Pernille B. Lærkegaard Hansen

Rapid Inhibition of Vasoconstriction in Renal Afferent Arterioles by Aldosterone

Abstract— Aldosterone has been suggested to elicit vessel contraction via a nongenomic mechanism. We tested this proposal in microdissected, perfused rabbit renal afferent arterioles. Aldosterone had no effect on internal diameter in concentrations from 10−10 to 10−5 mol/L, but aldosterone abolished the ability of 100 mmol/L KCl to induce vascular contraction. The inhibitory effect of aldosterone was observed from 1 pmol/L. The inhibitory effect was significant after 5 minutes and maximal after 20 minutes and was fully reversible. Actinomycin D (10−6 mol/L) prolonged the effect of aldosterone. The effect was abolished by the mineralocorticoid receptor antagonist spironolactone (10−7 mol/L) but not by the glucocorticoid receptor antagonist mifepristone (10−6 mol/L). The K+-mediated increase of intracellular calcium concentration in afferent arterioles was not affected by aldosterone. Mineralocorticoid receptor was detected by reverse transcription–polymerase chain reaction and immunohistochemistry in rat renal vasculature and rabbit endothelial cells. Inhibition of phosphatidylinositol (PI)-3 kinase with LY 294002 (3×10−6 mol/L) restored sensitivity to K+ in the presence of aldosterone, and afferent arterioles were immunopositive for PI-3 kinase subunit p110&agr;. Inhibition of NO formation by L-NAME (10−4 mol/L) or inhibition of soluble guanylyl cyclase with 1H-(1,2,4)Oxadiazolo[4,3-a]quinoxaline-1-one restored K+-induced vasoreactivity in the presence of aldosterone. Similar to aldosterone, the NO donor sodium nitroprusside inhibited K+-induced vascular contraction. Geldanamycin (10−6 mol/L), an inhibitor of heat shock protein 90, abolished aldosterone-induced vasorelaxation. We conclude that aldosterone inhibits depolarization-induced vasoconstriction in renal afferent arterioles by a rapid nongenomic mechanism that is initiated by mineralocorticoid receptor activation and involves PI-3 kinase, protein kinase B, and heat shock protein 90–mediated stimulation of NO generation.

Adenosine Induces Vasoconstriction through Gi-Dependent Activation of Phospholipase C in Isolated Perfused Afferent Arterioles of Mice

Adenosine induces vasoconstriction of renal afferent arterioles through activation of A1 adenosine receptors (A1AR). A1AR are directly coupled to Gi/Go, resulting in inhibition of adenylate cyclase, but the contribution of this signaling pathway to smooth muscle cell activation is unclear. In perfused afferent arterioles from mouse kidney, adenosine and the A1 agonist N(6)-cyclohexyladenosine, when added to the bath, caused constriction in the concentration range of 10(-9) to 10(-6) M (mean diameter: control, 8.8 +/- 0.3 micro m; adenosine at 10(-6) M, 2.8 +/- 0.5 micro m). Adenosine-induced vasoconstriction was stable for up to 30 min and was most pronounced in the most distal part of the afferent arterioles. Adenosine did not cause vasoconstriction in arterioles from A1AR-/- mice. Pretreatment with pertussis toxin (PTX) (400 ng/ml) for 2 h blocked the vasoconstricting action of adenosine or N(6)-cyclohexyladenosine. PTX pretreatment did not affect the constriction response to KCl, whereas the angiotensin II dose-response relationship was shifted rightward. Reverse transcription-PCR revealed expression of Gi but not Go in kidney cortex and preglomerular vessels. The phospholipase C inhibitor U73122 (4 micro M) blocked the constriction responses to both adenosine and angiotensin II. In contrast, the adenylate cyclase inhibitor SQ22536 (10 micro M) and the protein kinase A antagonist KT5720 (0.1 and 1 micro M) did not induce significant vasoconstriction of afferent arterioles. It is concluded that the constriction response to adenosine in afferent arterioles is mediated by A1AR coupled to a PTX-sensitive Gi protein and subsequent activation of phospholipase C, presumably through betagamma subunits released from Galphai.

