Induced Inositol Phosphate Accumulation Research Articles

Airway Smooth Muscle Relaxations Induced by Dipyrone

Background: Dipyrone differs from other nonsteroidal anti-inflammatory drugs with a distinctive spasmolytic effect; however, the mechanism of action is not clear yet. The possible mechanism behind this effect was investigated on airway smooth muscle tone in vitro. Method:The effect of dipyrone on inositol phosphate (IP) accumulation was evaluated on guinea pig trachea smooth muscle. Changes in intracellular free calcium levels and IP accumulation were evaluated in LTK8 cells.Results: Dipyrone (0.01, 0.1, 1 mmol/l) had a relaxing effect on histamine (0.02 mmol/l)- and adenosine triphosphate- (ATP) (0.01 mol/l)-induced contractions, but not potassium chloride (KCl)-stimulated contractions. This relaxing effect was not observed with in- domethacin. Indomethacin did not inhibit the relaxation response of dipyrone. In isolated tracheal smooth muscle, histamine- (0.02 mmol/l) and ATP (0.01 mmol/l)-induced IP accumulation was significantly inhibited by dipyrone (1 mmol/l). ATP (0.01 mmol/l)-induced IP accumulation was also significantly inhibited by dipyrone (0.01 mmol/l) in LTK8 cells. Dipyrone inhibited ATP-induced (0.01 and 0.1 mmol/l) intracellular calcium increase in LTK8 cells.Conclusion: Inhibition of the release of intracellular Ca²⁺ may play a role in the smooth muscle relaxing effect of dipyrone. The inhibitor effect of dipyrone on IP accumulation may be due to direct inhibition of phospholipase C (PLC) or impairment of the activation of PLC by G protein-coupled receptor (GPCR).

Inositol phosphate stimulation by LH requires the entire α Asn 56 oligosaccharide

Lentil lectin-bound, fucose-enriched hTSH was reported to stimulate both cAMP and inositol phosphate (IP) intracellular signalling pathways, whereas fucose-depleted hTSH stimulated only the cAMP pathway. Gonadotropins activate the cAMP pathway and in several studies higher concentrations activate the IP pathway. Since only the 10% of α subunit Asn 56 oligosaccharides (Asn 52 in humans) are fucosylated, the higher glycoprotein hormone concentrations required for IP pathway activation might be related to the abundance of competent hormone isoforms. Lentil lectin-fractionated equine (e)LHα and eFSHα preparations were combined with a truncated, des(121–149)eLHβ preparation. All four hybrid hormone preparations induced IP accumulation in porcine theca cells, suggesting that activation of the IP pathway was not dependent on fucosylation at α subunit Asn 56. However, the presence of Asn 56 carbohydrate was necessary for increased IP accumulation. Intact, rather than Asn 56-deglycosylated eLH preparations provoked a biphasic steroidogenic response by rat testis Leydig cells, suggesting that Gα i stimulation was also sensitive to loss of Asn 56 carbohydrate. While rat granulosa cells responded to human FSH preparations in a biphasic manner, a classical sigmoidal response was obtained to eFSH and Asn 56-deglycosylated eFSH, suggesting that the equine preparations did not activate Gα i. Purified oLHα Asn 56 oligosaccharides inhibited FSH-stimulated steroidogenesis in rat granulosa cell cultures indicating a direct role for carbohydrate in FSH action. The same carbohydrate preparation inhibited hCG-stimulated fluorescence energy transfer suggesting oligosaccharide involvement in activated LH receptor self-association.

Threonine 308 within a Putative Casein Kinase 2 Site of the Cytoplasmic Tail of Leukotriene B4 Receptor (BLT1) Is Crucial for Ligand-induced, G-protein-coupled Receptor-specific Kinase 6-mediated Desensitization

Desensitization of G-protein-coupled receptors may involve phosphorylation of serine and threonine residues. The leukotriene B(4) (LTB(4)) receptor (BLT1) contains 14 intracellular serines and threonines, 8 of which are part of consensus target sequences for protein kinase C (PKC) or casein kinase 2. In this study, we investigated the importance of PKC and GPCR-specific kinase (GRK) phosphorylation in BLT1 desensitization. Pretreatment of BLT1-transfected COS-7 cells with PKC activators caused a decrease of LTB(4)-induced inositol phosphate (IP) accumulation. This reduction was prevented with the PKC inhibitor, staurosporine, and not observed in cells expressing a BLT1 deletion mutant (G291stop) lacking the cytoplasmic tail. Moreover LTB(4)-induced IP accumulation was significantly inhibited by overexpression of GRK2, GRK5, and especially GRK6, in cells expressing wild type BLT1 but not in those expressing G291stop. GRK6-mediated desensitization correlated with increased phosphorylation of BLT1. The G319stop truncated BLT1 mutant displayed functional characteristics comparable with wild type BLT1 in terms of desensitization by GRK6, but not by PKC. Substitution of Thr(308) within a putative casein kinase 2 site to proline or alanine in the full-length BLT1 receptor prevented most of GRK6-mediated inhibition of LTB(4)-induced IP production but only partially affected LTB(4)-induced BLT1 phosphorylation. Our findings thus suggest that Thr(308) is a major residue involved in GRK6-mediated desensitization of BLT1 signaling.

