Donna H. Wang

TRPV1 Gene Knockout Impairs Postischemic Recovery in Isolated Perfused Heart in Mice

Background— Although pharmacological studies suggest that the transient receptor potential vanilloid type 1 (TRPV1) channels expressed in sensory nerve fibers innervating the heart may exert a cardioprotective effect, definitive evidence supporting such a notion is lacking. In addition, function and regulation of sensory neuropeptides, namely, calcitonin gene–related peptide (CGRP) and substance P (SP), in the face of challenges induced by cardiac injury in the presence or absence of the TRPV1 are largely unknown. Methods and Results— The hearts of gene-targeted TRPV1-null mutant (TRPV1−/−) mice or wild-type (WT) mice were perfused in a Langendorff apparatus in the presence or absence of capsazepine (a TRPV1 receptor antagonist), CGRP, CGRP8–37 (a CGRP receptor antagonist), SP, or RP67580 (a neurokinin-1 [NK1] receptor antagonist) when hearts were subjected to 40 minutes of ischemia and 30 minutes of reperfusion. Hemodynamic alterations and SP release measured by radioimmunoassay were assessed before and after ischemia/reperfusion injury of the heart. Expression of the NK1 receptor in the hearts of TRPV1−/− and WT mice were determined with the use of Western blot analyses. Impairment of postischemic recovery, defined by increased left ventricular end-diastolic pressure (LVEDP) and decreased left ventricular developed pressure (LVDP) and coronary flow (CF), was more severe in TRPV1−/− hearts than in WT hearts. Although it had no effect on postischemic recovery of TRPV1−/− hearts, blockade of the TRPV1 with capsazepine caused a most severe impairment of postischemic recovery in WT hearts compared with untreated WT and TRPV1−/− hearts. Exogenous CGRP and SP produced a significant improvement in postischemic recovery in both TRPV1−/− and WT hearts, and the maximal functional improvement in TRPV1−/− hearts was not different from that of WT hearts except that SP-induced increases in LVDP were larger in the former than in the latter. Blockade of the NK1 receptor with RP67580, but not blockade of the CGRP receptor with CGRP8–37, caused more severe impairment in postischemic recovery in both TRPV1−/− and WT hearts than in untreated hearts in both genotypes. The release of SP after ischemia/reperfusion injury was increased in both WT and TRPV1−/− hearts, albeit with a smaller magnitude of the increase in the latter. Capsazepine attenuated injury-induced SP release in WT but not TRPV1−/− hearts. There was no difference in the expression of the NK1 receptor between the 2 genotype hearts. Conclusions— Thus, our data show that (1) TRPV1 gene deletion decreases injury-induced SP release and impairs cardiac recovery function after ischemia/reperfusion injury; (2) TRPV1 gene deletion leads to reconditioning of the heart with improved postischemic recovery compared with that induced by acute TRPV1 blockade and in terms of cardiac response to exogenous SP; and (3) blockade of the NK1 but not CGRP receptors worsens postischemic recovery of hearts in both genotypes. Taken together, these data indicate that TRPV1 plays a role in protecting the heart from injury possibly via increasing SP release and that deletion of this receptor reconditions the heart for escaping, at least in part, from injury possibly via enhancing NK1 receptor function.

Anandamide-Induced Depressor Effect in Spontaneously Hypertensive Rats. Role of the Vanilloid Receptor

Abstract—To test the hypothesis that activation of the vanilloid receptor (VR1) contributes to the anandamide-induced depressor effect in spontaneously hypertensive rats (SHR), we used a selective VR1 antagonist capsazepine (CAPZ) and a selective cannabinoid type 1 receptor antagonist SR141716A in conjunction with a VR1 agonist capsaicin in both SHR and Wistar-Kyoto rats (WKY). Mean arterial pressure was increased in SHR compared with WKY (P <0.05). Intravenous administration of capsaicin caused a greater depressor response in SHR compared with WKY (P <0.05), which was blocked by ≈60% by CAPZ (P <0.05) in SHR only. Methanandamide caused a similar greater depressor response (P <0.05), which was blocked by ≈50% and 60% by CAPZ and SR141716A, respectively, in SHR (P <0.05) but not in WKY. Radioimmunoassay showed that methanandamide increased plasma calcitonin gene-related peptide (CGRP) levels from baseline in both SHR and WKY (P <0.05), with no difference between 2 strains. Western blot showed that protein expression for the calcitonin receptor–like receptor—but not receptor activity modifying protein 1, VR1, and cannabinoid type 1 receptors—was increased in mesenteric resistance arteries in SHR compared with WKY (P <0.05). These data indicate that in addition to activation of cannabinoid type 1, anandamide may serve as an endogenous compound to stimulate VR1, leading to a decrease in blood pressure via CGRP release from sensory nerve terminals. Increased mesenteric CGRP receptor expression in SHR may account for increased sensitivity of blood pressure to anandamide and may serve as a compensatory response to buffer the increase in blood pressure in SHR.

