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The perivascular sensory nerve (PvN) [Ca.sup.2+]-sensing receptor (CAR) is implicated in [Ca.sup.2+]induced relaxation of isolated, phenylephrine (PE)-contracted mesenteric arteries, which involves the vascular endogenous cannabinoid system. We determined the effect of inhibition of diacylglycerol (DAG) lipase (DAGL), phospholipase [A.sub.2] (PL[A.sub.2]), and cytochrome P-450 (CYP) on [Ca.sup.2+]-induced relaxation of PE-contracted rat ruesenteric arteries. Our findings indicate that [Ca.sup.2+]-induced vasorelaxation is not dependent on the endothelium. The DAGL inhibitor RHC 802675 (1 [micro]M) and the CYP and PL[A.sub.2] inhibitors quinacrine (5 [micro] M) (E[C.sub.50]: RHC 802675 2.8 [+ or -] 0.4 mM vs. control 1.4 [+ or -] 0.3 mM; quinacrine 4.8 [+ or -] 0.4 mM vs. control 2.0 [+ or -] 0.3 mM; n = 5) and arachidonyltrifluoromethyl ketone (AACOC[F.sub.3], 1 [micro] M) reduced [Ca.sup.2+]induced relaxation of mesenteric arteries. Synthetic 2-arachidonoylglycerol (2-AG) and glycerated epoxyeicosatfienoic acids (GEETs) induced concentration-dependent relaxation of isolated arteries. 2-AG relaxations were blocked by iberiotoxin (IBTX) (E[C.sub.50]: control 0.96 [+ or -] 0.14 nM, IBTX 1.3 [+ or -] 0.5 [micro] M) and miconazole (48 [+ or -] 3%), and 11,12-GEET responses were blocked by IBTX (E[C.sub.50]: control 55 [+ or -] 9 nM, IBTX 690 [+ or -] 96 nM) and SR-141716A. The data suggest that activation of the CaR in the PvN network by [Ca.sup.2+] leads to synthesis and/or release of metabolites of the CYP epoxygenase pathway and metabolism of DAG to 2-AG and subsequently to GEETs. The findings indicate a role for 2-AG and its metabolites in [Ca.sup.2+]-induced relaxation of resistance arteries; therefore this receptor may be a potential target for the development of new vasodilator compounds for antihypertensive therapy. [Ca.sup.2+]-sensing receptor; arachidonic acid; vasorelaxation

The rat dorsal root ganglion (DRG) [Ca.sup.2+]-sensing receptor (CAR) was stably expressed in-frame as an enhanced green fluorescent protein (EGFP) fusion protein in human embryonic kidney (HEK)293 cells, and is functionally linked to changes in intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]). RT-PCR analysis indicated the presence of the message for the DRG CaR cDNA. Western blot analysis of membrane proteins showed a doublet of 168-175 and 185 kDa, consistent with immature and mature forms of the CaR.EGFP fusion protein, respectively. Increasing extracellular [[Ca.sup.2+]] ([[[Ca.sup.2+]].sub.e]) from 0.5 to 1 mM resulted in increases in [[[Ca.sup.2+]].sub.i] levels, which were blocked by 30 [micro]M 2-aminoethyldiphenyl borate. [[[Ca.sup.2+]].sub.e]-response studies indicate a [Ca.sup.2+] sensitivity with an E[C.sub.50] of 1.75 [+ or -] 0.10 mM. NPS R-467 and [Gd.sup.3+] activated the CaR. When [[[Ca.sup.2+]].sub.e] was successively raised from 0.25 to 4 mM, peak [[[Ca.sup.2+]].sub.i], attained with 0.5 mM, was reduced by ~50%. Similar reductions were observed with repeated applications of 10 mM [Ca.sup.2+], 1 and 10 [micro]M NPS R-467, or 50 and 100 [micro]M [Gd.sup.3+], indicating desensitization of the response. Furthermore, [Ca.sup.2+] mobilization increased phosphorylated protein kinase C (PKC)[alpha] levels in the cells. However, the PKC activator, phorbol myristate acetate did not inhibit CaR-mediated [Ca.sup.2+] signaling. Rather, a spectrum of PKC inhibitors partially reduced peak responses to [Ca.sup.2+.sub.e]. Treatment of cells with 100 nM PMA for 24 h, to downregulate PKC, reduced [[Ca.sup.2+].sub.i] transients by 49.9 [+ or -] 5.2% (at 1 mM [Ca.sup.2+]) and 40.5 [+ or -] 6.5% (at 2 mM [Ca.sup.2+]), compared with controls. The findings suggest involvement of PKC in the pathway for [Ca.sup.2+] mobilization following CaR activation. desensitization; protein kinase C doi:10.1152/ajpcell.00404.2006

