Among the TRPC subfamily of TRP (classical transient receptor potential) channels, TRPC3, -6, and -7 are gated by signal transduction pathways that activate C-type phospholipases as well as by direct exposure to diacylglycerols. Since TRPC6 is highly expressed in pulmonary and vascular smooth muscle cells, it represents a likely molecular candidate for receptor-operated cation entry. To define the physiological role of TRPC6, we have developed a TRPC6-deficient mouse model. These mice showed an elevated blood pressure and enhanced agonist-induced contractility of isolated aortic rings as well as cerebral arteries. Smooth muscle cells of TRPC6-deficient mice have higher basal cation entry, increased TRPC-carried cation currents, and more depolarized membrane potentials. This higher basal cation entry, however, was completely abolished by the expression of a TRPC3-specific small interference RNA in primary TRPC6 ؊/؊ smooth muscle cells. Along these lines, the expression of TRPC3 in wild-type cells resulted in increased basal activity, while TRPC6 expression in TRPC6 ؊/؊ smooth muscle cells reduced basal cation influx. These findings imply that constitutively active TRPC3-type channels, which are up-regulated in TRPC6-deficient smooth muscle cells, are not able to functionally replace TRPC6. Thus, TRPC6 has distinct nonredundant roles in the control of vascular smooth muscle tone.The TRP (transient receptor potential) family of ion channels is a growing group of structurally and evolutionarily related cation channels formed of several subfamilies that include the TRPC, TRPM, and TRPV classes of channels (6, 22). TRP-type ion channels are presumed to be homo-or heterotetramers (13,14), each spanning the plasma membrane six times. The founding members of this channel family are the insect TRP and TRPL channels, which are responsible for photoreceptor depolarization in response to light. Mammalian TRPCs (C stands for canonical or classical) (23, 32) are the closest mammalian structural relatives of insect TRPs. Among the TRPC channels, TRPC3, -6, and -7 are 75% identical and gated by signal transduction pathways that activate C-type phospholipases (3, 32) as well as by direct exposure to diacylglycerols (DAG) (15). TRPC3, -6, and -7 interact physically and, upon coexpression, coassemble to form functional channels (14). Expression of TRPC3 and TRPC7 in HEK 293 cells, but not of TRPC6, reveals constitutively active cation channels that are permeable not only to monovalent but also to divalent cations such as Ca 2ϩ , Ba 2ϩ , and Mn 2ϩ (7,23,33). In contrast to members of other TRP families, the functional importance of most members of the TRPC subfamily is still poorly understood. A TRPC channel for which considerable evidence has accumulated for a specific role is TRPC6, which has been proposed to regulate smooth muscle function. The TRPC6 mRNA was originally isolated from mouse brain and was also identified in lung cells (4). By comparative biophysical characterization and gene suppression using antisense oligonucleot...

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Abstract-Nuclear factor kappa B (NF-B) participates in hypertension-induced vascular and target-organ damage. We tested whether or not endothelial cell-specific NF-B suppression would be ameliorative. We generated Cre/lox transgenic mice with endothelial cell-restricted NF-B super-repressor IB␣⌬N (Tie-1-⌬N mice) overexpression. We confirmed cell-specific IB␣⌬N expression and reduced NF-B activity after TNF-␣ stimulation in primary endothelial cell culture. To induce hypertension with target-organ damage, we fed mice a high-salt diet and N(omega)-nitro-Larginine-methyl-ester (L-NAME) and infused angiotensin (Ang) II. This treatment caused a 40-mm Hg blood pressure increase in both Tie-1-⌬N and control mice. In contrast to control mice, Tie-1-⌬N mice developed a milder renal injury, reduced inflammation, and less albuminuria. RT-PCR showed significantly reduced expression of the NF-B targets VCAM-1 and ICAM-1, compared with control mice. Thus, the data demonstrate a causal link between endothelial NF-B activation and hypertension-induced renal damage. We conclude that in vivo NF-B suppression in endothelial cells stops a signaling cascade leading to reduced hypertension-induced renal damage despite high blood pressure. Key Words: hypertension Ⅲ endothelium Ⅲ NF-B Ⅲ target-organ damage H ypertension is a risk factor for target-organ damage such as cardiac and renal disease. 1 Endothelial cell injury initiates adhesion molecule and chemokine expression promoting inflammation that contributes to the pathogenesis of hypertension-induced target organ damage. [2][3][4][5] Patients with severe hypertension and target-organ damage often show elevated angiotensin (Ang) II levels and reduced nitric oxide (NO) production. Ang II signaling blockade reduces blood pressure and blunts the development and progression of vascular disease in small and large vessels in experimental animal models and in humans. Ang II also elicits an inflammatory response in both endothelial cells 6 and vascular smooth muscle cells. 7,8 Numerous in vitro and in vivo studies demonstrated that Ang II activates the nuclear factor kappa B (NF-B), a major transcription factor in mediating inflammation and innate immunity. 4,6 -8 In resting cells, NF-B resides inactive in the cytoplasm by forming complexes with the inhibitor of B (I〉) proteins. Exposure to extracellular stimuli like TNF-␣, IL-1, reactive oxygen species, Ang II, and numerous other activators leads to rapid phosphorylation, site-specific ubiquitination, and subsequent degradation of the IB proteins by the 26S proteasome. 9 The resulting free NF-B molecules translocate to the nucleus and regulate target gene expression. The targets include genes involved in the control of cell proliferation, apoptosis, innate, and adaptive immune response. 10,11 NF-B and inflammation play an important role in the development of the target organ damage. 4,[12][13][14][15][16][17] However, inhibition of NF-B signaling could also be detrimental. 18,19 Furthermore, the ideal site for NF-B inhibition in the vesse...