Differential Regulation of Angiotensin II Receptor Subtypes in Rat Kidney by Low Dietary Sodium

This study was designed to determine whether expression of renal messenger RNA (mRNA) encoding the two known angiotensin II type 1 (AT1) receptor subtypes (AT1A and AT1B) can be regulated by dietary sodium. Seven-week-old male Wistar rats were fed a low-sodium diet (0.07%, n = 9) or a normal-sodium diet (0.5%, n = 9 [control]) for 14 days. A rat AT1 complementary DNA (cDNA) probe, which hybridizes to mRNA encoding both the AT1A and AT1B receptor subtypes, and cDNA probes, which are selective for AT1A or AT1B mRNA, were used in Northern blot or in situ hybridization analysis. By use of Northern blot analysis, renal mRNA levels for the AT1 and AT1A receptors in rats fed a low-sodium diet were found to be increased twofold (P < .05) compared with control. Because renal AT1B mRNA content was not detected by Northern blot analysis, quantitative image analysis of in situ hybridization with a digoxigenin-labeled cRNA probe made from AT1B cDNA was used. In situ hybridization analysis indicated that AT1B mRNA was expressed in the proximal and collecting tubules of the kidney in rats fed a normal-sodium diet. The low-sodium diet significantly decreased the percent positive staining area of AT1B mRNA in the renal cortex (5.51 +/- 0.77% versus 2.73 +/- 0.35%, P < .05) and medulla (4.76 +/- 0.70% versus 2.01 +/- 0.43%, P < .05) compared with the control diet.(ABSTRACT TRUNCATED AT 250 WORDS)

The vanilloid receptor and hypertension

AbstractMammalian transient receptor potential (TRP) channels consist of six related protein sub-families that are involved in a variety of pathophysiological function, and disease development. The TRPV1 channel, a member of the TRPV sub-family, is identified by expression cloning using the “hot” pepper-derived vanilloid compound capsaicin as a ligand. Therefore, TRPV1 is also referred as the vanilloid receptor (VR1) or the capsaicin receptor. VR1 is mainly expressed in a subpopulation of primary afferent neurons that project to cardiovascular and renal tissues. These capsaicin-sensitive primary afferent neurons are not only involved in the perception of somatic and visceral pain, but also have a “sensory-effector” function. Regarding the latter, these neurons release stored neuropeptides through a calcium-dependent mechanism via the binding of capsaicin to VR1. The most studied sensory neuropeptides are calcitonin gene-related peptide (CGRP) and substance P (SP), which are potent vasodilators and natriuretic/diuretic factors. Recent evidence using the model of neonatal degeneration of capsaicin-sensitive sensory nerves revealed novel mechanisms that underlie increased salt sensitivity and several experimental models of hypertension. These mechanisms include insufficient suppression of plasma renin activity and plasma aldosterone levels subsequent to salt loading, enhancement of sympathoexcitatory response in the face of a salt challenge, activation of the endothelin-1 receptor, and impaired natriuretic response to salt loading in capsaicin-pretreated rats. These data indicate that sensory nerves counterbalance the prohypertensive effects of several neurohormonal systems to maintain normal blood pressure when challenged with salt loading. The therapeutic utilities of vanilloid compounds, endogenous agonists, and sensory neuropeptides are also discussed.

Salt-sensitive hypertension induced by sensory denervation: introduction of a new model.

