Vasopressin stimulates the production of extracellular matrix by cultured rat mesangial cells

1. Mesangial expansion, an indicator of chronic glomerular diseases, occurs as a result of the excessive accumulation of extracellular matrix (ECM) proteins, such as type IV collagen. In order to investigate the ability of vasopressin (AVP), which causes mesangial cell proliferation and hypertrophy, to induce ECM production, an enzyme-linked immunosorbent assay was used to measure type I and IV collagen and fibronectin produced from cultured rat mesangial cells. 2. Addition of AVP (0.01-1000 nmol/L) caused a significant and concentration-dependent production of secreted and cell-associated ECM, type I collagen, type IV collagen and fibronectin by cultured rat mesangial cells. The AVP V(1A) receptor-selective antagonist YM218 (0.01-1000 nmol/L) potently and concentration-dependently inhibited the induced increase in ECM production caused by AVP, but the V(2) receptor-selective antagonist SR 121463A (0.1-1000 nmol/L) did not potently inhibit. 3. Vasopressin inhibited the synthesis of matrix metalloproteinase (MMP)-2, which degrades matrix proteins, including type IV collagen, and stimulated endothelin (ET)-1 secretion from mesangial cells. These effects were potently inhibited by YM218, but not by SR 121463A. 4. In addition, 10 nmol/L ET-1 inhibited the synthesis of MMP-2 and stimulated ECM production in mesangial cells. These effects were completely abolished by the ET(A) receptor-selective antagonist YM598 (1 micromol/L); however, the ET(B) receptor-selective antagonist BQ-788 (1 micromol/L) and the AVP receptor antagonists YM218 and SR 121463A did not inhibit ET-1-induced inhibition of MMP-2 synthesis and ECM production. In addition, AVP-induced inhibition of MMP-2 synthesis and ECM production were partly inhibited by YM598. 5. These findings indicate that AVP may modulate ECM production not only via a direct action on V(1A) receptors, but also through stimulation of ET-1 secretion. Vasopressin may contribute to the glomerular remodelling and ECM accumulation observed in glomerular diseases.     

Role of inositol (1,4,5)trisphosphate in epidermal growth factor-induced Ca2+ signaling in A431 cells

The effects of epidermal growth factor on Ca2+ signaling in A431 cells were investigated. Epidermal growth factor induced a transient Ca2+ signal in the absence of external Ca2+ and a sustained response in the presence of extracellular Ca2+, indicating an ability to mobilize intracellular Ca2+ as well as the ability to increase Ca2+ entry from the extracellular space. The Ca(2+)-ATPase inhibitor thapsigargin also activated Ca2+ entry, and neither epidermal growth factor nor the guanine nucleotide-dependent protein-linked receptor agonist bradykinin activated additional Ca2+ entry over that due to thapsigargin. In nominally Ca(2+)-free medium, the addition of bradykinin to A431 cells rapidly but transiently increased inositol 1,4,5-trisphosphate and, in parallel fashion, transiently increased cytosolic Ca2+. Unexpectedly, under these experimental conditions, epidermal growth factor elicited a small but significant Ca2+ signal after the addition of bradykinin. Experiments were designed to determine whether the Ca2+ response to epidermal growth factor after bradykinin results from mobilization of Ca2+ by an inositol 1,4,5-trisphosphate-independent mechanism. Epidermal growth factor stimulated additional inositol 1,4,5-trisphosphate formation in bradykinin-treated cells. Furthermore, the Ca2+ signals elicited by both bradykinin and epidermal growth factor were blocked in cells microinjected with the inositol 1,4,5-trisphosphate receptor antagonist heparin, whereas the intracellular Ca(2+)-ATPase inhibitor thapsigargin still mobilized Ca2+. Finally, histamine, a less efficacious guanine nucleotide-dependent protein-linked receptor agonist, as well as photolyzed, microinjected, caged inositol 1,4,5-trisphosphate, also mobilized Ca2+ after bradykinin. The results of this study show (i) that epidermal growth factor activates intracellular Ca2+ release as well as Ca2+ entry, the latter most likely resulting from an indirect effect due to the depletion of intracellular Ca2+ pools, (ii) that the actions of epidermal growth factor on Ca2+ homeostasis can be fully accounted for by inositol 1,4,5-trisphosphate formation, and (iii) that the ability of A431 cells to produce Ca2+ signals when epidermal growth factor is applied after bradykinin can be explained by the rapid and complete desensitization of the bradykinin stimulated phospholipase C activity.     