Vascular Smooth Muscle Cells Express the α1A Subunit of a P-/Q-Type Voltage-Dependent Ca2+Channel, and It Is Functionally Important in Renal Afferent Arterioles

In the present study, we tested whether the &agr;1A subunit, which encodes a neuronal isoform of voltage-dependent Ca2+ channels (VDCCs) (P-/Q-type), was present and functional in vascular smooth muscle and renal resistance vessels. By reverse transcription–polymerase chain reaction and Southern blotting analysis, mRNA encoding the &agr;1A subunit was detected in microdissected rat preglomerular vessels and vasa recta, in cultures of rat preglomerular vascular smooth muscle cells (VSMCs), and in cultured rat mesangial cells. With immunoblots, &agr;1A subunit protein was demonstrated in rat aorta, brain, aortic smooth muscle cells (A7r5), VSMCs, and mesangial cells. Immunolabeling with an anti-&agr;1A antibody was positive in acid-macerated, microdissected preglomerular vessels and in A7r5 cells. Patch-clamp experiments on aortic A7r5 cells showed 22±4% (n=6) inhibition of inward Ca2+ current by &ohgr;-Agatoxin IVA (10–8 mol/L), which in this concentration is a specific inhibitor of P-type VDCCs. Measurements of intracellular Ca2+ in afferent arterioles with fluorescence-imaging microscopy showed 32±9% (n=10) inhibition of the K+-induced rise in Ca2+ in the presence of 10–8 mol/L &ohgr;-Agatoxin IVA. In microperfused rabbit afferent arterioles, &ohgr;-Agatoxin IVA inhibited depolarization-mediated contraction with an EC50 of 10–17 mol/L and complete blockade at 10–14 mol/L. We conclude that the &agr;1A subunit is expressed in VSMCs from renal preglomerular resistance vessels and aorta, as well as mesangial cells, and that P-type VDCCs contribute to Ca2+ influx in aortic and renal VSMCs and are involved in depolarization-mediated contraction in renal afferent arterioles.

Prostaglandin E2 Induces Vascular Relaxation by E-Prostanoid 4 Receptor-Mediated Activation of Endothelial Nitric Oxide Synthase

The present experiments were designed to test the hypothesis that prostaglandin (PG) E2 causes vasodilatation through activation of endothelial NO synthase (eNOS). Aortic rings from mice with targeted deletion of eNOS and E-prostanoid (EP) receptors were used for contraction studies. Blood pressure changes in response to PGE2 were measured in conscious mice. Single doses of PGE2 caused concentration-dependent relaxations during contractions to phenylephrine (EC50=5*10−8 mol/L). Relaxation after PGE2 was absent in rings without endothelium and in rings from eNOS−/− mice and was abolished by NG-nitro-l-arginine methyl ester and the soluble guanylate cyclase inhibitor 1H1,2,4-oxadiazolo-[4,3-a]quinoxalin-1-one. In PGE2-relaxed aortic rings, the cGMP content increased significantly. PGE2-induced relaxations were abolished by the EP4 receptor antagonist AE3–208 (10−8 mol/L) and mimicked by an EP4 agonist (AE1–329, 10−7 mol/L) in the presence of endothelium and eNOS only. Relaxations were attenuated significantly in rings from EP4−/− mice but normal in EP2−/−. Inhibitors of the cAMP-protein kinase A pathway attenuated, whereas the inhibitor of protein phosphatase 1C, calyculin (10−8 mol/L), abolished the PGE2-mediated relaxation. In aortic rings, PGE2 dephosphorylated eNOS at Thr495. Chronically catheterized eNOS−/− mice were hypertensive (137±3.6 mm Hg, n=13, versus 101±3.9 mm Hg, n=9) and exhibited a lower sensitivity of blood pressure reduction in response to PGE2 compared with wild-type mice. There was no difference in the blood pressure response to nifedipine. These findings show that PGE2 elicits EP4 receptor-mediated, endothelium-dependent stimulation of eNOS activity by dephosphorylation at Thr495 resulting in guanylyl cyclase–dependent vasorelaxation and accumulation of cGMP in aortic rings.