. Autonomic nervous system and blood pressure regulation in RGS2-deficient mice. Am J Physiol Regul Integr Comp Physiol 288: R1134-R1142, 2005. First published January 20, 2005; doi:10.1152/ajpregu.00246.2004.-Regulator of G protein signaling (RGS2) deletion in mice prolongs signaling by G protein-coupled vasoconstrictor receptors and increases blood pressure. However, the exact mechanism of the increase in blood pressure is unknown. To address this question we tested autonomic nervous system function and blood pressure regulation in RGS2-deficient mice (RGS2 Ϫ/Ϫ). We measured arterial blood pressure and heart rate (HR) with telemetry, computed time and frequency-domain measures for blood pressure and HR variability (HRV) as well as baroreflex sensitivity [BRS-low frequency (LF)], and assessed environmental stress sensitivity. Mean arterial blood pressure (MAP) was ϳ10 mmHg higher in RGS2 Ϫ/Ϫ compared with RGS2 ϩ/ϩ mice, while HR was not different between the groups, indicating a resetting of the baroreceptor reflex. Atropine increased MAP more in RGS2 Ϫ/Ϫ than in RGS2 ϩ/ϩ mice while HR responses were not different. Urinary norepinephrine excretion was higher in RGS2 Ϫ/Ϫ than in RGS2 ϩ/ϩ mice. The blood pressure decrease following prazosin was more pronounced in RGS2 Ϫ/Ϫ mice than in RGS2 ϩ/ϩ mice. The LF and high-frequency (HF) power of HRV were reduced in RGS2 Ϫ/Ϫ compared with controls while BRS-LF and SBP-LF were not different. Atropine and atropine ϩ metoprolol markedly reduced the HRV parameters in the time (RMSSD) and frequency domain (LF, HF, LF/HF) in both strains. Environmental stress sensitivity was increased in RGS2 Ϫ/Ϫ mice compared with controls. We conclude that the increase in blood pressure in RGS2 Ϫ/Ϫ mice is not solely explained by peripheral vascular mechanisms. A central nervous system mechanism might be implicated by an increased sympathetic tone. This state of affairs could lead to a baroreceptor-HR reflex resetting, while BRS remains unimpaired.G protein-coupled receptors; RGS2-deficient mice; autonomic nervous system; heart rate variability; spectral analysis; baroreflex; telemetry G PROTEIN-COUPLED RECEPTORS (GPCRs) are important in cardiovascular regulation. Hormones such as norepinephrine, epinephrine, endothelin-1, thrombin, ANG II, and serotonin all bind to GPCRs to stimulate G protein-signaling cascades. The binding to G␣q-coupled receptors results in activation of a cascade that causes vasoconstriction (37). The duration and intensity of G␣q-coupled receptor signaling is regulated by GTPase-activating proteins (GAPs), which accelerate the return of the activated G␣ subunit to its inactive form. Regulators of G protein signaling (RGS) proteins are components of the G protein-coupled receptor signaling pathway, and RGS2 is a potent regulator of G␣q (10). RGS2 accelerates the rate of G protein deactivation by stimulating GTP hydrolysis. As a result, disruption of the RGS2 gene in mice increased blood pressure and markedly prolonged vasoconstrictor responses of the peripheral resistan...