Tumour necrosis factor-α enhances bradykinin-induced signal transduction via activation of Ras/Raf/MEK/MAPK in canine tracheal smooth muscle cells

Inhalation of tumour necrosis factor-α (TNF-α) induced a bronchial hyperreactivity to contractile agonists. However, the mechanisms of TNF-α involved in the pathogenesis of bronchial hyperreactivity were not completely understood. Therefore, we investigated the effect of TNF-α on bradykinin (BK)-induced inositol phosphate (IP) accumulation and Ca 2+ mobilization, and up-regulation of BK receptor density in canine cultured tracheal smooth muscle cells (TSMCs). Pretreatment of TSMCs with TNF-α potentiated BK-induced IP accumulation and Ca 2+ mobilization. However, there was no effect on the IP response induced by endothelin-1 (ET-1), 5-hydroxytryptamine (5-HT), and carbachol. Pretreatment with PDGF B-chain homodimer (PDGF-BB) also enhanced BK-induced IP response. These enhancements induced by TNF-α and PDGF-BB might be due to an increase in BK B 2 receptor density ( B max), since [ 3H]BK binding to TSMCs was inhibited by the B 2 selective agonist and antagonist, BK and Hoe 140, but not by the B 1 selective reagents. The enhancing effects of TNF-α and PDGF-BB were attenuated by PD98059 (an inhibitor of activation of MAPK kinase, MEK) and cycloheximide (an inhibitor of protein synthesis), suggesting that TNF-α may share a common signalling pathway with PDGF-BB via protein(s) synthesis in TSMCs. Furthermore, overexpression of dominant negative mutants, H-Ras-15A and Raf-N4, significantly suppressed p42/p44 mitogen-activated protein kinase (MAPK) activation induced by TNF-α and PDGF-BB and attenuated the effect of TNF-α on BK-induced IP response, indicating that Ras and Raf may be required for activation of these kinases. These results suggest that the augmentation of BK-induced responses produced by TNF-α might be, at least in part, mediated through activation of Ras/Raf/MEK/MAPK pathway in TSMCs.

Interaction between the partially insurmountable antagonist valsartan and human recombinant angiotensin II type 1 receptors.

The interaction between the AT1 receptor-selective antagonist valsartan, and its human receptor, was investigated by direct radioligand binding as well as by its inhibition of angiotensin II induced inositol phosphate accumulation in CHO cells expressing human recombinant AT1 receptors. Specific binding of [3H]-valsartan rapidly reached equilibrium at 37 degrees C. It was saturable and occurred to a homogeneous class of sites with a K(D) of 0.88+/-0.076. It was inhibited by other AT1 receptor antagonists with the same potency order as previously described for the binding of [3H]-angiotensin II and [3H]-candesartan to human AT1 receptors (i.e. candesartan > or = EXP3174 > valsartan = irbesartan = angiotensin II > losartan). When valsartan and angiotensin II were applied simultaneously to the CHO-AT1 cells. the antagonist caused a rightward shift of the angiotensin II concentration response curve. Hence, valsartan interacts with the AT1 receptor in a manner that is competitive with angiotensin II. Pre-incubation of the cells with 0.5, 5 and 50 nM valsartan caused an additional, concentration-dependent, up to 55% decline of the maximal response. The partial nature of this insurmountable inhibition by valsartan was confirmed by biphasic antagonist concentration-inhibition curves. These data reflect the co-existence of a fast reversible/surmountable as well as a tight binding/insurmountable valsartan receptor complex. In agreement, pre-incubation of the CHO-AT1 cells with 5 and 50 nM valsartan produced a partial inhibition of the angiotensin II induced increase of the free intracellular calcium concentration. [3H]-Valsartan dissociated from its receptors with a half-life of 17 min. In functional recovery experiments with valsartan-pre-treated cells, the angiotensin II-mediated response was half-maximally restored within approximately 30 min. These kinetic data suggest that the insurmountable inhibition by valsartan is related to its relatively slow dissociation from the human AT1 receptors.

Lipopolysaccharide enhances bradykinin-induced signal transduction via activation of Ras/Raf/MEK/MAPK in canine tracheal smooth muscle cells.

