High Salt Upregulates Ca2+-Sensing Receptor Expression and Ca2+-Induced Relaxation of Contracted Mesenteric Arteries from Dahl Salt-Sensitive Rats.

High Ca2+ lowers blood pressure in hypertension, but the mechanism is not clear. The missing link may be the perivascular sensory nerve Ca2+-sensing receptor (CaSR) that mediates a vasodilator system after activation by interstitial Ca2+ Our results show that high salt increased CaSR expression in mesenteric arteries as well as Ca2+ relaxation of contracted mesenteric arteries from salt-sensitive (SS) rats. The CaSR was expressed as a doublet (≈120-150 kDa) in arteries from animals fed a high-salt diet for 1-4 weeks. The higher molecular weight glycosylated protein increased in arteries from SS animals; however, expression of the low molecular mass high-mannose protein decreased over 4 weeks of feeding the diet. In tissues from salt-resistant (SR) rats, the diet decreased CaSR expression after 4 weeks. Ca2+ relaxation of mesenteric arteries under phenylephrine tone increased in SS rats but decreased in arteries from SR rats fed the high-salt diet. Ca2+-activated K+ channels have a larger role in Ca2+ relaxation of arteries in SR than SS rats. The data suggest that high salt epigenetically regulates the receptor at the translational level in vivo and that the in vitro effect of Ca2+ is on receptor trafficking and signaling. In conclusion, upregulated expression of the CaSR in salt sensitivity increased receptor-mediated vascular relaxation. These findings show that CaSR signaling may compensate for changes in the vasculature in salt-sensitive hypertension. SIGNIFICANCE STATEMENT: The perivascular sensory nerve Ca2+-sensing receptor (CaSR) mediates Ca2+ relaxation of isolated mesenteric arteries under tension. This receptor may therefore play a significant role in relaxation of resistance arteries in vivo, thus explaining the blood pressure-lowering effect of dietary Ca2+. The present studies describe the effect of high salt-induced upregulation of the CaSR in salt-sensitive rats and the roles played by Ca2+-activated K+ channels and nitric oxide in Ca2+ responses.

Protein Kinase C Downregulation Enhanced Extracellular Ca2+-Induced Relaxation of Isolated Mesenteric Arteries from Aged Dahl Salt-Sensitive Rats.

The Ca2+-sensing receptor (CaSR) detects small changes in extracellular calcium (Ca2+ e) concentration ([Ca2+]e) and transduces the signal into modulation of various signaling pathways. Ca2+-induced relaxation of isolated phenylephrine-contracted mesenteric arteries is mediated by the CaSR of the perivascular nerve. Elucidation of the regulatory mechanisms involved in vascular CaSR signaling may provide insights into the physiologic functions of the receptor and identify targets for the development of new treatments for cardiovascular pathologies such as hypertension. Protein kinase Cα (PKCα) is a critical regulator of multiple signaling pathways and can phosphorylate the CaSR leading to receptor desensitization. In this study, we used automated wire myography to investigate the effects of CaSR mutation and small-interfering RNA downregulation of PKCα on CaSR-mediated relaxation of phenylephrine-contracted mesenteric arteries from aged Dahl salt-sensitive (SS) rats on a low-salt diet. The data showed minimal relaxation responses of arteries to Ca2+ e in wild-type (SS) and CaSR mutant (SS-Casrem1Mcwi) rats. Mutation of the CaSR gene had no significant effect on relaxation. PKCα expression was similar in wild-type and mutant rats, and small-interfering RNA downregulation of PKCα and/or inhibition of PKC with the Ca2+-sensitive GÓ§ 6976 resulted in a >80% increase in relaxation. Significant differences in EC50 values were observed between treated and untreated controls (P < 0.05 analysis of variance). The results indicate that PKCα plays an important role in the regulation of CaSR-mediated relaxation of mesenteric arteries, and its downregulation or pharmacological inhibition may lead to an increased Ca2+ sensitivity of the receptor and reversal of age-related changes in vascular tone. SIGNIFICANCE STATEMENT: G protein-coupled CaSR signaling leads to the regulation of vascular tone and may, therefore, play a vital role in blood pressure regulation. The receptor has several PKC phosphorylation sites in the C-terminal intracellular tail that mediate desensitization. We have previously shown that activation of the CaSR in neuronal cells leads to PKC phosphorylation, indicating that protein kinase C is an important regulator of CaSR function. Therefore, PKC in the CaSR signaling pathway in mesenteric arteries is a potential target for the development of new therapeutic approaches to treat hypertension and age-related vascular dysfunction. The present studies show that small-interfering RNA downregulation of PKCα and pharmacological inhibition of PKC enhanced CaSR-mediated relaxation of phenylephrine-contracted mesenteric arteries from aged Dahl salt-sensitive rats.