Abstract-␣-2 Adrenoceptors are important in baroreflex regulation. We tested the impact of ␣-2 adrenoceptors on heart rate variability (HRV) and spontaneous baroreflex sensitivity (BRS) in conscious mice with telemetry (TA11PA-C20).Baseline beat-to-beat measurements (2 hours between 8:00 AM to 12:00 PM) were compared with measurements after intraperitoneal ␣-2 adrenoceptor blockade (yohimbine 2 mg/kg) and ␣-2 adrenoceptor stimulation (clonidine 1, 10, and 50 mg/kg). Blood pressure (BP) was 128Ϯ6/87Ϯ6 mm Hg and heart rate (HR) was 548Ϯ18 bpm at baseline. BRS, calculated with the cross-spectral method, was 1.2Ϯ0.1 ms/mm Hg at baseline. BP increased 20Ϯ2/13Ϯ2 mm Hg with yohimbine. HR increased by 158Ϯ23 bpm. BRS did not change. BP decreased 16Ϯ7/5Ϯ4 mm Hg with 1 mg/kg of clonidine and did not change with a higher dose. HR decreased with clonidine (176Ϯ28, 351Ϯ21, 310Ϯ29 bpm during 1, 10, and 50 mg/kg of clonidine, PϽ0.01). HRV (total powerϭ4629Ϯ465, 7002Ϯ440, and 6452Ϯ341 ms 2 during 1, 10, and 50 mg/kg of clonidine, PϽ0.01) and BRS were profoundly increased with clonidine (14Ϯ1, 13Ϯ1, and 10Ϯ1 ms/mm Hg, PϽ0.01). The effects of clonidine were abolished with atropine (2 mg/kg plus 50 mg/kg of clonidine) but not with metoprolol (4 mg/kg plus 50 mg/kg of clonidine). These data suggest that ␣-2 adrenoceptors exert a regulatory influence on autonomic cardiovascular control and baroreflex function. The effect of clonidine on baroreflex HR regulation is mediated by the parasympathetic nervous system. These murine data fit well with recent human observations regarding parasympathetic activation via ␣-2 adrenoceptors. Key Words: antihypertensive agents Ⅲ blood pressure Ⅲ heart rate Ⅲ baroreflex T he arterial baroreflex acts as an effective buffer of short-term blood pressure (BP) fluctuations and prevents excessive BP swings. 1 Adrenergic mechanisms in the brain have a major contribution to baroreflex function. ␣-2 Adrenoceptors may be particularly important in central nervous baroreflex regulation, both in animals and in humans. [2][3][4][5] Nonselective stimulation of ␣-2 adrenoceptors with clonidine elicited a depressor effect and enhanced baroreflex heart rate (HR) gain in human studies, 2 in rabbits, 6 and in dogs. 7 However, other studies in rats 8 and in humans 9 suggested that clonidine does not improve baroreflex HR gain. Species differences and different study protocols including anesthetic states may contribute to this controversy. We and others have recently shown the possibility to assess standard time and frequency domain measures of heart rate variability (HRV) and baroreflex sensitivity (BRS) testing by telemetric techniques in conscious mice. 10 -13 Therefore, the mouse is a well-suited animal model to further elucidate the functional role of ␣-2 adrenoceptors in more detail. 14 -16 The model may be particularly relevant given the large number of genetically modified mouse strains. Data on the effects of ␣-2 adrenoceptor manipulation on HRV and BRS in freely moving mice are still lacking. The aim of this ...