Bacterial lipopolysaccharide (LPS) was found to induce inflammatory responses and to enhance bronchial hyperreactivity to several contractile agonists. However, the implication of LPS in the pathogenesis of bronchial hyperreactivity was not completely understood. Therefore, in this study, we investigated the effect of LPS on mitogen-activated protein kinase (MAPK) activation associated with potentiation of bradykinin (BK)-induced inositol phosphates (IPs) accumulation and Ca(2+) mobilization in canine cultured tracheal smooth muscle cells (TSMCs). LPS stimulated phosphorylation of p42/p44 MAPK in a time- and concentration-dependent manner using a Western blot analysis against a specific phosphorylated form of MAPK antibody. Maximal stimulation of the p42 and p44 MAPK isoforms occurred after 7 min-incubation and the maximal effect was achieved with 100 microg ml(-1) LPS. Pretreatment of TSMCs with LPS potentiated BK-induced IPs accumulation and Ca(2+) mobilization. However, there was no effect on the IPs response induced by endothelin-1, 5-hydroxytryptamine, and carbachol. In addition, pretreatment with PDGF-BB enhanced BK-induced IPs response. These enhancements by LPS and PDGF-BB might be due to an increase in BK B(2) receptor density (B(max)) in TSMCs, characterized by competitive inhibition of [(3)H]-BK binding using B(1) and B(2) receptor-selective reagents. The enhancing effects of LPS and PDGF-BB were attenuated by PD98059, an inhibitor of MAPK kinase (MEK), suggesting that the effect of LPS may share a common signalling pathway with PDGF-BB in TSMCs. Furthermore, overexpression of dominant negative mutants, H-Ras-15A and Raf-N4, significantly suppressed p42/p44 MAPK activation induced by LPS and PDGF-BB, indicating that Ras and Raf may be required for activation of these kinases. These results suggest that the augmentation of BK-induced responses produced by LPS might be, at least in part, mediated through activation of Ras/Raf/MEK/MAPK pathway in TSMCs.

Amino Acids in the Second and Third Intracellular Loops of the Parathyroid Ca2+-sensing Receptor Mediate Efficient Coupling to Phospholipase C

To determine the role of amino acids in the second and third intracellular (IC) loops of the Ca(2+)-sensing receptor (CaR) in phospholipase C (PLC) activation, we mutated residues in these loops either singly or in tandem to Ala and assessed PLC activity by measuring high extracellular [Ca(2+)] ([Ca(2+)](o))-induced inositol phosphate accumulation and protein expression by immunoblotting and immunocytochemistry in human embryonic kidney 293 cells. Two CaR constructs in the second IC loop, F707A CaR and to a lesser extent L704A CaR, demonstrated reduced activation of PLC, despite levels of protein expression comparable with the wild-type (wt) CaR. Substitution of Tyr or His for Phe-707, but not Leu, Val, Glu, or Trp, partially restored the ability of high [Ca(2+)](o) to activate PLC. Eight residues in the third IC loop were involved in PLC signaling. The responses to high [Ca(2+)](o) in cells expressing CaRs with Ala substitutions at these sites were <35% of the wt CaR. The L798A, F802A, and E804A CaRs were dramatically impaired in their responses to [Ca(2+)](o) even up to 30 mm. Substitutions of Leu-798 with other hydrophobic residues (Ile, Val, or Phe), but not with acidic, basic, or polar residues, produced reduced responses compared with wt. Phe-802 could be replaced with either Tyr or Trp with partial retention of the ability to activate PLC. Glu-804 could only be substituted with Asp or Gln and maintain its signaling capacity. Cell surface expression of the CaRs mutated at Leu-798 and Phe-802 appeared normal compared with wt CaR. Cell surface CaR expression was, however, reduced substantially in cells expressing several mutants at position Glu-804 by confocal microscopy. These studies strongly implicate specific hydrophobic and acidic residues in the second and third IC loops of the parathyroid CaR (and potentially larger stretches of the third loop) in mediating efficient high [Ca(2+)](o)-induced PLC activation and or CaR expression.

Cellular signaling by the potent bronchoconstrictor endothelin-1 in airway smooth muscle.

The objective of this study was to determine whether the potent bronchoconstrictor endothelin-1 was coupled to the activation of the inositol phosphate and/or inhibition of the cyclic adenine monophosphate second messenger pathways in porcine airway smooth muscle. Prospective, controlled, in vitro, nonblinded study. University biochemical and molecular biological research laboratory. Pigs of both genders. Airway smooth muscle was dissected from the trachea of pigs exsanguinated under anesthesia. Airway smooth muscle from six animals preloaded with 3H-myoinositol was exposed to endothelin-1, carbachol (positive control) or vehicle for 30 mins. Some tissues were pretreated with antagonists selective for the ET(A) (BQ-485) and ET(B) (BQ-788) endothelin receptor subtypes. Newly synthesized 3H-inositol phosphates were recovered by column chromatography. Airway smooth muscle from an additional 7 pigs was homogenized and incubated in the presence of 32P-alpha-adenosine triphosphate, guanosine triphosphate (GTP) and either carbachol or endothelin to measure the inhibitory influence of carbachol (positive control) or endothelin on GTP-stimulated adenylyl cyclase activity. Newly synthesized 32P-cyclic adenosine monophosphate was isolated by sequential column chromatography over Dowex and alumina. Total inositol phosphates increased in porcine airway smooth muscle in response to either carbachol or endothelin. The endothelin receptor antagonist BQ-485 (ET(A) selective) but not BQ-788 (ET(B) selective) dose-dependently inhibited endothelin-1 induced inositol phosphate accumulation. In adenylyl cyclase assays, carbachol (positive control), but not endothelin-1, significantly inhibited GTP-stimulated adenylyl cyclase activity. Endothelin-1 couples to the activation of the inositol phosphate pathway via the ET(A) receptor subtype but does not couple to inhibition of the adenylyl cyclase pathway in porcine airway smooth muscle. The potent bronchoconstrictive effects of endothelin likely involve the acute activation of the inositol phosphate pathway in airway smooth muscle.

