Expression of nitric oxide synthase in kidney macula densa cells.

The distribution of nitric oxide synthase (NOS), the enzyme by which NO is generated from L-arginine, was investigated in rat kidney. The indirect immunofluorescence technique using a polyclonal antibody against type I NOS was applied, followed by the histochemical NADPH diaphorase staining technique on the same sections in order to demonstrate the enzymatic activity of NOS. Macula densa cells were strongly stained by both techniques, demonstrating abundant NOS in the cytoplasm of these cells. In addition, these findings were confirmed by nonradioactive in situ hybridization, thus demonstrating the corresponding messenger RNA in macula densa cells as well. Our findings provide the morphological basis for a possible role of NO as a mediator substance in signal transfer from distal tubular fluid to glomerular arterioles.     

Role of neuronal nitric oxide synthase in the macula densa.

BACKGROUND: There is evidence that macula densa nitric oxide (NO) inhibits tubuloglomerular feedback (TGF). However, TGF response is not altered in mice deficient in neuronal nitric oxide synthase (nNOS) (-/-). Furthermore, nNOS expression in the macula densa is inversely related to salt intake, yet micropuncture studies have shown that NOS inhibition potentiates TGF in rats on high sodium intake but not in rats on a low-salt diet. These inconsistencies may be due to confounding systemic factors, such as changes in circulating renin. To further clarify the role of macula densa nNOS in TGF response, independent of systemic factors, we tested the hypothesis that (1) TGF response is inversely related to sodium intake, and (2) during low sodium intake, NO produced by macula densa nNOS tonically controls the basal diameter of the afferent arteriole (Af-Art). METHODS: Af-Arts and attached macula densas were simultaneously microperfused in vitro. TGF response was determined by measuring Af-Art diameter before and after increasing NaCl in the macula densa perfusate. TGF response was studied in wild-type (+/+) and nNOS knockout mice (-/-), as well as in juxtaglomerular apparatuses (JGAs) from rabbits fed a low-, normal-, or high-NaCl diet. RESULTS: TGF responses were similar in nNOS +/+ and -/- mice. However, in nNOS +/+ mice, 7-nitroindazole (7-NI) perfused into the macula densa significantly potentiated the TGF response (P = 0.001), while in nNOS -/- mice, this potentiation was absent. In rabbits on three different sodium diets, TGF responses were similar and were potentiated equally by 7-NI. However, in JGAs from rabbits on a low-NaCl diet, adding 7-NI to the macula densa while perfusing it with low-NaCl fluid caused Af-Art vasoconstriction, decreasing the diameter by 14% (from 21.7 +/- 1.3 to 18.6 +/- 1.5 microm; P < 0.001). This effect was not observed in JGAs from rabbits fed a normal- (19.0 +/- 0.5 vs. 19.3 +/- 0.8 microm after 7-NI) or high-NaCl diet (18.6 +/- 0.7 vs. 18.4 +/- 0.7 microm). CONCLUSIONS: First, in this in vitro preparation, chronic changes in macula densa nNOS do not play a major role in the regulation of TGF. Compensatory mechanisms may develop during chronic alteration of nNOS that keep TGF relatively constant. Second, nNOS regulates TGF response acutely. Third, the results obtained in the +/+ and -/- mice also confirm that the effect of 7-NI is due to inhibition of macula densa nNOS. Finally, during low sodium intake (without induction of TGF), the regulation of basal Af-Art resistance by macula densa nNOS suggests that NO in the macula densa helps maintain renal blood flow during the high renin secretion caused by low sodium intake.     

Role of macula densa nitric oxide and cGMP in the regulation of tubuloglomerular feedback