A full-length cDNA encoding a [Ca.sup.2+]-sensing receptor (CaSR) expressed in rat dorsal root ganglia (DRG) was identified using rapid amplification of 5'-cDNA ends and primer extension and then cloned into the plasmid vector pCR3.1. The DNA sequence of the DRG CaSR was 99.9% homologous with published rat kidney CaSR in the coding region and 247 bp upstream of the start site but showed little homology 5' to this site, which maps to exonic junction I/II, supporting the hypothesis that CaSR message arises as a splice variant and showing tissue-to-tissue heterogeneity. Western blot revealed a doublet of 140 and 160 kDa in a thyroparathyroid preparation and a single 140-kDa band in DRG. Deglycosylation using N-glycanase increased the mobility of CaSR protein from both DRG and thyroparathyroid, whereas endo-H was without effect, indicating that the DGR CaSR is a mature form of the receptor. A DRG CaSR-pEGFP fusion product was constructed, and when transfected into HEK-293 cells, it was distributed at the cell membrane and resulted in extracellular [Ca.sup.2+] (0.5-3 mM)-evoked increases in intracellular [Ca.sup.2+], which in some instances exhibited oscillatory behavior. We conclude that DRG CaSR cDNA arises from tissue-specific alternative splicing of a single gene, that the amino acid sequence of DRG CaSR is homologous to other known CaSRs, and that the DRG CaSR undergoes differential posttranslational processing relative to the thyroparathyroid CaSR and is functionally active when transfected into a human-derived cell line. calcium; dorsal root ganglion; posttranscriptional modification; glycosylation

The hypothesis that Ca2+ in the rat duodenal submucosa varies through a range that is sufficient to activate this pathway is tested. The estimation of Ca2+ was done by in situ microdialysis method. Isometric tension responses of isolated mesenteric resistance arteries were studied by using wire myography. The results showed that duodenal Ca2+ undergoes dynamic changes over a range that activates the sensory nerve-linked dilator system. In addition, this system can link changes in local Ca2+ transport with alterations organ blood flow and regional resistance.

The effect of chronic sensory denervation on Ca2+-induced relaxation was examined. Mesenteric resistance arteries were isolated from rats treated as neonates with capsaicin, vehicle or saline. The effect of cumulative addition of Ca2+ was evaluated in vessels precontracted with 5 microM norepinephrine. Findings were in good agreement with the hypothesis that Ca2+-induced relaxation is mediated by activation of the Ca2+ receptor associated with capsaicin-sensitive perivascular neurons.

The effects of organ culture on vessel physical properties, force generation and myosin isoform expression were analyzed in cultured rat mesenteric resistance arteries. The magnitude of the maximal response of rat mesenteric resistance arteries to norepinephrine and K+ was significantly depressed by organ culture. Responses to alpha-adrenergic receptor activation was also attenuated in cultured rat mesenteric resistance arteries. Furthermore, 1alpha, 25-dihydroxyvitamin D3 exhibited differential effects on myosin heavy chain isoform expression.

The effects of traumatic brain injury (TBI) on cerebral artery contraction and relaxation were analyzed in anesthetized rats after moderate and severe midline fluid percussion TBI or sham injury. Analysis of isolated posterior and middle cerebral arteries from rats with midline fluid percussion TBI indicated the presence of unaltered intrinsic arterial contractile ability. Furthermore, TBI did not alter fundamental responses to applied agonists in rodent pial arterioles.