Inhibition of angiotensin II-induced inositol phosphate production by triacid nonpeptide antagonists in CHO cells expressing human AT1 receptors.

The aim of the present work is to describe the inhibitory properties of LY301875 and LY303336, two polysubstituted 4-aminoimidazole AT1 receptor antagonists, on CHO cells expressing human recombinant AT1 receptors. The binding of [3H]-angiotensin II to intact cells as well as to angiotensin II induced inositol phosphate accumulation is measured. Both antagonists inhibit specific [3H]-angiotensin II binding to AT1 receptors in these cells, with IC50 values of 5.9 and 5.2 nM, respectively. Preincubation of the cells with LY301875 results in a decline of up to 80% of the maximal angiotensin II-stimulated inositol phosphate (IP) production. A near complete decline of the maximal response is observed for LY303336. This insurmountable inhibition is attenuated for both antagonists when losartan is included during the preincubation of the cells. Functional recovery experiments, in which antagonist-preincubated cells are washed and exposed to fresh media, suggest that the insurmountable inhibition by LY301875 and LY303336 is related to their relatively slow dissociation from the AT1 receptors. As already described for losartan and the derived insurmountable AT1 antagonists candesartan. EXP3174, and irbesartan, coincubation experiments reveal that LY301875 and LY303336 interact with the AT1 receptor in a manner that is competitive with angiotensin II.

Forskolin Inhibits 5-Hydroxytryptamine-Induced Phosphoinositide Hydrolysis and Ca 2+ Mobilisation in Canine Cultured Aorta Smooth Muscle Cells

The effect of forskolin on 5-hydroxytryptamine (5-HT)-induced inositol phosphate (IP) and Ca 2+ mobilisation was investigated in canine cultured aorta smooth muscle cells (ASMCs). Pretreatment of ASMCs with forskolin attenuated 5-HT-induced IP accumulation and Ca 2+ mobilisation in a time- and concentration-dependent manner. The half-maximal effects (pEC 50) of forskolin to attenuate IP and Ca 2+ responses to 5-HT occurred at concentrations of 6.28 and 6.64, respectively. Pretreatment of ASMCs with cholera toxin caused a similar inhibition on 5-HT-induced responses. Even after treatment with forskolin for 24 h, the 5-HT-induced responses were still inhibited. The inhibitory effect of forskolin resulted from both a depression of the maximal response and a shift to the right of the concentration-effect curves of 5-HT in these responses. The water-soluble forskolin analogue L-858051 [7-deacetyl-7β-(γ-N-methylpiperazino)-butyryl forskolin] significantly inhibited the 5-HT-stimulated IP accumulation. In contrast, the addition of 1,9-dideoxy forskolin, an inactive forskolin analogue, had little effect on IP response. Moreover, SQ-22536 [9-(tetrahydro-2-furanyl)-9-H-purin-6-amine], an inhibitor of adenylate cyclase, and both H-89 [N-(2-aminoethyl)-5-iosquinolinesulphonamide] and HA-1004 [N-(2-guanidinoethyl)-5-iosquinolinesulphonamide], inhibitors of cAMP-dependent protein kinase (PKA), attenuated the ability of forskolin to inhibit the 5-HT-stimulated accumulation of IP in ASMCs. These results indicate that activation of cAMP/PKA might inhibit the 5-HT-stimulated IP accumulation and consequently reduce Ca 2+ mobilisation, or inhibit both responses independently.

Fluorescent pseudo-peptide linear vasopressin antagonists: design, synthesis, and applications.

