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. 2010 Sep;120(9):3267-79.
doi: 10.1172/JCI41348. Epub 2010 Aug 2.

Increased catecholamine secretion contributes to hypertension in TRPM4-deficient mice

Ilka Mathar  1 , Rudi Vennekens, Marcel Meissner, Frieder Kees, Gerry Van der Mieren, Juan E Camacho Londoño, Sebastian Uhl, Thomas Voets, Björn Hummel, An van den Bergh, Paul Herijgers, Bernd Nilius, Veit Flockerzi, Frank Schweda, Marc Freichel
Affiliations

Increased catecholamine secretion contributes to hypertension in TRPM4-deficient mice

Ilka Mathar et al. J Clin Invest. 2010 Sep.

Abstract

Hypertension is an underlying risk factor for cardiovascular disease. Despite this, its pathogenesis remains unknown in most cases. Recently, the transient receptor potential (TRP) channel family was associated with the development of several cardiovascular diseases linked to hypertension. The melastatin TRP channels TRPM4 and TRPM5 have distinct properties within the TRP channel family: they form nonselective cation channels activated by intracellular calcium ions. Here we report the identification of TRPM4 proteins in endothelial cells, heart, kidney, and chromaffin cells from the adrenal gland, suggesting that they have a role in the cardiovascular system. Consistent with this hypothesis, Trpm4 gene deletion in mice altered long-term regulation of blood pressure toward hypertensive levels. No changes in locomotor activity, renin-angiotensin system function, electrolyte and fluid balance, vascular contractility, and cardiac contractility under basal conditions were observed. By contrast, inhibition of ganglionic transmission with either hexamethonium or prazosin abolished the difference in blood pressure between Trpm4-/- and wild-type mice. Strikingly, plasma epinephrine concentration as well as urinary excretion of catecholamine metabolites were substantially elevated in Trpm4-/- mice. In freshly isolated chromaffin cells, lack of TRPM4 was shown to cause markedly more acetylcholine-induced exocytotic release events, while neither cytosolic calcium concentration, size, nor density of vesicles were different. We therefore conclude that TRPM4 proteins limit catecholamine release from chromaffin cells and that this contributes to increased sympathetic tone and hypertension.