RANTES stimulates Ca2+ mobilization and inositol trisphosphate (IP3) formation in cells transfected with G protein-coupled receptor 75

Background and purpose:     

Mass changes of inositol(1,4,5)trisphosphate in trachealis muscle following agonist stimulation

Agonist-stimulated changes in D-Ins(1,4,5)P3 has been examined in tracheal smooth muscle using a specific radioreceptor assay utilizing the binding of [3H] or [32P]Ins(1,4,5)P3 to a bovine adrenal cortex preparation. Carbachol produced a maximal increase in Ins(1,4,5)P3 concentration after 5 s (basal: 12.9 +/- 0.8; stimulated: 27 +/- 1.5 pmol/mg protein) with an EC50 of 6.7 +/- 0.8 microM. With longer stimulation periods Ins(1,4,5)P3 rapidly returned to basal values by 30 s. This is the first report of mass measurements of Ins(1,4,5)P3 in smooth muscle.     

Myo-inositol(1,4,5)trisphosphorothioate binds to specific [3H]inositol(1,4,5)trisphosphate sites in rat cerebellum and is resistant to 5-phosphatase

D-Myo-inositol(1,4,5)trisphosphorothioate, a synthetic analogue of inositol(1,4,5)trisphosphate was shown to bind with a relatively high affinity to specific sites on rat cerebellar membranes labelled with [3H]inositol(1,4,5)trisphosphate. Use of this binding assay has also established that unlike the trisphosphate, the trisphosphorothioate is completely resistant to a specific 5-phosphatase prepared from human erythrocytes. The ability of this novel analogue to release intracellular Ca2+ has already been reported and it offers considerable potential in the investigation of phosphoinositide-linked receptors.     

The effect of PPADS as an antagonist of inositol (1,4,5)trisphosphate induced intracellular calcium mobilization.

1. Organ bath experiments and measurements of prostanoids were performed to investigate the presence of nitric oxide synthase in venous smooth muscle and its interaction with cyclo-oxygenase. 2. In rings of canine saphenous vein without endothelium, the inhibitor of cyclo-oxygenase, indomethacin (10 microM), induced contraction. NG-nitro-L-arginine (100 microM) (L-NOARG), an inhibitor of nitric oxide synthase did not affect the tone of rings of canine saphenous vein when administered alone. However, in the presence of indomethacin L-NOARG (100 microM) induced further contraction. 3. Similar results were obtained in response to NG-monomethyl-L-arginine (L-NMMA)(300 microM or NG-nitro-L-arginine methylester (L-NAME)(100 microM). 4. When rings of canine saphenous vein without endothelium were contracted with phenylephrine (1 microM) instead of indomethacin, neither L-NOARG or L-NMMA induced further contraction. 5. When rings of canine saphenous vein without endothelium were contracted with noradrenaline (0.3 microM) in the presence of indomethacin (10 microM) plus L-NOARG (100 microM), a relaxation to L-arginine was observed. Transient relaxations to superoxide dismutase (150 u ml-1) were observed in all rings. 6. When rings of saphenous vein without endothelium were incubated with lipopolysaccharide (LPS) (100 micrograms ml-1) or interleukin-1 beta (10 u ml-1) the concentration-contraction curve to noradrenaline was not affected. 7. Rings without endothelium released prostaglandin E2 and prostaglandin I2, as measured by radioimmunoassay. The basal production was abolished by indomethacin and not affected by L-NOARG. 8. These results suggest that when cyclo-oxygenase is inhibited, a nitric oxide synthase activity is revealed in rings of canine saphenous vein without endothelium.     

Amyloid beta-(1-40) stimulates cyclic GMP production via release of kinins in primary cultured endothelial cells.

Increased beta-amyloid production is believed to play a central role in the pathogenesis of Alzheimer's disease. Amyloid is deposited not only in the brain of Alzheimer patients as senile plaques but also in the cerebral vessel wall leading to cerebral amyloid angiopathy. Freshly solubilised amyloid beta-(1-40) was previously reported to exert a vasoconstrictor effect. We investigated whether amyloid beta-(1-40) affects the nitric oxide (NO)/cyclic GMP pathway in primary cultured endothelial cells from bovine aorta and rat coronary microvessels. Surprisingly, a significant increase in cyclic GMP production after incubation with freshly dissolved amyloid beta-(1-40) was found. The stimulation of cyclic GMP production could be inhibited by the bradykinin B(2) receptor antagonist icatibant, the NO synthase inhibitor N-omega-nitro-L-arginine, the serine protease inhibitor 3, 4-dichloroisocoumarin and the selective plasma kallikrein inhibitor Pefabloc PK, suggesting activation of the plasma kallikrein-kinin system. This is supported by a three- to four-fold increase in kinins in the supernatant of both types of endothelial cells after incubation with amyloid beta-(1-40) at concentrations of 10(-7) and 10(-6) mol/l.     