Abstract-We adapted telemetry and sequence analysis employed in humans to mice and measured heart rate variability and the spontaneous baroreflex sensitivity in angiotensin II type 2 (AT 2 ) receptor-deleted (AT 2 Ϫ/Ϫ) and wild-type (AT 2 ϩ/ϩ) mice with either deoxycorticosterone acetate (DOCA)-salt hypertension or N-nitro-L-arginine methylester hydrochloride (L-NAME) hypertension. Mean arterial pressure leveled during the day at 101Ϯ1 mm Hg and during the night at 109Ϯ1 mm Hg in AT 2 receptor-deleted mice, compared with 98Ϯ2 mm Hg (day) and 104Ϯ2 mm Hg (night) in wild-type mice. Mean arterial pressure increased in AT 2 receptor-deleted mice with L-NAME to 114Ϯ1 mm Hg (day) and 121Ϯ1 mm Hg (night), compared with 105Ϯ2 mm Hg (day) and 111Ϯ2 mm Hg (night), respectively. DOCA-salt also increased day and night blood pressures in AT 2 receptor-deleted mice to a greater degree than in wild-type mice. Heart rate variability in the time and frequency domain was not different between AT 2 receptor-deleted mice and AT 2 receptor-deleted mice at baseline. Systolic blood pressure variability in the low frequency band was lower in AT 2 receptor-deleted mice (0.6Ϯ0.1 ms 2 versus 3.9Ϯ1.3 ms 2 ) than in wild-type mice. Baroreceptor-heart rate reflex sensitivity was significantly increased in AT 2 receptor-deleted mice compared with wild-type mice (3.4Ϯ0.6 versus 2.1Ϯ0.5 ms/mm Hg). These differences remained after DOCA-salt and L-NAME treatments. We conclude that activation of the AT 2 receptor impairs arterial baroreceptor reflex function, probably by a central action. These data support the existence of an inhibitory central effect of the AT 2 receptor on baroreflex function. Although not yet as clearly delineated as the AT 1 receptor, the AT 2 receptor may be responsible for counterregulating the effects induced by the AT 1 receptor. 1 In accord with this view, disruption of the AT 2 receptor caused increased blood pressure in mice and increased vasopressor responses to Ang II. [1][2][3][4][5] In experimental and human hypertension, the sensitivity of the baroreceptor control of heart rate is reduced and has been associated with an overactivity of the renin-angiotensin system. 6 Endogenous Ang II affects the baroreceptor function through the AT 1 receptor 7,8 and the AT 2 receptor via central tonic inhibitory effects. 9,10 Therefore, the AT 2 receptor gene-disrupted (Ϫ/Ϫ) mouse could be a useful model for studying the involvement of the AT 2 receptor in baroreceptor function. In man, the combined computer analysis of spontaneous blood pressure and heart rate fluctuations allows the assessment of baroreflex function without invasive or provocative interventions. We adapted telemetry to AT 2 receptor Ϫ/Ϫ and wild-type (ϩ/ϩ) mice to study blood pressure and continuous spontaneous baroreflex function in the mouse. To our knowledge, this report is the first in which these clinically accepted methods are applied to mice. MethodsExperiments were performed on 8 adult male AT 2 receptor-deleted (AT 2 Ϫ/Ϫ) and 7 male wild-type (AT 2 ϩ/...

The regulator of G protein signaling (RGS) 2, a GTPase-activating protein, is activated via the nitric oxide (NO)-cGMP pathway and thereby may influence blood pressure regulation. To test that notion, we measured mean arterial blood pressure (MAP) and heart rate (HR) with telemetry in N(omega)-nitro-l-arginine methyl ester (l-NAME, 5 mg l-NAME/10 ml tap water)-treated RGS2-deficient (RGS2(-/-)) and RGS2-sufficient (RGS2(+/+)) mice and assessed autonomic function. Without l-NAME, RGS2(-/-) mice showed during day and night a similar increase of MAP compared with controls. l-NAME treatment increased MAP in both strains. nNOS is involved in this l-NAME-dependent blood pressure increase, since 7-nitroindazole increased MAP by 8 and 9 mmHg (P < 0.05) in both strains. The l-NAME-induced MAP increase of 14-15 mmHg during night was similar in both strains. However, the l-NAME-induced MAP increase during the day was smaller in RGS2(-/-) than in RGS2(+/+) (11 +/- 1 vs. 17 +/- 2 mmHg; P < 0.05). Urinary norepinephrine and epinephrine excretion was higher in RGS2(-/-) than in RGS2(+/+) mice. The MAP decrease after prazosin was more pronounced in l-NAME-RGS2(-/-). HR variability parameters [root mean square of successive differences (RMSSD), low-frequency (LF) power, and high-frequency (HF) power] and baroreflex sensitivity were increased in RGS2(-/-). Atropine and atropine plus metoprolol markedly reduced RMSSD, LF, and HF. Our data suggest an interaction between RGS2 and the NO-cGMP pathway. The blunted l-NAME response in RGS2(-/-) during the day suggests impaired NO signaling. The MAP increases during the active phase in RGS2(-/-) mice may be related to central sympathetic activation and increased vascular adrenergic responsiveness.

The AT2 receptor is not essential for development of L-NAME-induced cardiac hypertrophy, fibrosis and concomitant changes in left ventricular performance. In contrast, the AT2 receptor offers a protective effect.