Fluoresceinyl and rhodamyl groups have been coupled by an amide link to side-chain amino groups at positions 1, 6, and 8 of pseudo-peptide linear vasopressin antagonists (Manning et al. Int. J. Pept. Protein Res. 1992, 40, 261-267) through different positions on the fluorophore, to give tetraethylrhodamyl-DTyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2 (2), 4-HOPh(CH2)2CO-DTyr(Me)-Phe-Gln-Asn-Lys(5-carboxyfl uoresceinyl)-Pro-A rg-NH2 (4), 4-HOPh(CH2)2CO-DTyr(Me)-Phe-Gln-Asn-Lys(5- or 6-carboxytetramethylrhodamyl)-Pro-Arg-NH2 (5, 6), 4-HOPh(CH2)2CO-DTyr(Me)-Phe-Gln-Asn-Arg-Pro-Lys(5- or 6- carboxyfluoresceinyl)-NH2 (8, 9), and 4-HOPh(CH2)2CO-DTyr(Me)-Phe-Gln-Asn-Arg-Pro-Lys(5- or 6- carboxytetramethylrhodamyl)-NH2 (10, 11). The closer to the C-terminus the fluorophore, the higher the affinities of the fluorescent derivatives for the human vasopressin V1a receptor transfected in CHO cells. The compound 10 has a Ki of 70 pM, as determined by competition experiments with [125I]-4-HOPhCH2CO-DTyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-NH2. It showed a good selectivity for human V1a receptor versus human OT (Ki = 1.2 nM), human vasopressin V1b (Ki approximately 27 nM), and human vasopressin V2 (Ki > 5000 nM) receptor subtypes. All fluorescent analogues were antagonists as shown by the inhibition of vasopressin induced inositol phosphate accumulation. These fluorescent ligands are efficient for labeling cells expressing the human V1a receptor subtype, as shown by flow cytofluorometric experiments or fluorescence microscopy. They are also appropriate tools for structural analysis of the vasopressin receptors by fluorescence.

Distinction between surmountable and insurmountable selective AT1 receptor antagonists by use of CHO-K1 cells expressing human angiotensin II AT1 receptors.

1. CHO-K1 cells that were stably transfected with the gene for the human AT1 receptor (CHO-AT1 cells) were used for pharmacological studies of non-peptide AT1 receptor antagonists. 2. In the presence of 10 mM LiCl, angiotensin II caused a concentration-dependent and long-lasting increase of inositol phosphates accumulation with an EC50 of 3.4 nM. No angiotensin II responses are seen in wild-type CHO-K1 cells. 3. [3H]-Angiotensin II bound to cell surface AT1 receptors (dissociates under mild acidic conditions) and is subject to rapid internalization. 4. Non-peptide selective AT1 antagonists inhibited the angiotensin II (0.1 microM) induced IP accumulation and the binding of [3H]-angiotensin II (1 nM) with the potency order: candesartan > EXP3174 > irbesartan > losartan. Their potencies are lower in the presence of bovine serum albumin. 5. Preincubation with the insurmountable antagonist candesartan decreased the maximal angiotensin II induced inositol phosphate accumulation up to 94% and, concomitantly, decreased the maximal binding capacity of the cell surface receptors. These inhibitory effects were half-maximal for 0.6 nM candesartan and were attenuated by simultaneous preincubation with 1 microM losartan indicating a syntopic action of both antagonists. 6. Losartan caused a parallel rightward shift of the angiotensin II concentration-response curves and did not affect the maximal binding capacity. EXP3174 (the active metabolite of losartan) and irbesartan showed a mixed-type behavior in both functional and binding studies. 7. Reversal of the inhibitory effect was slower for candesartan as compared with EXP3174 and irbesartan and it was almost instantaneous for losartan, suggesting that the insurmountable nature of selective AT1 receptor antagonists in functional studies was related to their long-lasting inhibition.

Two different P2Y receptors linked to steroidogenesis in bovine adrenocortical cells.

Both extracellular adenosine 5'-triphosphate (ATP) and uridine 5'-triphosphate (UTP) induced corticoid production (steroidogenesis) concentration-dependently in bovine adrenocortical cells (BA cells). Pertussis toxin (PTX, approx. 2 microg/ml) partially inhibited (approx. 55% inhibition) extracellular ATP (100 microM)-induced steroidogenesis in BA cells. However, PTX did not inhibit extracellular UTP (100 microM)-induced steroidogenesis. Both ATP- and UTP-induced steroidogeneses were significantly inhibited by suramin (50-200 microM). These effects were inhibited significantly by reactive blue-2 (more than 100 microM) and pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (more than 100 microM). Both nucleotides (1-100 microM) induced inositol phosphates accumulation and intracellular Ca2+ mobilization, but PTX did not inhibit them. The RT-PCR procedure identified only P2Y2-receptor mRNA in BA cells. These results suggest that extracellular ATP induces steroidogenesis via a unique P2 receptor linked to PTX-sensitive guanine nucleotide-binding protein (G-protein), while extracellular UTP induces steroidogenesis via P2 receptor linked to PTX-insensitive G-protein. Thus, it was concluded that at least two different P2Y-like receptors linking to steroidogenesis exist in BA cells.