Nitric-oxide synthase knockout modulates Ca²âº-sensing receptor expression and signaling in mouse mesenteric arteries.

Extracellular calcium (Ca²âº(e))-induced relaxation of isolated, phenylephrine (PE)-contracted mesenteric arteries is dependent on an intact perivascular sensory nerve network that expresses the Ca²âº-sensing receptor (CaSR). Activation of the receptor stimulates an endocannabinoid vasodilator pathway, which is dependent on cytochrome P450 and phospholipase A2 but largely independent of the endothelium. In the present study, we determined the role of nitric oxide (NO) in perivascular nerve CaSR-mediated relaxation of PE-contracted mesenteric resistance arteries isolated from mice. Using automated wire myography, we studied the effects of NO synthase (NOS) gene knockout (NOS(-/-)) and pharmacologic inhibition of NOS on Ca²âº(e)-induced relaxation of PE-contracted arteries. Endothelial NOS knockout (eNOS(-/-)) upregulates but neuronal NOS knockout (nNOS(-/-)) downregulates CaSR expression. NOS(-/-) reduced maximum Ca²âº(e)-induced relaxation with no change in EC50 values, with eNOS(-/-) having the largest effect. The responses of vessels to calindol and Calhex 231 indicate that the CaSR mediates relaxation. L-N5-(1-iminoethyl)-ornithine reduced Ca²âº(e)-induced relaxation of PE-contracted arteries from C57BL/6 control mice by ≈38% but had a smaller effect in vessels from eNOS(-/-) mice. 7-Nitroindazole had no significant effect on relaxation of arteries from NOS(-/-) mice, but both N(G)-nitro-L-arginine methylester and N(G)-monomethyl-L-arginine significantly reduced the relaxation maxima in all groups. Interestingly, the nNOS-selective inhibitor S-methyl-L-thiocitrulline significantly increased the EC50 value by ≈60% in tissues from C57BL/6 mice but reduced the maximum response by ≈80% in those from nNOS(-/-) mice. Ca²âº-activated big potassium channels play a major role in the process, as demonstrated by the effect of iberiotoxin. We conclude that CaSR signaling in mesenteric arteries stimulates eNOS and NO production that regulates Ca²âº(e)-induced relaxation.

Different effects of angiotensin receptor blockade on end-organ damage in salt-dependent and salt-independent hypertension.

BACKGROUND: Although angiotensin II type 1 receptor blockers have emerged as effective antihypertensive agents, it is not known how efficacious these agents are in treating hypertension-associated target organ damage. METHODS AND RESULTS: The present study was undertaken to compare the effect of angiotensin type 1 receptor inhibition on the progression of the organ damage observed in 2 models of hypertension, namely, salt-sensitive and nitric oxide synthase inhibition-mediated hypertension. Effective (16.4 micromol/kg) and ineffective (0.8 to 4.9 micromol/kg) antihypertensive doses of candesartan cilexetil were initiated after hypertension was established. Both low- and high-dose candesartan cilexetil significantly reduced cardiac and renal damage in the nitric oxide synthase inhibitor model of hypertension (P < 0.05 versus untreated); however, high-dose candesartan caused a significant increase in renal damage in the Dahl salt-sensitive model of hypertension (P < 0.05 versus untreated). Interestingly, the beneficial end-organ effects of candesartan in the nitric oxide synthase inhibition model were independent of sustained antihypertensive actions of candesartan, whereas the exacerbation of renal injury with candesartan in the Dahl salt-sensitive model was inversely related to its blood pressure-lowering effect. CONCLUSIONS: These data show that angiotensin type 1 blockade reduces injury in the l-nitroarginine methyl ester model but increases tissue injury in the salt-sensitive model. These data suggest that angiotensin II via angiotensin type 1 receptor activation contributes to organ damage in nitric oxide-deficient salt-independent hypertension but is protective in salt-induced hypertension. These data further suggest that (1) renal injury may evolve independently of blood pressure and (2) the effectiveness of an antihypertensive agent in ameliorating renal injury may depend on the etiology of the hypertension.