Inhibitory effect of diazepam on muscarinic receptor-stimulated inositol 1,4,5-trisphosphate production in rat parotid acinar cells

1. This study examined the effect of diazepam (DZP) on phosphoinositide turnover, which plays an important role in the regulation of salivary secretion, in rat parotid acinar cells. 2. DZP (10(-9) M to 10(-5) M), a potent agonist of both central- and peripheral-type benzodiazepine receptors, dose-dependently decreased inositol 1,4,5-trisphosphate IP3 production stimulated by carbachol, a muscarinic receptor agonist, in the cells. 3. DZP produced a maximum inhibitory response at a concentration of 10(-5) M, with IP3 production decreased to 63% of maximal levels. The concentration inducing half maximal inhibition of IP3 production was approximately 3.5 x 10 (-8) M. 4. An inhibitory response to DZP was produced by a short-term pretreatment (<3 min) of the cells and prevented by antagonist and competing ligand for the central- and peripheral-type benzodiazepine receptors, flumazenil and PK 11195, respectively. 5. DZP showed a non-competitive inhibition of carbachol-stimulated IP3 production. It did not directly inhibit the activities of GTP-binding regulatory proteins and phosphatidylinositol 4,5-bisphosphate-specific phospholipase C (PLC) in the parotid gland membranes, though choline chloride inhibited PLC activity. 6. DZP (10(-5) M) attenuated the increase in the intracellular Ca2+ concentration ([Ca(2+)](i)) in the cells following stimulation of the muscarinic and alpha(1)-adrenoceptors. 7. These results suggest that in the parotid acinar cells, DZP inhibits muscarinic receptor-stimulated IP3 production through benzodiazepine receptors and that PLC activity which produces IP3 is inhibited by chloride. The decreases in IP3 and [Ca(2+)](i) in the cells may be connected with the suppression of salivary secretion induced by DZP.     

Bradykinin down-regulates LPS-induced eosinophil accumulation in the pleural cavity of mice through type 2-kinin receptor activation: a role for prostaglandins.

1. The role of both exogenously administered and endogenously generated bradykinin (BK) on LPS-induced eosinophil accumulation in the mice pleural cavity was investigated by means of treatment with BK selective receptor agonists/antagonists and captopril. 2. Intrathoracic (i.t.) injection of LPS (250 ng cavity(-1)) induced eosinophil influx at 24 h as previously described (Bozza et al., 1993). Pretreatment with the B1 receptor antagonist des-Arg9-[leu-8]BK (0.025 and 0.25 nmol cavity(-1)) showed no effect on this phenomenon, whereas pretreatment with the B2 receptor antagonists, NPC 17731 (0.025 and 0.25 nmol cavity(-1)) or HOE 140 (2.5 nmol cavity(-1)), increased LPS-induced eosinophil influx. Accordingly, pretreatment with captopril at 10 mg kg(-1) i.p., inhibited eosinophil infiltration induced by LPS in the pleural cavity, suggesting that endogenous BK is down-regulating LPS-induced eosinophil accumulation. 3. BK administered at 15 and 25 nmol cavity(-1), i.t. or i.p. also inhibited LPS-induced eosinophil accumulation. BK alone had no effect on the basal number of leucocytes in the pleural or peritoneal cavity in doses up to 25 nmol cavity(-1). Nevertheless, when injected at doses of 50 and 100 nmol cavity(-1) BK induced leucocyte influx characterized by neutrophil and eosinophil accumulation at 24 h. 4. Similarly to what was observed with BK, a specific B2 receptor agonist, Tyr8BK, administered at 0.25 nmol cavity(-1) i.p., significantly inhibited the eosinophil influx induced by LPS. 5. The mechanism by which B2 receptor agonists inhibit LPS-induced eosinophil accumulation was investigated by pretreating the animals with indomethacin or a selective cyclooxygenase-2 inhibitor, NS-398. Pretreatment with either indomethacin or NS-398 had no effect on eosinophil influx induced by LPS alone, but those drugs were able to restore the LPS-induced eosinophil influx in Tyr8BK (0.25 nmol cavity(-1)) injected mice. 6. In conclusion, endogenously generated bradykinin seems to modulate, through activation of B2 receptors, eosinphil accumulation induced by LPS via a mechanism dependent on prostanoid synthesis.     