Angiotensin II Regulates microRNA-132/-212 in Hypertensive Rats and Humans

MicroRNAs (miRNAs), a group of small non-coding RNAs that fine tune translation of multiple target mRNAs, are emerging as key regulators in cardiovascular development and disease. MiRNAs are involved in cardiac hypertrophy, heart failure and remodeling following cardiac infarction; however, miRNAs involved in hypertension have not been thoroughly investigated. We have recently reported that specific miRNAs play an integral role in Angiotensin II receptor (AT1R) signaling, especially after activation of the Gαq signaling pathway. Since AT1R blockers are widely used to treat hypertension, we undertook a detailed analysis of potential miRNAs involved in Angiotensin II (AngII) mediated hypertension in rats and hypertensive patients, using miRNA microarray and qPCR analysis. The miR-132 and miR-212 are highly increased in the heart, aortic wall and kidney of rats with hypertension (159 ± 12 mm Hg) and cardiac hypertrophy following chronic AngII infusion. In addition, activation of the endothelin receptor, another Gαq coupled receptor, also increased miR-132 and miR-212. We sought to extend these observations using human samples by reasoning that AT1R blockers may decrease miR-132 and miR-212. We analyzed tissue samples of mammary artery obtained from surplus arterial tissue after coronary bypass operations. Indeed, we found a decrease in expression levels of miR-132 and miR-212 in human arteries from bypass-operated patients treated with AT1R blockers, whereas treatment with β-blockers had no effect. Taken together, these data suggest that miR-132 and miR-212 are involved in AngII induced hypertension, providing a new perspective in hypertensive disease mechanisms.

Control of renin secretion from rat juxtaglomerular cells by cAMP-specific phosphodiesterases.

We tested the hypothesis that cGMP stimulates renin release through inhibition of the cAMP-specific phosphodiesterase 3 (PDE3) in isolated rat juxtaglomerular (JG) cells. In addition, we assessed the involvement of PDE4 in JG-cell function. JG cells expressed PDE3A and PDE3B, and the PDE3 inhibitor trequinsin increased cellular cAMP content, enhanced forskolin-induced cAMP formation, and stimulated renin release from incubated and superfused JG cells. Trequinsin-mediated stimulation of renin release was inhibited by the permeable protein kinase A antagonist Rp-8-CPT-cAMPS. PDE4C was also expressed, and the PDE4 inhibitor rolipram enhanced cellular cAMP content. Dialysis of single JG cells with cAMP in whole-cell patch-clamp experiments led to concentration-dependent, biphasic changes in cell membrane capacitance (Cm) with a marked increase in Cm at 1 &mgr;mol/L, no net change at 10 &mgr;mol/L, and a decrease at 100 &mgr;mol/L cAMP. cGMP also had a dual effect on Cm at 10-fold higher concentration compared with cAMP. Trequinsin, milrinone, and rolipram mimicked the effect of cAMP on Cm. Trequinsin, cAMP, and cGMP enhanced outward current 2- to 3-fold at positive membrane potentials. The effects of cAMP, cGMP, and trequinsin on Cm and cell currents were abolished by inhibition of protein kinase A with Rp-cAMPs. We conclude that degradation of cAMP by PDE3 and PDE4 contributes to regulation of renin release from JG cells. Our data provide evidence at the cellular level that stimulation of renin release by cGMP involves inhibition of PDE3 resulting in enhanced cAMP formation and activation of the cAMP sensitive protein kinase.

Prostaglandin F2alpha elevates blood pressure and promotes atherosclerosis.

Little is known about prostaglandin F2α in cardiovascular homeostasis. Prostaglandin F2α dose-dependently elevates blood pressure in WT mice via activation of the F prostanoid (FP) receptor. The FP is expressed in preglomerular arterioles, renal collecting ducts, and the hypothalamus. Deletion of the FP reduces blood pressure, coincident with a reduction in plasma renin concentration, angiotensin, and aldosterone, despite a compensatory up-regulation of AT1 receptors and an augmented hypertensive response to infused angiotensin II. Plasma and urinary osmolality are decreased in FP KOs that exhibit mild polyuria and polydipsia. Atherogenesis is retarded by deletion of the FP, despite the absence of detectable receptor expression in aorta or in atherosclerotic lesions in Ldlr KOs. Although vascular TNFα, inducible nitric oxide enzyme and TGFβ are reduced and lesional macrophages are depleted in the FP/Ldlr double KOs, this result reflects the reduction in lesion burden, as the FP is not expressed on macrophages and its deletion does not alter macrophage cytokine generation. Blockade of the FP offers an approach to the treatment of hypertension and its attendant systemic vascular disease.

Superparamagnetic iron oxide polyacrylic acid coated γ-Fe2O3 nanoparticles do not affect kidney function but cause acute effect on the cardiovascular function in healthy mice.