BACKGROUND: Previous studies have suggested that nitric oxide (NO) produced within cells of the macula densa (MD) modulates tubuloglomerular feedback (TGF). We tested the hypothesis that NO produced in the MD acts locally as an autacoid to activate soluble guanylate cyclase and cGMP-dependent protein kinase in the MD itself. METHODS: Rabbit afferent arterioles (Af-Arts) and attached MD were simultaneously microperfused in vitro. The TGF response was determined by measuring the Af-Art diameter before and after increasing NaCl in the MD perfusate (from 17 mmol/L of Na and 2 of Cl to 65 mmol/L of Na and 50 of Cl). TGF was studied before (control TGF) and after inhibiting components of the NO-cGMP-dependent cascade in the tubular or vascular compartment. RESULTS: Increasing NaCl concentration in the MD perfusate decreased the Af-Art diameter by 3.2 +/- 0.5 microm (from 18.5 +/- 1.3 to 15.4 +/- 1.3 microm, P < 0.001). Adding a soluble guanylate cyclase inhibitor (LY83583) to the MD increased TGF response to 6.3 +/- 1.1 microm (P < 0.031 vs. control TGF). Similarly, when cGMP-dependent protein kinase was inhibited with KT5823, TGF was augmented from 2.6 +/- 0.3 to 4.0 +/- 0.7 microm (P < 0.023). An analogue of cGMP in the MD reversed the TGF-potentiating effect of both 7-nitroindazole (7NI; an nNOS inhibitor) and LY83583. Inhibition of MD guanylate cyclase did not alter the effect of acetylcholine (a NO-cGMP-dependent vasodilator) on the Af-Art. Perfusing the Af-Art with the guanylate cyclase inhibitor did not potentiate TGF, suggesting that the effect of NO occurred at the MD via a cGMP-dependent mechanism. To determine whether the effect of NO in the MD was entirely mediated by cGMP, TGF was studied after giving (1) LY83583 or (2) LY83583 plus 7NI. Adding the nNOS inhibitor to the MD did not potentiate the TGF response further. CONCLUSIONS: We concluded the following: (1) NO produced by the MD inhibits TGF via stimulation of soluble guanylate cyclase, generating cGMP and activating cGMP-dependent protein kinase; (2) NO acts on the MD itself rather than by diffusing to the Af-Art; and (3) most, if not all, of the effect of NO in the MD is due to a cGMP-dependent mechanism rather than to other NO mediators.     

Changes of cell volume and nitric oxide concentration in macula densa cells caused by changes in luminal NaCl concentration

The luminal NaCl concentration ([NaCl]) at the macula densa (MD) controls both tubuloglomerular feedback (TGF) and renin release. Nitric oxide (NO) inhibits TGF sensitivity to a great extent. The NO concentration in the MD cells is not known. This study measured this concentration in MD cells with confocal microscopy in the isolated perfused thick ascending limb using a NO-sensitive fluorophore 4,5-diaminofluorescein (DAF-2). Calcein was used to measure cell volume changes. The loop perfusion fluid was a modified Ringer solution containing 10, 35, or 135 mM NaCl with a constant total osmolarity (290 mOsm), and the bath was perfused with the 135 mM NaCl solution. The results show that MD cell volume and NO concentration measured with DAF-2 DA increased considerably with increasing luminal [NaCl] and with calcium-free solutions in the lumen and bath. L-arginine (5 mM) increased NO concentration in the MD cells by 30%. 7-nitroindazole could totally inhibit the NO production caused by L-arginine and by increased luminal [NaCl]. In conclusion, the MD cell volume changes caused by the changes of luminal [NaCl] were quantitatively measured, and it was found that increasing the luminal [NaCl] resulted in an increase in cell volume. It was also found that NO formation in MD cells could be measured with DAF-2 and that NO production was increased through neuronal NO synthase activation with an increased luminal [NaCl]. An increased NO production will inhibit the vasoconstriction induced by the TGF and at the same time will reduce TGF sensitivity.     

VEGF regulation of endothelial nitric oxide synthase in glomerular endothelial cells