To test the novel hypothesis that neonatal degeneration of capsaicin-sensitive sensory nerves causes the rat to respond to a salt load with a significant and sustained rise in blood pressure, newborn Wistar rats were given 50 mg/kg capsaicin subcutaneously on the 1st and 2nd day of life. Control rats were treated with vehicle. Immediately after the weanling period, male rats were divided into 4 groups and fed different sodium diets for 2 weeks: capsaicin pretreatment plus high sodium diet (4%, CAP-HS), capsaicin plus normal sodium diet (0.5%, CAP-NS), control plus high sodium diet (CON-HS), and control plus normal sodium diet (CON-NS). Both tail-cuff systolic blood pressure and mean arterial pressure with anesthesia were significantly higher in CAP-HS than in CAP-NS, CON-HS, and CON-NS (P<0.05), but they were not different among the latter 3 groups. Radioimmunoassay revealed that levels of calcitonin gene related peptide in dorsal root ganglia were markedly decreased by capsaicin treatment (P<0.05). Twenty-four-hour urine volume and urine sodium excretion were significantly lower in CAP-HS than in CON-HS but were higher in CAP-HS and CON-HS compared with CAP-NS and CON-NS (P<0.05). Urine potassium excretion was not different among the 4 groups. Thus, this study provides the first evidence that neonatal degeneration of capsaicin-sensitive sensory nerves renders the rat salt-sensitive in terms of blood pressure regulation. Furthermore, our data suggest that neonatal capsaicin treatment may impair renal sodium and water excretion responses to high sodium intake. This model will provide a novel experimental paradigm for exploring underlying molecular mechanisms linked with salt-sensitive hypertension and sensory nerve function.Abstract —To test the novel hypothesis that neonatal degeneration of capsaicin-sensitive sensory nerves causes the rat to respond to a salt load with a significant and sustained rise in blood pressure, newborn Wistar rats were given 50 mg/kg capsaicin subcutaneously on the 1st and 2nd day of life. Control rats were treated with vehicle. Immediately after the weanling period, male rats were divided into 4 groups and fed different sodium diets for 2 weeks: capsaicin pretreatment plus high sodium diet (4%, CAP-HS), capsaicin plus normal sodium diet (0.5%, CAP-NS), control plus high sodium diet (CON-HS), and control plus normal sodium diet (CON-NS). Both tail-cuff systolic blood pressure and mean arterial pressure with anesthesia were significantly higher in CAP-HS than in CAP-NS, CON-HS, and CON-NS ( P <0.05), but they were not different among the latter 3 groups. Radioimmunoassay revealed that levels of calcitonin gene–related peptide in dorsal root ganglia were markedly decreased by capsaicin treatment ( P <0.05). Twenty-four-hour urine volume and urine sodium excretion were significantly lower in CAP-HS than in CON-HS but were higher in CAP-HS and CON-HS compared with CAP-NS and CON-NS ( P <0.05). Urine potassium excretion was not different among the 4 groups. Thus, this study provides the first evidence that neonatal degeneration of capsaicin-sensitive sensory nerves renders the rat salt-sensitive in terms of blood pressure regulation. Furthermore, our data suggest that neonatal capsaicin treatment may impair renal sodium and water excretion responses to high sodium intake. This model will provide a novel experimental paradigm for exploring underlying molecular mechanisms linked with salt-sensitive hypertension and sensory nerve function.

N-oleoyldopamine, a novel endogenous capsaicin-like lipid, protects the heart against ischemia-reperfusion injury via activation of TRPV1.