The calciotropic steroid, 1-alpha,25-dihydroxycholecalciferol (1,25(OH2)D3), accelerates the intracellular Ca2+ mobilization which leads to increased force generation in resistance arteries. Smooth muscle cells have increased levels of intracellular Ca2+. The 1,25(OH2)D3 induces active stress and the activity of verapamil-sensitive Ca2+ channels in the mesenteric resistance arteries. Basal-sensitive Ca2+ channels have increased Ca2+ mobilization and active stress.

1,25-dihydroxyvitamin D3 (1,25{OH}2D3) is important to understand because of its elevation in many conditions associated with heart dysfunction. 1,25{OH}2D3 enhances the expression of the myosin light chain and heavy chain in the aorta, but there is differential distribution and 1,25(OH)2D3 responsiveness of the smooth muscle isoform of the light chain along the arterial tree. The increase in light chain is due to an increase in the smooth muscle isoform of the protein, showing the differentiating effect of 1,25(OH)2D3 in the aorta. 1,25(OH)2D3 also enhances stress generation in the resistant artery.

A study of mesentric arteries isolated from Wistar rats using wire myography and a fura-based method to unravel the mechanism by which extracellular Ca(super 2+) controls vascular smooth muscle force generation revealed that Ca(sub 0) regulates force generation by depressing myofilament Ca(super 2+) sensitivity and lowering Ca(sub i) after to activation of a calcium activated potassium channel. The results comply with the theory that enhancing extracellular Ca(super 2+) over a bodily concentration range downregulates force generation by altering the Ca(sub i)-force relationship.

Mupanomunda, Maria M., Bing Tian, Norio Ishioka, and Richard D. Bukoski. Renal interstitial [Ca.sup.2+]. Am J Physiol Renal Physiol 278: F644-F649, 2000.--Renal interstitial fluid [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.isf]) was measured in anesthetized Wistar rats by using in situ microdialysis. During perfusion of 20 cm of the proximal small intestine with [Ca.sup.2+]-free buffer, renal [[[Ca.sup.2+]].sub.isf] was 1.63 [+ or -] 0.19 mmol/l in the cortex (n = 6) and 1.93 [+ or -] 0.12 mmol/l in the medulla (n = 5, P = 0.223). When [Ca.sup.2+] in the intestinal lumen was increased to 3 mmol/l, no change was seen in total or ionized serum [Ca.sup.2+] ([S.sub.Ca]), urinary [Ca.sup.2+] excretion ([U.sub.Ca]), or [Ca.sup.2+] in a microdialysate of the kidney cortex. Increasing intestinal [Ca.sup.2+] further, to 6 mmol/l, was without effect on [S.sub.Ca] but significantly increased [U.sub.Ca] by 38% and microdialysate [Ca.sup.2+] by 36% (1.25 [+ or -] 0.0.09 vs. 1.70 [+ or -] 0.14 mmol/l, n = 4, P [is less than] 0.05). Intravenous infusion of 28 ng [multiplied by] [kg.sup.-1] [multiplied by] [min.sup.-1] of parathyroid hormone for 1 h during perfusion of the intestinal lumen with 1 mmol/ [Ca.sup.2+] caused a 7-10% rise in [S.sub.Ca], a 40% fall in [U.sub.Ca], and a 32% increase in microdialysate [Ca.sup.2+] (1.32 [+ or -] 0.13 vs. 1.74 [+ or -] 0.13 mmol/l, n = 6, P [is less than] 0.05). Interlobar arteries with a mean diameter of 120 [micro]m were studied by using a wire myograph to determine whether changes in extracellular [Ca.sup.2+] affect muscle tone. When precontracted with 5 [micro]mol/l serotonin, the arteries relaxed in response to cumulative addition of [Ca.sup.2+] (1-5 mmol/l) with an [ED.sub.50] value for [Ca.sup.2+] of 3.30 [+ or -] 0.08 mmol/l, n = 3. These data demonstrate that [[[Ca.sup.2+]].sub.isf] changes dynamically during manipulation of whole-animal [Ca.sup.2+] homeostasis and that intrarenal arteries relax in response to extracellular [Ca.sup.2+] varied over the range measured in vivo. kidney; calcium; microdialysis; relaxation