BACKGROUND: Vascular endothelial growth factor (VEGF) regulation of endothelial nitric oxide synthase (eNOS) and signaling pathways involved have not been well studied in glomerular endothelial cells (GENCs). METHODS: GENCs grown from tsA58 Immortomice were used. Immunoblotting and in-cell Western blot analysis were employed to assess changes in VEGF receptor signaling pathway and eNOS phosphorylation of ser1177. Immunokinase assay and immunoblotting with phosphospecific antibodies were performed to assess activity of kinases. RESULTS: VEGF rapidly induced tyrosine phosphorylation of type 1 and type 2 VEGF receptors. Physical association between VEGF-receptor 2 (VEGF-R2) and insulin receptor substrate (IRS-1) and phosphatidylinositol 3'-kinase (PI3K) was induced by VEGF, which augmented PI3K activity in VEGF-R2 immunoprecipitates. VEGF stimulated Akt phosphorylation in a PI3K-dependent manner. VEGF increased eNOS phosphorylation on Ser1177. Activation of eNOS was associated with nitric oxide generation as measured by medium nitrite content. Signaling mechanisms involved in VEGF stimulation of eNOS were explored. VEGF-induced eNOS phosphorylation was abolished by SU1498, a VEGF-R2 inhibitor, LY294002, a PI3K inhibitor, and infection of cells with an adenovirus carrying a dominant negative-mutant of Akt, demonstrating the requirement of the VEGF-R2/IRS-1/PI3K/Akt axis for activation of eNOS. VEGF also activated extracellular signal-regulated protein kinase (ERK) in a time-dependent manner; and VEGF-stimulated eNOS phosphorylation on Ser1177 was prevented by PD098059, an upstream inhibitor of ERK, demonstrating that ERK was involved in VEGF regulation of eNOS. ERK phosphorylation was abolished by LY294002, suggesting ERK was downstream of PI3K in VEGF-treated GENC. CONCLUSIONS: Our data demonstrate that in GENC, VEGF stimulates VEGF-R2/IRS-1/PI3K/Akt axis to regulate eNOS phosphorylation on Ser1177 in conjunction with the ERK signaling pathway.     

Neuronal nitric oxide synthase mediates halothane-induced cerebral microvascular dilation

BACKGROUND: The causes of volatile anesthetic-induced cerebral vasodilation include direct effects on smooth muscle and indirect effects via changes in metabolic rate and release of mediators from vascular endothelium and brain parenchyma. The role of nitric oxide and the relative importance of neuronal and endothelial nitric oxide synthase (nNOS and eNOS, respectively) are unclear. METHODS: Rat brain slices were superfused with oxygenated artificial cerebrospinal fluid. Hippocampal arteriolar diameters were measured using computerized videomicrometry. Vessels were preconstricted with prostaglandin F2alpha (PGF2alpha; halothane group) or pretreated with 7-nitroindazole sodium (7-NINA, specific nNOS inhibitor, 7-NINA + halothane group) or N-nitro-L-arginine methylester (L-NAME; nonselective NOS inhibitor, L-NAME + halothane group) and subsequently given PGF2alpha to achieve the same total preconstriction as in the halothane group. Increasing concentrations of halothane were administered and vasodilation was calculated as a percentage of preconstriction. RESULTS: Halothane caused significant, dose-dependent dilation of hippocampal microvessels (halothane group). Inhibition of nNOS by 7-NINA or nNOS + eNOS by L-NAME similarly attenuated halothane-induced dilation at 0.6, 1.6, and 2.6% halothane. The dilation (mean +/- SEM) at 1.6% halothane was 104 +/- 10%, 65 +/- 6%, and 51 +/- 9% in the halothane, 7-NINA + halothane and L-NAME + halothane groups, respectively. The specificity of 7-NINA was confirmed by showing that acetylcholine-induced dilation was not inhibited by 7-NINA but was converted to constriction by L-NAME. CONCLUSIONS: At clinically relevant concentrations, halothane potently dilates intracerebral arterioles. This dilation is mediated, in part, by neuronally derived nitric oxide. Endothelial NOS does not play a major role in halothane-induced dilation of hippocampal microvessels.     

Expression and functional role of nitric oxide synthase isoforms in human osteoblast-like cells

Previous studies have shown evidence of constitutive and cytokine-inducible nitric oxide (NO) synthase activity in cultured osteoblast-like cells from various species. Although cytokine-induced NO production has been found to inhibit osteoblast growth, the role of constitutive NO production in regulating osteoblast function is less clear and the isoforms of nitric oxide synthase (NOS) that are expressed by human osteoblasts have not been determined. Here, we investigated NOS expression in cultured human osteoblast-like cells and studied the effects of constitutive and cytokine-induced NO on osteoblast growth and differentiation. Low levels of NO were produced constitutively by osteoblast-like cells as reflected by analysis of medium nitrite concentrations, and evidence of ecNOS mRNA, protein, and bioactivity was found in primary osteoblasts (hOBs), TE85, and MG63 osteosarcoma cells. None of the osteoblast-like cells expressed nNOS, however, and iNOS was produced only by hOB cells after stimulation with the cytokines IL-1beta, TNF-alpha, and IFN-gamma. The NOS inhibitor, L-NMMA, did not affect growth or alkaline phosphatase activity in unstimulated osteoblasts. Incubation of hOB cells with cytokines inhibited growth and stimulated alkaline phosphatase activity and these effects were abrogated by L-NMMA. Cytokines also inhibited growth of TE85 cells and MG63 cells, but these effects appeared to be NO independent because they were not influenced by L-NMMA. Our experiments show that human osteoblasts constitutively produce NO through the ecNOS pathway, but demonstrate that this does not appear to exert an appreciable effect on osteoblast growth or differentiation under basal conditions. In contrast, IL-1beta, TNF-alpha, and IFN-gamma exerted growth-inhibiting and differentiation-inducing effects on osteoblasts that were partly NO dependent, indicating that NO may act predominantly as a modulator of cytokine-induced effects on osteoblast function.     