Characterization of a B2-bradykinin receptor in rat renal mesangial cells

The specific binding of bradykinin (BK) was investigated using membrane fractions from mesangial cells in primary culture, a cloned cell line, and in intact adherent cells with three different radiolabelled BK analogues: ¹²⁵I-[Tyr⁰]BK, ¹²⁵I-[Tyr⁵]BK and ¹²⁵I-[Tyr⁸]BK. The best radioligand was ¹²⁵I-[Tyr⁰]BK, and assay conditions were determined to ensure maximal stable binding. Binding appeared to be reversible and not to be inhibited by a wide variety of protease inhibitors including converting enzyme inhibitor and phosphoramidon. The maximum density of binding sites (B_max) was about 88 ± 18 fmol/mg protein, which is equivalent to about 6000 sites/cell, and the dissociation constant averaged 2 nM. No significant difference in B_max was observed between membranes from cells in primary culture and those from cloned cells. Of the BK analogues tested, unmodified BK exhibited the highest inhibition constant (close to 10⁻¹⁰ M). No displacement of ¹²⁵I-[Tyr⁰]BK was observed in the presence of the B₁ agonist des-Arg⁹-BK or several unrelated peptides, including atrial natriuretic factor and angiotensin I and II, whereas 50% inhibition of binding was achieved with the B₂ antagonist [D-Arg,Hyp³,D-Phe⁷]BK (10⁻⁹ M). Addition of BK for 3 min to the incubation medium of cloned mesangial cells induced a dose- and time-dependent increase in PGE₂ unlike des-Arg⁹-BK, which showed no such effect. The secretion was strongly inhibited by prior incubation with the B₂ antagonist [D-Arg,Hyp³,D-Phe⁷]BK. The pharmacological profile of the binding site determined with various BK agonists and antagonists, and the stimulating effect of binding site activation on prostaglandin release strongly suggest that B₂-kinin-like receptors are present in rat mesangial cells.     

Stimulation by phaclofen of inositol 1,4,5-triphosphate production in cultured neurons from chick tectum

The influence of gamma-aminobutyric acid (GABA) receptor agonists and antagonists on the intracellular concentration of inositol 1,4,5-triphosphate (IP3) was examined in neuronal cultures of embryonic chick tectum. GABA and selective agonists of its receptors were inactive, whereas phaclofen, a GABA-B receptor antagonist, increased IP3 levels in a concentration-dependent manner. This effect occurred at phaclofen concentrations much lower than those required to affect GABA-B receptors. It is concluded that phaclofen, in addition to its known effect at GABA-B receptors, is also active at an as-yet undefined site.     

Dextran sulfate inhibits the inositol 1,4,5-trisphosphate-induced Ca2+ release from skinned and cultured smooth muscle cells.

Dextran sulfate inhibited the inositol 1,4,5-trisphosphate (IP3)-induced decrease in 45Ca2+ content, in a dose-dependent manner, in saponin-skinned and primary cultured smooth muscle cells from the rat aorta. The maximum inhibition was observed at 3-10 mg/ml, and the IC50 was about 173 micrograms/ml. Dextran sulfate also inhibited the IP3-induced increase in 45Ca2+ efflux rate, but did not affect the caffeine-induced Ca2+ release. Dextran sulfate inhibited the specific binding of [4,5-32P]IP3 to the skinned cells, thereby indicating that it may have an effect on the IP3 receptor. Dextran (without the sulfates) had no inhibitory effect on either the IP3-induced Ca2+ release or on the specific binding of [4,5-32P]IP3. Thus, sulfate groups on the molecule of dextran sulfate may play an important role in the inhibition of the IP3-induced Ca2+ release from intracellular stores.     

Regulation of inositol 1,4,5-trisphosphate metabolism by guanine nucleotides in membranes of cultured newborn rat cardiomyocytes.

