEM of 5 preparations, respectively. Christian Grünberger et al. Circ Res. 2006;99:1197-1206 Copyright © American Heart Association, Inc. All rights reserved.

Figure 5. A, mRNA expression of AC5 and AC6 under control conditions (no siRNA), after transfection with control siRNA and siRNA directed against AC5 and AC6. Figure 5. A, mRNA expression of AC5 and AC6 under control conditions (no siRNA), after transfection with control siRNA and siRNA directed against AC5 and AC6. B, Protein expression of AC5 in response to transfection with control siRNA and after transfection with siRNA directed against AC5 and AC6. *P<0.05 vs control siRNA, #P<0.01 vs control siRNA; n=4 respectively. Christian Grünberger et al. Circ Res. 2006;99:1197-1206 Copyright © American Heart Association, Inc. All rights reserved.

Figure 6. Effects of forskolin 5 μmol/L and isoproterenol 100 nmol/L on cAMP levels of renin-producing As4.1 cells with or without endothelin 10 nmol/L or thapsigargin 1 μmol/L. Figure 6. Effects of forskolin 5 μmol/L and isoproterenol 100 nmol/L on cAMP levels of renin-producing As4.1 cells with or without endothelin 10 nmol/L or thapsigargin 1 μmol/L. Cells were transfected with control siRNA, siRNA directed against AC5 or AC6, or a combination of AC5 and AC6 siRNA for 72 hours before stimulation. *P<0.05 vs vehicle, #P<0.01 vs vehicle; n=6. Christian Grünberger et al. Circ Res. 2006;99:1197-1206 Copyright © American Heart Association, Inc. All rights reserved.

Figure 7. Effects of forskolin (5 μmol/L) and isoproterenol (100 nmol/L) on renin release (percentage of total renin content) from As4.1 cells with or without endothelin (10 nmol/L) or thapsigargin (1 μmol/L). Figure 7. Effects of forskolin (5 μmol/L) and isoproterenol (100 nmol/L) on renin release (percentage of total renin content) from As4.1 cells with or without endothelin (10 nmol/L) or thapsigargin (1 μmol/L). Cells were transfected with control siRNA, siRNA directed against AC5 or AC6, or a combination of AC5 and AC6 siRNA for 72 hours before stimulation. *P<0.05 vs vehicle, #P<0.01 vs vehicle; n=5. Christian Grünberger et al. Circ Res. 2006;99:1197-1206 Copyright © American Heart Association, Inc. All rights reserved.

Figure 8. Effects of angiotensin II 3 nmol/L on the stimulation of renin secretion by isoproterenol 30 nmol/L (closed circles) or 8-br-cAMP 250 μmol/L (open circles). Figure 8. Effects of angiotensin II 3 nmol/L on the stimulation of renin secretion by isoproterenol 30 nmol/L (closed circles) or 8-br-cAMP 250 μmol/L (open circles). An angiotensin II infusion started either before (bottom) or after isoproterenol or 8-br-cAMP administration (top); n=5 each. For statistics, see text. Christian Grünberger et al. Circ Res. 2006;99:1197-1206 Copyright © American Heart Association, Inc. All rights reserved.