一氧化氮(NO)合酶和NO在延迟性异种移植排斥反应中的作用

目的利用小鼠至大鼠异位心脏移植模型,研究诱导性一氧化氮合酶(iNOS)和受体血清一氧化氮(NO)在延迟性异种移植排斥反应(DXR)中的作用.方法将大鼠随机分为4组A组(6只),空白对照;B组(5只),来氟米物(Lef)+环孢素A(CsA);C组(6只),氨基胍;D组(6只),氨基胍+Lef+CsA.利用免疫组织化学染色检测CD68和NOS2,原位杂交技术检测iNOS mRNA表达.于移植前3 d和移植心脏排斥时分别采集血清检测NO含量.结果所有被排斥心脏中均见巨噬细胞(MФ)浸润,Lef+CsA显著延长移植心脏存活(与A和C组相比,P＜0.05),单用氨基胍使移植心脏存活(3 83±1.47)d(与A组比较,P＜0.05),氨基胍联用Lef和CsA使移植心脏存活(8.67±1.76)d(与A、B和C组比较,P＜0.05).发生DXR时浸润的MФ均有NOS2蛋白和mRNA阳性表达,且不受氨基胍影响.发生DXR时大鼠血清NO水平较移植前显著升高(P＜0.01),氨基胍可显著降低排斥时NO水平.结论小鼠至大鼠心脏移植发生DXR时浸润的MФ表达iNOS增多,且血清NO升高.抑制iNOS活性,降低NO水平可显著延长移植物存活时间,提示iNOS和NO是DXR发生的可能机制之一.     

Localization and role of nitric oxide synthase and endogenous nitric oxide synthase inhibitors in the rabbit lower urinary tract

PURPOSE: We investigated the possible role of endogenous methylarginine derivatives, such as NG-monomethyl-L-arginine (L-NMMA), asymmetrical NG, NG-dimethyl-L-arginine (ADMA) and symmetrical NG, N'G-dimethyl-L-arginine (SDMA), for regulating nitric oxide synthase in the rabbit lower urinary tract. MATERIALS AND METHODS: Strips of detrusor, trigone and proximal urethra were processed for the determination of endogenous methylarginines and L-arginine by automated high performance liquid chromatography. We also compared nitric oxide synthase activity and nitric oxide mediated functional responses to electrical field stimulation in the 3 regions. Nitric oxide synthase activity was measured by determining the conversion of [3H] L-arginine to [3H] L-citrulline. RESULTS: L-NMMA, ADMA and SDMA were detectable by high performance liquid chromatography in the detrusor, trigone and proximal urethra. Electrical field stimulation induced nitric oxide mediated prominent relaxation in the trigone and proximal urethra, while no relaxation response was observed in the detrusor. Exogenously applied 1 to 100 microM. L-NMMA and 1 to 100 microM. ADMA but not 100 microM. SDMA dependently inhibited Ca2+ dependent nitric oxide synthase activity in all regions, and electrical field stimulation induced relaxation in the trigone and proximal urethra. Inhibition with L-NMMA and ADMA was blocked in the presence of 3 mM. L-arginine but not by 3 mM. D-arginine. CONCLUSIONS: Three methylarginines and nitric oxide synthase are localized throughout the rabbit lower urinary tract. Endogenous L-NMMA and ADMA may be involved in regulating nitric oxide biosynthesis of the micturition reflex in the normal or disease state.     

