Comparative expression of the extracellular calcium-sensing receptor in the mouse, rat, and human kidney.

The calcium-sensing receptor (CaSR) was cloned over 20 years ago and functionally demonstrated to regulate circulating levels of parathyroid hormone by maintaining physiological serum ionized calcium concentration ([Ca(2+)]). The receptor is highly expressed in the kidney; however, intrarenal and intraspecies distribution remains controversial. Recently, additional functions of the CaSR receptor in the kidney have emerged, including parathyroid hormone-independent effects. It is therefore critical to establish unequivocally the localization of the CaSR in the kidney to relate this to its proposed physiological roles. In this study, we determined CaSR expression in mouse, rat, and human kidneys using in situ hybridization, immunohistochemistry (using 8 different commercially available and custom-made antibodies), and proximity ligation assays. Negative results in mice with kidney-specific CaSR ablation confirmed the specificity of the immunohistochemistry signal. Both in situ hybridization and immunohistochemistry showed CaSR expression in the thick ascending limb, distal tubule, and collecting duct of all species, with the thick ascending limb showing the highest levels. Within the collecting ducts, there was significant heterogeneity of expression between cell types. In the proximal tubule, lower levels of immunoreactivity were detected by immunohistochemistry and proximity ligation assays. Proximity ligation assays were the only technique to demonstrate expression within glomeruli. This study demonstrated CaSR expression throughout the kidney with minimal discrepancy between species but with significant variation in the levels of expression between cell and tubule types. These findings clarify the intrarenal distribution of the CaSR and enable elucidation of the full physiological roles of the receptor within this organ.

The vascular Ca2+-sensing receptor regulates blood vessel tone and blood pressure.

The extracellular calcium-sensing receptor CaSR is expressed in blood vessels where its role is not completely understood. In this study, we tested the hypothesis that the CaSR expressed in vascular smooth muscle cells (VSMC) is directly involved in regulation of blood pressure and blood vessel tone. Mice with targeted CaSR gene ablation from vascular smooth muscle cells (VSMC) were generated by breeding exon 7 LoxP-CaSR mice with animals in which Cre recombinase is driven by a SM22α promoter (SM22α-Cre). Wire myography performed on Cre-negative [wild-type (WT)] and Cre-positive (SM22α)CaSR(Δflox/Δflox) [knockout (KO)] mice showed an endothelium-independent reduction in aorta and mesenteric artery contractility of KO compared with WT mice in response to KCl and to phenylephrine. Increasing extracellular calcium ion (Ca(2+)) concentrations (1-5 mM) evoked contraction in WT but only relaxation in KO aortas. Accordingly, diastolic and mean arterial blood pressures of KO animals were significantly reduced compared with WT, as measured by both tail cuff and radiotelemetry. This hypotension was mostly pronounced during the animals' active phase and was not rescued by either nitric oxide-synthase inhibition with nitro-l-arginine methyl ester or by a high-salt-supplemented diet. KO animals also exhibited cardiac remodeling, bradycardia, and reduced spontaneous activity in isolated hearts and cardiomyocyte-like cells. Our findings demonstrate a role for CaSR in the cardiovascular system and suggest that physiologically relevant changes in extracellular Ca(2+) concentrations could contribute to setting blood vessel tone levels and heart rate by directly acting on the cardiovascular CaSR.

Adenomatous human parathyroid cells exhibit impaired sensitivity to L-amino acids.

CONTEXT: Primary hyperparathyroidism, which occurs most commonly in patients with adenomatous disease of a single parathyroid gland, arises as a result of impaired extracellular Ca(2+) (Ca(2+)(o))-dependent feedback on PTH secretion, a process mediated by the calcium-sensing receptor (CaR). OBJECTIVE: Because the Ca(2+)(o) sensitivity of the CaR is positively modulated by L-amino acids, we decided to investigate whether the impaired feedback of PTH secretion in adenomatous parathyroid cells might arise from decreased sensitivity to L-amino acids. DESIGN: Samples of normal and adenomatous human parathyroid cells were prepared by collagenase treatment and then exposed in vitro to various concentrations of Ca(2+)(o) or the CaR-active amino acid, L-phenylalanine (L-Phe). SETTING AND PATIENTS: Excess normal parathyroid tissue was obtained from parathyroid autotransplants at the time of thyroid surgery. Samples of adenomatous tissue were obtained from histologically confirmed parathyroid adenomas. MAIN OUTCOME MEASURES: The primary measure was sensitivity of Ca(2+)(o)-dependent PTH secretion to the amino acid L-Phe. The secondary measure was sensitivity of Ca(2+)(o)-dependent intracellular Ca(2+) mobilization to L-Phe. RESULTS: Parathyroid adenomas exhibited reduced sensitivity to the CaR-active amino acid L-Phe, which affected both Ca(2+)(o)-dependent PTH secretion and Ca(2+)(o)-dependent intracellular Ca(2+) mobilization as a measure of CaR-dependent signaling in parathyroid cells. CONCLUSIONS: Impaired L-amino acid sensing by calcium-sensing receptors in adenomatous parathyroid cells contributes to the loss of feedback control of PTH secretion in primary hyperparathyroidism. The CaR's amino acid binding site may be exploited as a target in the medical treatment of primary and perhaps other forms of hyperparathyroidism.

Physiological significance of L-amino acid sensing by extracellular Ca(2+)-sensing receptors.