N-oleoyldopamine (OLDA), a bioactive lipid originally found in the mammalian brain, is an endovanilloid that selectively activates the transient receptor potential vanilloid type 1 (TRPV1) channel. This study tests the hypothesis that OLDA protects the heart against ischemia and reperfusion (I/R) injury via activation of the TRPV1 in wild-type (WT) but not in gene-targeted TRPV1-null mutant (TRPV1(-/-)) mice. Hearts of WT or TRPV1(-/-) mice were Langendorffly perfused with OLDA (2 x 10(-9) M) in the presence or absence of CGRP8-37 (1 x 10(-6) M), a selective calcitonin gene-related peptide (CGRP) receptor antagonist; RP-67580 (1 x 10(-6) M), a selective neurokinin-1 receptor antagonist; chelerythrine (5 x 10(-6) M), a selective protein kinase C (PKC) antagonist; or tetrabutylammonium (TBA, 5 x 10(-4) M), a nonselective K(+) channel antagonist, followed by 35 min of global ischemia and 40 min of reperfusion (I/R). Left ventricular end-diastolic pressure (LVEDP), left ventricular developed pressure (LVDP), coronary flow (CF), and left ventricular peak positive dP/dt (+dP/dt) were evaluated after I/R. OLDA improved recovery of cardiac function after I/R in WT but not TRPV1(-/-) hearts by increasing LVDP, CF, and +dP/dt and by decreasing LVEDP. CGRP8-37, RP-67580, chelerythrine, or TBA abolished the protective effect of OLDA in WT hearts. Radioimmunoassay showed that the release of substance P (SP) and CGRP after OLDA treatment was higher in WT than in TRPV1(-/-) hearts, which was blocked by chelerythrine or TBA. Thus OLDA exerts a cardiac protective effect during I/R injury in WT hearts via CGRP and SP release, which is abolished by PKC or K(+) channel antagonists. The protective effect of OLDA is void in TRPV1(-/-) hearts, supporting the notion that TRPV1 mediates OLDA-induced protection against cardiac I/R injury.

Angiotensin II inhibits HCO 3 absorption via a cytochrome P-450-dependent pathway in MTAL

The role of ANG II in the regulation of ion reabsorption by the renal thick ascending limb is poorly understood. Here, we demonstrate that ANG II (10-8 M in the bath) inhibits [Formula: see text] absorption by 40% in the isolated, perfused medullary thick ascending limb (MTAL) of the rat. The inhibition by ANG II was abolished by pretreatment with eicosatetraynoic acid (10 μM), a general inhibitor of arachidonic acid metabolism, or 17-octadecynoic acid (10 μM), a highly selective inhibitor of cytochrome P-450 pathways. Bath addition of 20-hydroxyeicosatetraenoic acid (20-HETE; 10-8 M), the major P-450 metabolite in the MTAL, inhibited [Formula: see text] absorption, whereas pretreatment with 20-HETE prevented the inhibition by ANG II. The addition of 15-HETE (10-8 M) to the bath had no effect on [Formula: see text]absorption. The inhibition of [Formula: see text]absorption by ANG II was reduced by >50% in the presence of the tyrosine kinase inhibitors genistein (7 μM) or herbimycin A (1 μM). We found no role for cAMP, protein kinase C, or NO in the inhibition by ANG II. However, addition of the exogenous NO donor S-nitroso- N-acetylpenicillamine (SNAP; 10 μM) or the NO synthase (NOS) substratel-arginine (1 mM) to the bath stimulated [Formula: see text] absorption by 35%, suggesting that NO directly regulates MTAL[Formula: see text] absorption. Addition of 10-11 to 10-10 M ANG II to the bath did not affect [Formula: see text] absorption. We conclude that ANG II inhibits [Formula: see text]absorption in the MTAL via a cytochrome P-450-dependent signaling pathway, most likely involving the production of 20-HETE. Tyrosine kinase pathways also appear to play a role in the ANG II-induced transport inhibition. The inhibition of [Formula: see text]absorption by ANG II in the MTAL may play a key role in the ability of the kidney to regulate sodium balance and extracellular fluid volume independently of acid-base balance.

Transient Receptor Potential Vanilloid Gene Deletion Exacerbates Inflammation and Atypical Cardiac Remodeling After Myocardial Infarction