Mesenteric artery contraction and relaxation studies using automated wire myography.

Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance. These small vessels of between 100-400 µm in diameter function primarily in directing blood flow to various organs according to the overall requirements of the body. The rat mesenteric artery has a diameter greater than 100 µm. The myography technique, first described by Mulvay and Halpern(1), was based on the method proposed by Bevan and Osher(2). The technique provides information about small vessels under isometric conditions, where substantial shortening of the muscle preparation is prevented. Since force production and sensitivity of vessels to different agonists is dependent on the extent of stretch, according to active tension-length relation, it is essential to conduct contraction studies under isometric conditions to prevent compliance of the mounting wires. Stainless steel wires are preferred to tungsten wires because of oxidation of the latter, which affects recorded responses(3).The technique allows for the comparison of agonist-induced contractions of mounted vessels to obtain evidence for normal function of vascular smooth muscle cell receptors. We have shown in several studies that isolated mesenteric arteries that are contracted with phenylyephrine relax upon addition of cumulative concentrations of extracellular calcium (Ca(2+)(e;)). The findings led us to conclude that perivascular sensory nerves, which express the G protein-coupled Ca(2+)-sensing receptor (CaR), mediate this vasorelaxation response. Using an automated wire myography method, we show here that mesenteric arteries from Wistar, Dahl salt-sensitive(DS) and Dahl salt-resistant (DR) rats respond differently to Ca(2+)(e;). Tissues from Wistar rats showed higher Ca(2+)-sensitivity compared to those from DR and DS. Reduced CaR expression in mesenteric arteries from DS rats correlates with reduced Ca(2+)(e;)-induced relaxation of isolated, pre-contracted arteries. The data suggest that the CaR is required for relaxation of mesenteric arteries under increased adrenergic tone, as occurs in hypertension, and indicate an inherent defect in the CaR signaling pathway in Dahl animals, which is much more severe in DS. The method is useful in determining vascular reactivity ex vivo in mesenteric resistance arteries and similar small blood vessels and comparisons between different agonists and/or antagonists can be easily and consistently assessed side-by-side(6,7,8).

The effect of salt on renal damage in eNOS-deficient mice.

African Americans have an increased incidence of end-stage renal disease and are characterized as having reduced bioavailability of nitric oxide and salt-sensitivity. We propose that endothelial nitric oxide synthase (eNOS) knockout mice (eNOS(-/-)) are a suitable model of hypertension-associated renal injury as seen in African Americans. Therefore, the purpose of this study was to determine whether older eNOS(-/-) mice have hypertension-associated renal injury and if dietary salt modulates this injury. Six-month-old eNOS(-/-) mice were placed on 0.12%, 0.45% or 8% NaCl diet for 8 weeks and blood pressure measured weekly; kidneys were collected for pathology evaluation and scoring at the end of the 8-week period. Mice deficient of eNOS were hypertensive at baseline compared with control mice in all three groups (128+/-3 vs. 112+/-3, P<0.05). Blood pressure was significantly elevated from baseline in eNOS(-/-) on 0.45 and 8% salt diets (P<0.02). The composite renal pathology scores for eNOS(-/-) mice were significantly greater than wild-type mice, indicating high salt intake exacerbates the injury (P<0.001 vs. normal salt diet). eNOS(-/-) mice may be used as a model of salt-induced and hypertension-associated renal injury as seen in African Americans.