Membranes of cultured newborn rat cardiomyocytes contain enzymatic activities that regulate the formation and the breakdown of inositol 1,4,5-trisphosphate (1,4,5-IP3). GTP gamma S increased the rate of exogenous [3H]phosphatidyl 4,5-bisphosphate ([3H]PIP2) hydrolysis (EC50: 40 microM). This effect was dependent on the presence of deoxycholate and maximal at 2 mM deoxycholate. GTP gamma S increased the efficacy of phospholipase C (PLC) (by 2.3-fold), but did not alter the apparent affinity of the enzyme for PIP2. Other nucleotides, GDP beta S and ATP gamma S, and pyrophosphate also stimulated PIP2 hydrolysis, while AlF4- was ineffective. The effect of GTP gamma S was not inhibited by GDP beta S. The agonists norepinephrine and thrombin, which by themselves had no effect, did not potentiate the response to GTP gamma S. In contrast, 1,4,5-IP3 hydrolysis was decreased by GTP gamma S (EC50: 100 microM) as well as by other nucleotides and by pyrophosphate, but not by AlF4-. GDP beta S did not antagonize the GTP gamma S-induced inhibition of IP3 hydrolysis. These results suggest that GTP can stimulate the hydrolysis of exogenous PIP2 by an action on membrane-bound PLC at a site beyond the G protein activating PLC and inhibit the hydrolysis of 1,4,5-IP3 by a mechanism common to all nucleotides. Thus, GTP can regulate 1,4,5-IP3 metabolism by stimulating its formation and inhibiting its breakdown.     

Dexamethasone potentiates production of inositol trisphosphate evoked by endothelin-1 in vascular smooth muscle cells.

To investigate the possibility of participation of glucocorticoids in the action of endothelin-1 (ET-1), the effect of glucocorticoids on ET-1-induced inositol trisphosphate (IP3) production was studied in cultured vascular smooth muscle cells (VSMC). ET-1 transiently increased IP3 in a dose-dependent manner. Pretreatment with 1 microM dexamethasone for 48 h shifted the dose-response curve of ET-1-induced IP3 production to the left; i.e., it significantly reduced the half-maximal effective concentration of ET-1 (from 50 to 10 nM). This action of dexamethasone required a minimum of 12 h of incubation. A glucocorticoid antagonist, RU 38486, completely blocked this effect. To elucidate the interaction with prostaglandin, we used indomethacin, a potent inhibitor of prostaglandin synthesis. Treatment with 1 microM indomethacin shifted the dose-response curve of ET-1-induced IP3 production to the left, but this shift was significantly less than that observed after treatment with dexamethasone and no additive effect between indomethacin and dexamethasone was noted. Glucocorticoids potentiate the IP3 production response to ET-1 in cultured VSMC, and this action of glucocorticoids depends partially on prostaglandin inhibition. These results suggest that glucocorticoids play an important role in modulation of ET-1 action.     

Inhibition of inositol 1,4,5-trisphosphate formation by cyclic GMP in cultured aortic endothelial cells of the pig.

1. In cultured endothelial cells of the pig the endothelium-derived relaxing factor (EDRF) releasing agent thrombin (2 u ml-1) caused a significant increase in basal levels of both guanosine 3':5'-cyclic monophosphate (cyclic GMP) and inositol 1,4,5-trisphosphate (IP3). This increase was time dependent, with peak levels occurring at 2 min and returning towards basal values after 5 min. 2. Pretreatment of the cells with the EDRF inhibitors haemoglobin (1 microM) or L-NG-nitro arginine (50 microM) significantly reduced the cyclic GMP response to thrombin. Both agents also resulted in significant elevations in basal levels of IP3. The IP3 response to thrombin was significantly enhanced at all time points by haemoglobin and at 5 min for L-NG-nitro arginine, when compared with the response to thrombin alone. 3. Pretreatment of the cells with either sodium nitroprusside (10 microM) or atrial natriuretic peptide (1 microM) caused a significant elevation of basal cyclic GMP levels. Although subsequent exposure to thrombin caused a further increase in cyclic GMP, which together with the rise induced by the previous two agents was significantly greater than the increase caused by thrombin alone, the incremental increase induced by thrombin was markedly less in the presence of nitroprusside or atrial natriuretic peptide. Both these agents, as well as 8-bromo cyclic GMP, resulted in a significant suppression of the IP3 response to thrombin. 4. These findings show that one mechanism for the inhibitory effect of cyclic GMP on EDRF release from endothelium may be through the inhibition of IP3 formation in response to EDRF releasing agents.     

醛固酮及其受体拮抗剂对肾小球系膜细胞增殖的影响

目的 研究醛固酮(ALD)及其受体拮抗剂螺内酯(SPI)对大鼠肾小球系膜细胞增殖和细胞周期的影响.方法 应用MTT法检测系膜细胞增殖,用流式细胞术检测细胞周期各时相的百分比.结果 MTT结果显示,ALD抑制肾小球系膜细胞增殖(与对照组比较P＜0.01),并具有一定的剂量依赖和时间依赖性,SPI能拮抗其作用.流式细胞术结果显示,ALD作用于系膜细胞24 h,G1期细胞数增多,S期细胞数减少(与对照组比较,P＜0.01).结论 ALD通过调控细胞周期抑制肾小球系膜细胞增殖.