Two Different P2Y Receptors Linked to Steroidogenesis in Bovine Adrenocortical Cells

Both extracellular adenosine 5′-triphosphate (ATP) and uridine 5′-triphosphate (UTP) induced corticoid production (steroidogenesis) concentration-dependently in bovine adrenocortical cells (BA cells). Pertussis toxin (PTX, approx. 2 μg/ml) partially inhibited (approx. 55% inhibition) extracellular ATP (100 μM)-induced steroidogenesis in BA cells. However, PTX did not inhibit extracellular UTP (100 μM)-induced steroidogenesis. Both ATP- and UTP-induced steroidogeneses were significantly inhibited by suramin (50-200 μΜ). These effects were inhibited significantly by reactive blue-2 (more than 100 μΜ) and pyridoxal-phosphate-6-azophenyl-2’,4’-disulphonic acid (more than 100 μΜ). Both nucleotides (1-100 μΜ) induced inositol phosphates accumulation and intracellular Ca2+ mobilization, but PTX did not inhibit them. The RT-PCR procedure identified only P2Y2-receptor mRNA in BA cells. These results suggest that extracellular ATP induces steroidogenesis via a unique P2 receptor linked to PTX-sensitive guanine nucleotide-binding protein (G-protein), while extracellular UTP induces steroidogenesis via P2 receptor linked to PTX-insensitive G-protein. Thus, it was concluded that at least two different P2Y-like receptors linking to steroidogenesis exist in BA cells.

Effect of forskolin on endothelin-induced phosphoinositide hydrolysis and calcium mobilization in cultured canine tracheal smooth muscle cells.

1. The effects of increase in intracellular adenosine 3':5'-cyclic monophosphate (cAMP) on endothelin-1 (ET-1)-induced generation of inositol phosphates (IPs) and increase in intracellular Ca2+ ([Ca2+]i) were investigated in canine cultured tracheal smooth muscle cells (TSMCs). 2. Pretreatment of TSMCs with either cholera toxin (CTX; 10 microg ml(-1), 4 h), forskolin (10 microM, 30 min), or dibutyryl cAMP (1 mM, 30 min) inhibited ET-1-stimulated Ca2+ mobilization (by 23 +/- 5%, n = 8) and IPs accumulation (by 32 +/- 6%, n = 4). While after treatment with forskolin for 24 h, the cells retained the ability to respond to ET-1-induced Ca2+ mobilization to the same extent as the control group. 3. Forskolin (1-100 microM) inhibited the ET-1-induced increase in [Ca2+]i, but the lower concentrations had little effect on this response. The inhibitory effects of these agents produced both depression of the maximal response and a shift to the right of the concentration-response curve of ET-1 without changing the -logEC50 values. 4. The water-soluble forskolin analogue L-858051, 7-deacetyl-7beta-(gamma-N-methylpiperazino)-butyryl forskolin, significantly inhibited ET-1-stimulated IPs accumulation. In contrast, the addition of 1,9-dideoxy forskolin, an inactive analogue of forskolin, had little effect on stimulated responses. Moreover, SQ-22536, 9-(tetrahydro-2-furanyl)-9H-purin-6-amine, an inhibitor of adenylate cyclase, and both H-89, N-(2-aminoethyl)-5-isoquinolinesulfonamide, and HA-1004, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide, inhibitors of cAMP-dependent protein kinase (PKA), attenuated the ability of forskolin to inhibit ET-1-induced IPs accumulation. These results suggest that activation of cAMP/PKA was involved in these inhibitory effects of forskolin. 5. The locus of this inhibition of forskolin treatment on AlF4(-)-stimulated IPs accumulation was investigated in canine TSMCs. The AlF4(-)-induced IPs accumulation was inhibited by forskolin, supporting that G protein(s) are directly activated by AlF4- and uncoupled to phospholipase C by forskolin treatment. 6. We conclude that cAMP elevating agents inhibit ET-1-stimulated generation of IPs and Ca2+ mobilization in canine cultured TSMCs. Since generation of IPs and increases in [Ca2+]i are very early events in the activation of ET-1 receptors, attenuation of these events by cAMP elevating agents might well contribute to the inhibitory effect of cAMP on tracheal smooth muscle function.

Association of the G protein alpha(q)/alpha11-subunit with cytoskeleton in adrenal glomerulosa cells: role in receptor-effector coupling.

In 3-day primary cultures of rat glomerulosa cells, a 30-min pre-incubation with either 10 microM colchicine (a microtubule-disrupting agent) or 10 microM cytochalasin B (a microfilament-disrupting agent) decreased angiotensin II (Ang II)-induced inositol phosphate accumulation by 50%. Moreover, both drugs decreased inositol phosphate production induced by fluoroaluminate (a nonspecific activator of all G proteins), indicating that both microtubules and microfilaments are essential for phospholipase C activation. Analysis of microfilament- and microtubule-enriched fractions and immunoprecipitation of actin and tubulin revealed that the alpha(q)/alpha11-subunit of the G(q/11) protein was associated with both structures. Ang II stimulation induced a rapid translocation of alpha(q)/alpha11, microfilaments, and microtubules to the membrane and induced a time-dependent increase in the level of alpha(q)/alpha11 associated with both microfilaments and microtubules. Moreover, double immunofluorescence staining clearly showed a colocalization of the alpha(q)/alpha11-subunit of the G(q/11) coupling protein and microfilament distribution. These associations and plasma membrane redistribution under Ang II stimulation indicate that microfilaments and microtubules are both involved in phospholipase C activation and inositol phosphate production. Moreover, our results indicate that the alpha(q)/alpha11 protein is closely associated with cytoskeletal elements and is found both at the plasma membrane level as well as on intracellular stress fibers.

