Extracellular calcium (Ca 21 e )-induced relaxation of isolated, phenylephrine (PE)-contracted mesenteric arteries is dependent on an intact perivascular sensory nerve network that expresses the Ca 21 -sensing receptor (CaSR). Activation of the receptor stimulates an endocannabinoid vasodilator pathway, which is dependent on cytochrome P450 and phospholipase A 2 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 2/2 ) and pharmacologic inhibition of NOS on Ca mice. 7-Nitroindazole had no significant effect on relaxation of arteries from NOS 2/2 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 EC 50 value by 60% in tissues from C57BL/6 mice but reduced the maximum response by 80% in those from nNOS 2/2 mice. Ca 21 -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 Ca 21 e -induced relaxation.

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 . Video LinkThe video component of this article can be found at http://www.jove.com/details.php?id=3119 2. Perform a mid-line laparotomy to expose mesenteric bed. 3. Using scissors, remove about 85 cm of intestine with feeding vasculature with the superior mesenteric artery. Cut the proximal end of intestinal section close to the pylorus and the distal end near the ileo-coecal junction. Segments of the intestine are isolated from rats that are deeply anesthetized with isoflurane and euthanized by open-chest cardiac puncture. 4. Placed excised section in a coated petri dish ...

We interpret these data to mean that (i) short-term thiazide treatment does not reduce blood pressure by restoring renal cortical interstitial fluid calcium concentration and (ii) a decrease in renal cortical interstitial fluid calcium may not contribute to the increased renal vasoconstriction seen in salt-sensitivity.

High Ca 21 lowers blood pressure in hypertension, but the mechanism is not clear. The missing link may be the perivascular sensory nerve Ca 21 -sensing receptor (CaSR) that mediates a vasodilator system after activation by interstitial Ca 21 . Our results show that high salt increased CaSR expression in mesenteric arteries as well as Ca 21 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. Ca 21 relaxation of mesenteric arteries under phenylephrine tone increased in SS rats but decreased in arteries from SR rats fed the high-salt diet. Ca 21 -activated K 1 channels have a larger role in Ca 21 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 Ca 21 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 STATEMENTThe perivascular sensory nerve Ca 21 -sensing receptor (CaSR) mediates Ca 21 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 Ca 21 . The present studies describe the effect of high salt-induced upregulation of the CaSR in salt-sensitive rats and the roles played by Ca 21activated K 1 channels and nitric oxide in Ca 21 responses.

Ca2+e‐induced relaxation of mesenteric artery is dependent on the perivascular nerve (PvN) Ca2+‐sensing receptor (CaSR). The CaSR stimulates a cannabinoid vasodilator pathway, with a small (~20%) nitric oxide (NO)‐dependent component. Wire myography of phenylephrine (PE)‐contracted mesenteric arteries was used to determine the source of NO contributing to CaSR‐mediated relaxation in NO synthase (NOS) gene knockout mice in combination with NOS inhibitors. . Endothelial NOS gene knockout (eNOS−/−) up‐regulates, whereas neuronal NOS gene knockout (nNOS−/−) down‐regulates CaSR protein expression. nNOS−/− reduced maximum Ca2+ e‐induced relaxation by ~ 34% with no change in EC50 values, consistent with down‐regulation of the receptor as the non‐selective NOS inhibitor, L‐N5‐(1‐Iminoethyl)‐Ornithine (10 μM) reduced Ca2+ e‐induced relaxation in C57BL/6 control mice by ~ 38% (p < 0.05), but not eNOS−/− mice. The nNOS inhibitor, 7‐Nitroindazole had no effect on arteries from nNOS−/− mice. Both NG‐Nitro‐L‐Arginine Methylester and NG‐Monomethyl‐L‐Arginine reduced Ca2+‐induced relaxation maxima by about 55%, 50% and 30% in C57BL/6, nNOS−/− and eNOS−/− mice, respectively. Thus eNOS down‐regulates the CaSR in mesenteric arteries and contributes to the NO‐mediated Ca2+‐induced relaxation, but nNOS is not a major contributor.Support: HL064761, HL059868, MD000175 & HL099139