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Figures

Figure 1
Figure 1. Elevated blood pressure in TRPM4-deficient mice.
(A) Western blot of protein fractions from aortic endothelial cells (MAEC), atrium, ventricle, kidney, and adrenal gland of WT (+/+) and TRPM4-deficient (–/–) mice using TRPM4-specific antibody 578. (B–E) Time course of MAP (*P < 0.01, B), SBP (*P < 0.05) and DBP (*P < 0.01) (C), and HR (D) in WT (black, n = 7–13) and Trpm4–/– mice (red, n = 8–15) after implantation of the blood pressure transmitter. (E) Averaged MAP, SBP, and DBP from the period of day 9 to day 13 after implantation (*P < 0.01).
Figure 2
Figure 2. Circadian rhythm of blood pressure, HR, and locomotor activity in TRPM4-deficient mice.
Circadian rhythm of MAP (A), HR (B), and locomotor activity (C) of WT (black, n = 7–13) and Trpm4–/– mice (red, n = 8–15) averaged from day 9 to day 13 after implantation of the blood pressure transmitter. The time period between 6:00 p.m. and 6:00 a.m. is marked in gray. (D) Activity-dependent analysis of MAP, SBP, and DBP blood pressure from day 9 to day 13 after operation; *P < 0.01. Blood pressure values at a locomotor activity score above 9 (active) or below 9 (nonactive) were analyzed.
Figure 3
Figure 3. RAAS and fluid balance in Trpm4–/– mice.
(A) Renin secretion rate in the perfusate of isolated kidneys from WT (black) and Trpm4–/– (red, n = 3 per genotype) mice before and after administration of isoproterenol (10 nM) and Ang II (0.1–1.0 nM). (B) Renal renin mRNA expression normalized to β-actin mRNA in WT (n = 5) and Trpm4–/– mice (n = 6) determined via real-time PCR (left panel); and plasma renin activity (right panel) in Trpm4–/– (n = 25) and WT mice (n = 23). (C) Plasma aldosterone concentrations in WT and Trpm4–/– (n = 18 per genotype) mice. (D–H) Analysis of fluid and electrolyte balance. Electrolyte excretion (WT, n = 21; Trpm4–/–, n = 22) in 24-hour urine (D), serum electrolyte concentrations (n = 8 per genotype, E), hematocrit (n = 8 per genotype, F), plasma volume (n = 5 per genotype, G), and 24-hour urine volume (WT, n = 21; Trpm4–/–, n = 22; *P < 0.05, H).
Figure 4
Figure 4. Vascular reactivity in Trpm4–/– mice.
(A) MAP and HR after i.p. injection (at time point 0 minutes) of PE (1.0 mg/kg BW; WT, black, n = 7; Trpm4–/– red, n = 9). (B) Concentration-response curve of PE-induced contraction of isolated aortic rings (WT n = 7, Trpm4–/– n = 8). (C) Time course of vascular resistance of the vasculature of isolated hind limbs calculated from perfusion pressure and perfusate flow (inset); bolus injections (200 μl) of increasing PE concentrations (in μM) are indicated by arrowheads. (D) Dose-response curves of changes in flow and vascular resistance after application of PE in the perfused hind limb vasculature (n = 9 per genotype). (E) Averaged changes in perfusate flow after bolus injections (200 μl) of increasing epinephrine concentrations (in μM; n = 6 per genotype). (F) Changes in vascular resistance of the perfused hind limb after bolus injections (200 μl) of increasing acetylcholine concentrations (n = 7 per genotype). (G and H) Analysis of pressure-induced vascular resistance. Average change in vascular resistance (G) using physiological KH solution (black, red) or Ca2+-free KH (gray, dark red) as perfusate in WT (upper panel, n = 11) or Trpm4–/– mice (lower panel, n = 10); intravascular pressure is indicated. (H) Analysis of the myogenic response in dependence of the intravascular pressure calculated from measurement in G.
Figure 5
Figure 5. Neurogenic mechanisms contribute to hypertension in Trpm4–/– mice.
(A) Time course of MAP before and after inhibition of ganglionic transmission by hexamethonium (20 mg/kg i.p.) in Trpm4–/– (red, n = 9) and WT (black, n = 7) mice. (B and C) Averaged peak response of MAP to injection of hexamethonium (*P < 0.01) (B) or prazosin (1 mg/kg i.p.; WT, n = 7, Trpm4–/–, n = 9; *P < 0.05, C). (D) Plasma epinephrine and norepinephrine concentrations in WT and Trpm4–/– mice (n = 14 per genotype, *P < 0.01). (E) Catecholamine metabolite concentration in urine collected for 24 hours from WT (n = 15; for VMA, n = 6) and Trpm4–/– mice (n = 14; for VMA, n = 8; *P < 0.05).
Figure 6
Figure 6. Adrenal gland phenotype in Trpm4–/– mice.
Increased rate of acetylcholine-induced catecholamine release from TRPM4-deficient chromaffin cells. (A) Representative examples of H&E-stained adrenal gland sections from WT and Trpm4–/– mice. Scale bars: 100 μm (upper panels) and 20 μm (lower panels). (B) Amplification of Trpm4 and Hprt transcripts by RT-PCR from RNA of cell clusters dissected from the adrenal medulla. neg Trpm4 and neg HPRT indicate amplifications without template cDNA. (C) Representative traces of carbon fiber amperometry and simultaneous measurements of intracellular calcium concentration ([Ca2+]cyt) in chromaffin cells from WT and Trpm4–/– mice. (D) Averaged [Ca2+]cyt before (baseline) and at indicated time points after application of acetylcholine (ACh; 10 μM) in chromaffin cells from WT (n = 9) and Trpm4–/– mice (n = 14). (E) Number of amperometric events under basal conditions during 5 minutes before stimulation in WT (n = 9) and Trpm4–/– (n = 14) chromaffin cells. (F) Box plot (left) and cumulative analysis (right) of amperometric events after stimulation with 10 μM acetylcholine in WT (n = 9) and Trpm4–/– (n = 14) chromaffin cells. *P < 0.01; WT and Trpm4–/– values in the cumulative probability plot are significantly different (Kolmogorov-Smirnov test, P = 0.015).
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