The peripheral NK-1/NK-2 receptor antagonist MDL 105,172A inhibits tachykinin-mediated respiratory effects in guinea-pigs.

1. Stimulation of sensory nerves causes release of tachykinins, including substance P (SP) and neurokinin A (NKA), which produce a variety of respiratory effects via NK-1 and NK-2 receptors, respectively. Hence, development of a compound which could potently and equivalently antagonize both receptors was pursued. 2. MDL 105,172A ((R)-1-[3-(3,4-dicholorophenyl)-1-(3,4,5-trimethoxybenzoyl)- 3-pyrrolidinyl]-4- phenyl-piperidine-4-morpholinecarboxamide) exhibited high affinity for NK-1 (4.34 nM) and NK-2 (2.05 nM) receptors. In vitro, the compound antagonized SP (pA2 = 8.36) or NKA (pA2 = 8.61)-induced inositol phosphate accumulation in tachykinin monoreceptor cell lines. 3. In anaesthetized guinea-pigs, MDL 105,172A inhibited SP-induced plasma protein extravasation (ED50 = 1 mg kg-1, i.v.) and [beta-Ala8]NKA 4-10-induced bronchoconstriction (ED50 = 0.5 mg kg-1, i.v.) indicating NK-1 and NK-2 antagonism, respectively. 4. Capsaicin was used to elicit respiratory effects in anaesthetized and conscious guinea-pigs; the latter were inhibited by MDL 105,172A following i.v. (ED50 = 1 mg kg-1) or oral (ED50 = 20 mg kg-1) administration. Hence, MDL 105,172A can inhibit pulmonary responses to tachykinins released endogenously in the airways. 5. At doses up to 200 mg kg-1, p.o., MDL 105,172A failed to inhibit repetitive hind paw tapping induced by i.c.v GR 73632, and NK-1 selective agonist, in gerbils, whereas CP-99,994 (0.87 mg kg-1, s.c.) completely ablated the effect. These data suggest that MDL 105,172A does not penetrate the central nervous system (CNS) and its tachykinin antagonism is restricted to the periphery. 6. MDL 105,172A is a non-peptide, potent, equivalent antagonist of NK-1 and NK-2 receptors. Its ability to inhibit both exogenously administered as well as endogenously released tachykinins support its use in examining the role of sensory neuropeptides in diseases associated with neurogenic inflammation including asthma.

Influences of hypothyroidism on lipid composition and inositol lipid-linked receptors responsiveness and protein kinase C (PKC) activity in the cerebral cortex of Lewis rats.

The influence of hypothyroidism (HO) induced by treatment with propylthiouracil on lipid composition, receptor responsiveness of M1-muscarinic receptors (M1AChRs) and metabotropic glutamate receptors (mGluRs) as well as on protein kinase C (PKC) activity was investigated in the cerebral cortex of Lewis rats. HO did not influence the lipid composition. There was a significant 2-fold increase of efficacy and 6-fold decrease of potency of carbachol-induced inositol phosphate (IP) accumulation in HO, with respect to control rats. The efficacy of trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid (ACPD)-induced IP accumulation was also higher in HO (by 50%), without differences in EC50 values. The activities of soluble calcium-dependent and calcium-independent PKC were higher in HO than in control rats (both roughly 30%); membrane-associated PKCs were not modified. The data indicate that HO induces an increased responsiveness of M1AChRs and mGluRs and a rise in the soluble PKC activity that could be available and ready for translocation.

The effect of cyclic AMP elevating agents on bradykinin‐ and carbachol‐induced signal transduction in canine cultured tracheal smooth muscle cells