Chronic diabetic nephropathy: role of inducible nitric oxide synthase

Nitric oxide (NO) is a multifunctional mediator that has been implicated in the short-term hemodynamic alterations that occur in acute streptozocin (STZ)-induced diabetes. We investigated the role of NO produced by inducible nitric oxide synthase (iNOS) in chronic STZ diabetic nephropathy. Diabetes was induced in C57BL/6 and iNOS knockout (KO) mice with two intraperitoneal injections of STZ, 100 mg/kg. Animals were maintained without insulin treatment for 40 weeks. There were no significant differences between the strains in blood urea nitrogen (BUN), serum creatinine or glucose concentration, or urinary protein excretion during the entire observation period. Urinary nitrite + nitrate excretion was significantly lower in iNOS KO mice compared to control animals at all time points; in C57 mice, urinary nitrite declined progressively with more prolonged duration of diabetes. Renal hypertrophy (kidney weight/body weight) was noted in both strains of mice. However, histopathological assessment of renal tissue specimens at 16 and 40 weeks demonstrated increased mesangial hypercellularity and expansion as well as more prominent tubulointerstitial fibrosis in iNOS KO versus C57 mice. These changes were accompanied by increased interstitial deposition of type I collagen at 16 and 40 weeks in iNOS KO mice. Glomerular basement membrane staining for type IV collagen was also increased at 40 weeks in diabetic iNOS KO mice. While iNOS protein was undetectable in any of the kidney specimens obtained from either strain, eNOS was present throughout the course of chronic STZ diabetes. Moreover, eNOS expression was significantly increased by approximately 40% at 16 and 40 weeks of observation in iNOS KO versus C57 mice. There was no difference in renal cortical malondialdehyde content between the strains early or late in the disease course. In time control animals, there was no evidence of renal histopathological damage in iNOS KO or C57 mice after 40 weeks. We conclude that iNOS-derived NO modulates glomerulosclerosis and tubulointerstitial fibrosis in chronic STZ nephropathy. This action is probably a result of the direct actions of NO on the synthesis and degradation of extracellular matrix proteins. Received: 28 February 2001 / Revised: 10 August 2001 / Accepted: 13 August 2001     

Possible role of adenosine in macula densa control of glomerular hemodynamics.

BACKGROUND: The macula densa (MD), a plaque of specialized tubular epithelial cells, senses changes in tubular NaCl concentration and sends a signal(s) that controls the resistance of the glomerular afferent arteriole (Af-Art). This mechanism, called tubuloglomerular feedback (TGF), is thought to be important in the homeostasis of body fluids and electrolytes. Our aim was to determine the range of NaCl concentrations in tubular fluid at the MD that would elicit the Af-Art response. In addition, we examined the possible involvement of adenosine in transmitting the signal from the MD to the Af-Art. METHODS: Rabbit Af-Arts and attached MD were simultaneously microperfused in vitro, keeping pressure in the Af-Art at 60 mm Hg. RESULTS: Increasing the Na+/Cl- concentration of the MD perfusate from 26/7 to 41/22 mEq/L decreased the luminal diameter of the terminal Af-Art segment by 10 +/- 4% (N=9; P < 0.01). The response was maximal at 55/36 mEq/L (18 +/- 6%), so that further elevation of NaCl concentration had no additional effect (20 +/- 6% at 84/65 mEq/L). When FK838 (10(-6) mol/L), a specific adenosine A1 receptor antagonist, was added to both Af-Art perfusate and bath, Af-Art constriction was completely abolished. The maximum response was 20 +/- 3% before FK838 and 0.6 +/- 1% afterward (N=12). Adding adenosine at 10(-8) mol/L to both bath and perfusate significantly augmented Af-Art constriction induced by increased NaCl at the MD (P < 0.01); however, adding 10-8 to 10-6 mol/L adenosine to the MD perfusate had no effect regardless of the NaCl concentration at the MD. CONCLUSIONS: These results demonstrate that MD control of Af-Art resistance is induced by relatively low NaCl concentrations at the MD, and that activation of the adenosine A1 receptor in the vascular and interstitial space (but not the tubular lumen) may be essential for signal transmission from the MD to the Af-Art.     