The calcium-sensing receptor is a multimodal, multimetabolic sensor that mediates the feedback-dependent control of whole body calcium metabolism. Remarkably, in addition to its role in Ca(2+)(o) (extracellular Ca(2+)) sensing, the CaR (Ca(2+)-sensing receptor) also responds to L-amino acids. L-amino acids appear to activate, predominantly, a signalling pathway coupled with intracellular Ca(2+) mobilization, require a threshold concentration of Ca(2+)(o) for efficacy and sensitize the receptor to activation by Ca(2+)(o). Here, we review the evidence that the CaR, like other closely related members of the class 3 GPCR (G-protein-coupled receptor) family including GPRC6A, is a broad-spectrum amino acid-sensing receptor, consider the nature of the signalling response to amino acids and discuss its physiological significance.

Effect of alpha-adrenergic agonist and antagonist infusion on the umbilical and uterine circulations of pregnant sheep.

The effects of the alpha-adrenergic agonists, norepinephrine and methoxamine, and the alpha-antagonist, phenoxybenzamine, on umbilical and uterine blood flows, fetal and maternal heart rates, arterial and venous pressures were examined in near-term chronic sheep preparations. Norepinephrine injection or methoxamine infusion to either fetus or ewe resulted n a respective unilateral fetal or maternal pressur response associated with bradycardial. Uterine blood flow decreased significantly with alpha-agonist administration to either fetus or ewe. Umbilical blood flow did not change with either, but an increase in calculated umbilicalo vascular resistance did occur after fetal administrations of the alpha-agonist. Thus, both the uterine and umbilical vascular beds are responsive to alpha-agonism, but maternal uteroplacental perfusion appears to be more sensitive. alpha-Blockade in either fetus or mother produced no significant changes in umbilical or uterine blood flows or fetal or maternal perfusion pressure suggesting that basal alpha-adrenergic tone is unnecessary for normal maintenance of either fetal or maternal uteroplacental circulation.

Effects of indomethacin on sheep uteroplacental circulations and sensitivity to angiotensin II.

Indomethacin, a prostaglandin synthetase inhibitor, injected simultaneously into the abdominal aorta of the ewe and her fetus at 125 days' gestation causes vasoconstriction of both the uterine and umbilical circulations. There is no change in these hemodynamic effects of indomethacin in the presence of phenoxybenzamine, an alpha-adrenergic antagonist. When the indomethacin experiment is repeated seven to 10 days later, a uterine vasoconstriction 10 per cent greater than that found earlier occurs, but there is no change from control umbilical vascular resistance. The mean maternal arterial pressure response (20 mm. Hg) to angiotensin II with and without indomethacin also was examined at the same time in gestation. Indomethacin infusion to the ewe significantly reduces the pressor dose (nanograms per kilogram per minute) by 42 +/- 4 per cent. Thus, in pregnant sheep in the third trimester, endogenous prostaglandins appear to be: (1) partial determinants of basal blood flow in both the uterine and umbilical circulations and (2) modulators of the vasoconstrictive effects of angiotensin II in the ewe.

Vasoconstrictive effects of prostaglandins in sheep placental circulations.

Changes in the uterine and umbilical circulations after intra-aortic injection of prostaglandin E2 (PGE2) or protaglandin F2alpha (PGF2alpha) to either the fetus or the mother have been examined in 13 ewes at late gestation. PGF2alpha vien to either the fetus or the mother is vasoconstrictive in both uterine and umbilical circulations. PGE2 given to the fetus results in a dissociated response with vasodilation of the uterine circulation and vasoconstriction of the umbilical circulation. PGE2 given to the mother causes a uterine contraction that precludes specific hemodynamic analysis. Therefore, exposure of either maternal or fetal ovine circulation to exogenous prostaglandins in associated with acute changes in both placental circulations. Similar responses to pertubations in endogenous prostaglandin levels may exist in pregnant sheep.

Effects of prolonged infusion of beta-adrenergic agonists on uterine and umbilical blood flow in pregnant sheep.

We have extended our evaluation of the effects of three beta-adrenergic agents in near-term pregnant sheep to include a period of infusion three times longer than previously studied. This extension has provided the following information: (1) Initial depression of uterine blood flow and mean arterial pressure associated with administration of ritodrine or salbutamol abate with time despite continued drug infusion; (2) the uterine hyperemia associated with salbutamol and fenoterol are drug-related rather than postinfusion-related phenomena; (3) increased uterine vascular resistance is found with ritodrine, and decreased uterine vascular resistance occurs with salbutamol and fenoterol.

Effects of salbutamol and isoxsuprine on uterine and umbilical blood flow in pregnant sheep.

The effects of salbutamol and isoxsuprine upon uterine artery blood flow (UtBF) and umbilical vein blood flow (UmBF) were investigated in near-term, nonlaboring chronic sheep preparations. During both intravenous salbutamol and isoxsuprine infusions to the ewe, UtBF and mean maternal arterial pressure decreased significantly. Also, dose-related maternal tachycardia, hyperglycemia, and relative acidemia occurred. There were no significant changes in UmBF, mean fetal arterial pressure, or fetal heart rate (FHR) during salbutamol infusions, but UmBF and FHR increased during isoxsuprine infusions. During the 120 minute postinfusion recovery period, UtBF rose significantly after the salbutamol infusions but not after the isoxsupine infusions. The effects and structure-activity relationship of these two drugs are comparable to those of ritodrine and fenoterol, two other beta-adrenergic agonists.