The transient receptor potential vanilloid (TRPV1) channels expressed in sensory afferent fibers innervating the heart may be activated by protons or endovanilloids released during myocardial ischemia (MI), leading to angina. Although our previous in vitro data indicate that TRPV1 activation may preserve cardiac function after ischemia-reperfusion injury, the underlying mechanisms are largely unknown. To test the hypothesis that TRPV1 modulates inflammatory and early remodeling processes to prevent cardiac functional deterioration after myocardial infarction, TRPV1-null mutant (TRPV1−/−) and wild-type (WT) mice were subjected to left anterior descending coronary ligation or sham operation. The infarct size was greater in TRPV1−/− than in WT mice (P<0.001) 3 days after MI, and the mortality rate was higher in TRPV1−/− than in WT mice (P<0.05) 7 days after MI. The levels of plasma cardiac troponin I; cytokines, including tumor necrosis factor-α, interleukin-1β, and interleukin-6; chemokines, including monocyte chemoattractant protein-1 and macrophage inflammatory protein-2; and infiltration of inflammatory cells, including neutrophils, macrophages, and myofibroblasts; as well as collagen contents, were greater in TRPV1−/− than in WT mice (P<0.05) in the infarct area on days 3 and 7 after MI. Changes in left ventricular geometry led to increased end-systolic and -diastolic diameters and reduced contractile function in TRPV1−/− compared with WT mice. These data show that TRPV1 gene deletion results in excessive inflammation, disproportional left ventricular remodeling, and deteriorated cardiac function after MI, indicating that TRPV1 may prevent infarct expansion and cardiac injury by inhibiting inflammation and abnormal tissue remodeling.

VR1-Mediated Depressor Effects During High-Salt Intake. Role of Anandamide

This study was designed to test the hypothesis that increased sensitivity of blood pressure to anandamide (AEA), an endocannabinoid compound, occurs during high-salt intake, which can be blocked by a selective vanilloid receptor 1(VR1) antagonist, capsazepine (CAPZ). Intravenous administration of a metabolically stable analog, methanandamide (MethA), dose-dependently decreased mean arterial pressure (MAP) in conscious rats fed a high-sodium diet (HS) for 3 weeks but it had a minimal effect in normal sodium (NS)-treated rats. The MethA-induced decrease in MAP was significantly attenuated but not abolished by CAPZ, or a selective cannabinoid receptor 1 (CB1) antagonist, SR141716A, administered separately in HS-treated rats. The MethA-induced depressor effect was prevented by the combined administration of CAPZ and SR141716A in HS-treated rats. Likewise, administration of capsaicin, a selective VR1 receptor agonist, dose-dependently decreased MAP in both HS- and NS-treated rats. The depressor effect of capsaicin was more profound in HS-treated rats, which was prevented by CAPZ. Western blot showed that expression of VR1 but not CB1 in mesenteric arteries was increased in HS-treated compared with NS-treated rats. Therefore, these data show that: (1) HS upregulates mesenteric VR1 expression; (2) HS increases sensitivity of blood pressure to AEA; and (3) HS-induced enhancement of the depressor effect of AEA can be prevented only when both VR1 and CB1 receptors are blocked. These results indicate that AEA contributes to the prevention of salt induced increases in blood pressure via, at least in part, activating the VR1 receptor.

Development of Hypertension and Kidney Hypertrophy in Transgenic Mice Overexpressing ARAP1 Gene in the Kidney

Angiotensin II regulates blood pressure via activation of the type 1 receptor. We previously identified a novel angiotensin II type 1 receptor–associated protein and demonstrated that it promotes receptor recycling to the plasma membrane. To delineate the pathophysiological function of the ARAP1 in the kidneys, we generated transgenic mice that overexpress rat ARAP1 cDNA specifically in proximal tubules and tested the hypothesis that proximal tubule-specific overexpression of ARAP1 causes hypertension. Two lines of male transgenic mice, 650 and 670, displayed kidney-specific transgene expression. Systolic blood pressure was significantly elevated by ≈20 to 25 mm Hg in these lines of mice at 20 weeks of age compared with their nontransgenic litter mates. Urine volume, but not water intake, was significantly decreased in both lines compared with nontransgenic controls. The kidney/body weight ratio was significantly increased in both lines compared with their nontransgenic litter mates at 12 and 20 weeks of age. In contrast, no difference was observed in the ratio of brain, spleen, heart, and testis to body weight between male transgenic and nontransgenic animals. Inhibitions of the renin–angiotensin system completely normalized the systolic blood pressure of transgenic mice. Moreover, low salt intake prevented the development of hypertension, whereas high salt intake exacerbated the increase in blood pressure in transgenic mice. Therefore, our data show that proximal tubule-specific overexpression of ARAP1 leads to hypertension, suggesting that renal ARAP1 plays an important role in the regulation of blood pressure and renal function via activation of the intrarenal renin–angiotensin system.