A protective role for endothelial nitric oxide synthase in glomerulonephritis

In acute glomerulonephritis (GN), increased nitric oxide (NO) production occurs, suggesting a pathophysiological role for NO in the disease process. Although NO potentially could have both toxic as well as protective effects, its exact role in the pathophysiology of GN is unclear and may depend on the NOS isoform generating NO. The protective effects of NO such as prevention of leukocyte and platelet activation and adhesion have been attributed to NO generated by endothelial nitric oxide synthase (eNOS). Evidence for a beneficial role for eNOS includes the demonstration of reduced eNOS expression in experimental models of GN as well as human biopsy specimens that is mostly likely due to endothelial cell necrosis. Reduced NO production in GN also may occur through reaction of NO with superoxide anions or the myeloperoxidase (MPO)/hypochlorous acid (HOCL) system. Further evidence has been provided by the observation that in several experimental models of GN, glomerular injury is exacerbated following treatment with non-selective NO inhibitors. Finally, the development of GN is severely aggravated in mice lacking a functional gene for eNOS as compared to wild-type mice, providing direct support for a protective role of eNOS-derived NO in acute GN.     

体外培养的人腹主动脉瘤血管平滑肌细胞中一氧化氮的生成和诱生型一氧化氮合酶的表达

目的探讨体外培养的人腹主动脉瘤 (AAA)血管平滑肌细胞 (SMC)及其表达诱生型一氧化氮合酶 (iNOS)和培养液中生成一氧化氮 (NO)的情况。方法 0 0 2 %Ⅰ型胶原酶消化法进行AAA SMC原代培养 ,平滑肌α 肌动蛋白 (α SMA)鉴定SMC并绘制AAA SMC的增殖曲线 ;免疫细胞化学方法检测AAA SMC中iNOS蛋白的表达 ,并测定原代及 2代细胞培养液中亚硝酸盐和硝酸盐的浓度之和 (NO2 - NO3 -,NOX)。结果酶消化法成功培养AAA SMC ,α SMA阳性率为 4 5 %± 5 8% ,传代培养发现AAA SMC增殖力有限 ;AAA SMC中iNOS蛋白阳性率 86 7%± 4 6 % ,细胞培养液中存在高浓度的NOX。结论AAA SMC存在异常增殖但增殖力有限 ,且细胞可能存在表型变化 ;AAA SMC中iNOS蛋白的高表达及NO的过量生成 ,提示由SMC生成的过量NO可能在腹主动脉瘤发病机制中具有重要作用。     

Purinergic receptor signaling at the basolateral membrane of macula densa cells

Purinergic receptors are important in the regulation of renal hemodynamics; therefore, this study sought to determine if such receptors influence macula densa cell function. Isolated glomeruli containing macula densa cells, with and without the cortical thick ascending limb, were loaded with the Ca(2+) sensitive indicators, Fura Red (confocal microscopy) or fura 2 (conventional video image analysis). Studies were performed on an inverted microscope in a chamber with a flow-through perfusion system. Changes in cytosolic calcium concentration ([Ca(2+)](i)) from exposed macula densa plaques were assessed upon addition of adenosine, ATP, UTP, ADP, or 2-methylthio-ATP (2- MeS-ATP) for 2 min added to the bathing solution. There was no change in [Ca(2+)](i) with addition of adenosine (10(-7) to 10(-3) M). UTP and ATP (10(-4) M) caused [Ca(2+)](i) to increase by 268 +/- 40 nM (n = 21) and 295 +/- 53 nM (n = 21), respectively, whereas in response to 2MesATP and ADP, [Ca(2+)](i) increased by only 67 +/- 13 nM (n = 8) and 93 +/- 36 nM (n = 14), respectively. Dose response curve for ATP (10(-7) to 10(-3) M) added in bath showed an EC(50) of 15 microM. No effect on macula densa [Ca(2+)](i) was seen when ATP was added from the lumen. ATP caused similar increases in macula densa [Ca(2+)](i) in the presence or absence of bath Ca(2+) and addition of 5 mM ethyleneglycotetraacetic acid (EGTA). Suramin (an antagonist of P2X and P2Y receptors) completely inhibited ATP-induced [Ca(2+)](i) dynamics. Also, ATP-Ca(2+) responsiveness was prevented by the phospholipase C inhibitor, U-73122, but not by its inactive analog, U-73343. These results suggest that macula densa cells possess P2Y(2) purinergic receptors on basolateral but not apical membranes and that activation of these receptors results in the mobilization of Ca(2+).