1. The effects of cholera toxin (CTX), forskolin and dibutyryl cyclic AMP on bradykinin (BK)- and carbachol-induced accumulation of inositol phosphates (IPs) and Ca2+ mobilization were investigated in canine cultured tracheal smooth muscle cells (TSMCs). The BK-induced responses were mediated via a G protein which was not inhibited by CTX or pertussis toxin treatment. 2. BK-stimulated IPs accumulation and Ca2+ mobilization were potentiated by CTX (10 micrograms ml-1) pretreatment which was time-dependent. Maximal increase of these responses occurred after 24 h treatment with CTX. The concentration-effect relationship of BK-induced responses were shifted to the left and BK was substantially more effective in CTX-treated cells than in the control cells. This enhancing effect of CTX did not occur with carbachol. 3. Short-term (< 4 h) treatment with forskolin (10 microM) or dibutyryl cyclic AMP (1 mM) failed to accentuate BK-induced responses, but long-term (> 4 h) treatment of TSMCs with these agents mimicked the enhancing effect of CTX, suggesting that CTX-induced enhancement of BK responsiveness might be due to a rise in cyclic AMP. 4. Prolonged treatment of TSMCs with these agents was accompanied by an increase in cell surface [3H]-BK binding sites, which was inhibited by concurrent incubation with cycloheximide, an inhibitor of protein biosynthesis. Cycloheximide also abolished the potentiating actions of CTX, forskolin, and dibutyryl cyclic AMP on BK-induced IPs formation and Ca2+ mobilization. 5. The locus of this enhancement was further investigated by examining the effects of CTX, forskolin and dibutyryl cyclic AMP on A1F4(-)-induced IPs accumulation in canine TSMCs. AIF4-induced IPs accumulation was not affected by CTX, forskolin, or dibutyryl cyclic AMP treatment, supporting the contention that this stimulatory effect is located at the BK receptor level.6. These results demonstrate that the augmentation of BK-induced IPs accumulation and Ca2+mobilization produced by CTX, forskolin and dibutyryl cyclic AMP involves a cyclic AMP-dependent mechanism which is induced by a sustained increase in the level of intracellular cyclic AMP. CTX and forskolin may promote an increase of the synthesis of BK receptors, and thereby enhance BK-induced responses.

5‐Hydroxytryptamine receptor‐mediated phosphoinositide hydrolysis in canine cultured tracheal smooth muscle cells

1. 5-Hydroxytryptamine (5-HT) has been shown to induce contraction of tracheal smooth muscle. However, the mechanisms of action of 5-HT are not known. We therefore investigated the effects of 5-HT on phospholipase C (PLC)-mediated phosphoinositide (PI) hydrolysis and its regulation in canine cultured tracheal smooth muscle cells (TSMCs) labelled with [3H]-inositol. 5-HT-induced inositol phosphates (IPs) accumulation was time- and dose-dependent with a half-maximal response (EC50) and a maximal response at 0.38 +/- 0.05 and 10 microM, respectively. 2. Ketanserin and mianserin (10 and 100 nM), 5-HT2 receptor antagonists, were equipotent in blocking the 5-HT-induced IPs accumulation with pKB values of 8.46 and 8.21, respectively. In contrast, the dose-response curves of 5-HT-induced IPs accumulation were not shifted until the concentrations of NAN-190 and metoclopramide (5-HT1A and 5-HT3 receptor antagonists, respectively) were increased up to 10 microM. 3. Pretreatment of TSMCs with pertussis toxin or cholera toxin did not inhibit the 5-HT-induced IPs accumulation, but partially inhibited the AlF(4-)-induced IPs response. 4. Stimulation of IPs accumulation by 5-HT required the presence of external Ca2+ and was blocked by EGTA. The addition of Ca2+ (3-620 nM) to digitonin-permeabilized TSMCs directly stimulated IPs accumulation. A further Ca(2+)-dependent increase in IPs accumulation was obtained by inclusion of either guanosine 5'-O-(3-thiotriphoshate) (GTP gamma S) or 5-HT. The combination of GTP gamma S and 5-HT elicited an additive effect on IPs accumulation. 5. Treatment with phorbol 12-myristate 13-acetate (PMA, 1 microM, 30 min) abolished the 5-HT-induced IPs accumulation. The concentrations of PMA that gave a half-maximal and maximal inhibition of 5-HT-induced IPs accumulation were 2.2 +/- 0.4 nM and 1 microM, n = 3, respectively. The protein kinase C (PKC) activator, 4 alpha-phorbol 12,13-didecanoate, at 1 microM, did not influence this response. The inhibitory effect of PMA was reversed by staurosporine, a PKC inhibitor, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. 6. The site of this inhibition was further investigated by examining the effect of PMA on AlF(4-)-induced IPs accumulation in canine TSMCs. AlF(4-)-stimulated IPs accumulation was inhibited by PMA treatment, suggesting that the effect of PMA is distal to the 5-HT receptor. 7. Acetylcholine-induced IPs accumulation was completely inhibited by atropine, but not affected by ketanserin or mianserin, suggesting that 5-HT-induced IPs accumulation is not due to release of acetylcholine.8. These results demonstrate that 5-HT directly stimulates PLC-mediated PI hydrolysis via a pertussis toxin- and cholera toxin-insensitive GTP binding protein in canine TSMCs and that this coupling process is negatively regulated by PKC. 5-HT2 receptors may be predominantly mediating IPs accumulation and presumably IP-induced Ca2+ release may function as the transducing mechanism for 5-HT stimulated contraction of tracheal smooth muscle.