This study describes the distribution of intravenously injected polyacrylic acid (PAA) coated γ-Fe(2)O(3) NPs (10 mg kg(-1)) at the organ, cellular and subcellular levels in healthy BALB/cJ mice and in parallel addresses the effects of NP injection on kidney function, blood pressure and vascular contractility. Magnetic resonance imaging (MRI) and transmission electron microscopy (TEM) showed accumulation of NPs in the liver within 1h after intravenous infusion, accommodated by intracellular uptake in endothelial and Kupffer cells with subsequent intracellular uptake in renal cells, particularly the cytoplasm of the proximal tubule, in podocytes and mesangial cells. The renofunctional effects of NPs were evaluated by arterial acid-base status and measurements of glomerular filtration rate (GFR) after instrumentation with chronically indwelling catheters. Arterial pH was 7.46±0.02 and 7.41±0.02 in mice 0.5 h after injections of saline or NP, and did not change over the next 12 h. In addition, the injections of NP did not affect arterial PCO(2) or [HCO(3)(-)] either. Twenty-four and 96 h after NP injections, the GFR averaged 0.35±0.04 and 0.35±0.01 ml min(-1) g(-1), respectively, values which were statistically comparable with controls (0.29±0.02 and 0.33±0.1 ml(-1) min(-1) 25 g(-1)). Mean arterial blood pressure (MAP) decreased 12-24 h after NP injections (111.1±11.5 vs 123.0±6.1 min(-1)) associated with a decreased contractility of small mesenteric arteries revealed by myography to characterize endothelial function. In conclusion, our study demonstrates that accumulation of superparamagnetic iron oxide nanoparticles does not affect kidney function in healthy mice but temporarily decreases blood pressure.

T-type voltage-gated calcium channels regulate the tone of mouse efferent arterioles

Voltage-gated calcium channels are important for the regulation of renal blood flow and the glomerular filtration rate. Excitation-contraction coupling in afferent arterioles is known to require activation of these channels and we studied their role in the regulation of cortical efferent arteriolar tone. We used microdissected perfused mouse efferent arterioles and found a transient vasoconstriction in response to depolarization with potassium; an effect abolished by removal of extracellular calcium. The T-type voltage-gated calcium channel antagonists mibefradil and nickel blocked this potassium-induced constriction. Further, constriction by the thromboxane analogue U46619 was significantly inhibited by mibefradil at a concentration specific for T-type channels. Using PCR, we found that two channel subtypes, Ca(v)3.1 and Ca(v)3.2, were expressed in microdissected efferent arterioles. Ca(v)3.1 was found by immunocytochemistry to be located in mouse efferent arterioles, human pre- and postglomerular vasculature, and Ca(v)3.2 in rat glomerular arterioles. Inhibition of endothelial nitric oxide synthase by L-NAME or its deletion by gene knockout changed the potassium-elicited transient constriction to a sustained response. Low concentrations of nickel, an agent that blocks Ca(v)3.2, had a similar effect. Thus, T-type voltage-gated calcium channels are functionally important for depolarization-induced vasoconstriction and subsequent dilatation in mouse cortical efferent arterioles.

Coexpression of Voltage-Dependent Calcium Channels Cav1.2, 2.1a, and 2.1b in Vascular Myocytes

Voltage-dependent Ca2+ channels Cav1.2 (L type) and Cav2.1 (P/Q type) are expressed in vascular smooth muscle cells (VSMCs) and are important for the contraction of renal resistance vessels. In the present study we examined whether native renal VSMCs coexpress L-, P-, and Q-type Ca2+ currents. The expression of both Cav2.1a (P-type) and Cav2.1b (Q-type) mRNA was demonstrated by RT-PCR in renal preglomerular vessels from rats and mice. Immunolabeling was performed on A7r5 cells, renal cryosections, and freshly isolated renal VSMCs with anti-Cav1.2 and anti-Cav2.1 antibodies. Conventional and confocal microscopy revealed expression of both channels in all of the smooth muscle cells. Whole-cell patch clamp on single preglomerular VSMCs from mice showed L-, P-, and Q-type currents. Blockade of the L-type currents by calciseptine (20 nmol/L) inhibited 35.6±3.9% of the voltage-dependent Ca2+ current, and blocking P-type currents (&ohgr;-agatoxin IVA 10 nmol/L) led to 20.2±3.0% inhibition, whereas 300 nmol/L of &ohgr; agatoxin IVA (blocking P/Q-type) inhibited 45.0±7.3%. In rat aortic smooth muscle cells (A7r5), blockade of L-type channels resulted in 28.5±6.1% inhibition, simultaneous blockade of L-type and P-type channels inhibited 58.0±11.8%, and simultaneous inhibition of L-, P-, and Q-type channels led to blockade (88.7±5.6%) of the Ca2+ current. We conclude that aortic and renal preglomerular smooth muscle cells express L-, P-, and Q-type voltage-dependent Ca2+ channels in the rat and mouse.