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PubMed1995

Modulation of ADP-stimulated inositol phosphate metabolism in rat alveolar macrophages by oxidative stress.

The present study examined alterations of adenosine-5-diphosphate (ADP)-stimulated inositol phosphate (IP3) metabolism and the respiratory burst of rat alveolar macrophages (AM) under oxidant stress using the model oxidant, tert-butyl hydroperoxide (tBOOH). Isolated AM were maintained in a system with an air-liquid interface, which approximates the lung environment. tBOOH produced a dual effect on the ADP-stimulated respiratory burst of these AM. Pretreatment of these AM with 50 microM tBOOH for 15 min led to a significant enhancement of the respiratory burst while significant inhibition was observed with 200 microM tBOOH. Treatment of AM with 100 microM ADP led to a significant increase in the generation of IP3, reaching a maximum at 30 s. In contrast, treatment of AM with 50 or 200 microM tBOOH did not stimulate IP3 generation during a 15-min period. Pretreatment of AM with 50 microM tBOOH for 15 min had no effect on ADP-stimulated IP3 generation. Preincubation with 200 microM tBOOH significantly inhibited generation of IP3 in response to ADP stimulation; however, this probably did not contribute to inhibition of the respiratory burst. The results suggest that production of IP3 may participate in stimulation of the respiratory burst by ADP but that enhancement of the respiratory burst by 50 microM tBOOH probably did not involve alteration of IP3 production.

Adenosine Diphosphate↗
PubMed1993

Thyrotropin, like luteinizing hormone (LH) and chorionic gonadotropin (CG), increases cAMP and inositol phosphate levels in cells with recombinant human LH/CG receptor.

Glycoprotein hormones and their receptors are each structurally related; thus, ligand-receptor cross reactivity may exist in pathologic situations, i.e., high human chorionic gonadotropin (hCG) levels in patients has been suggested to activate the thyrotropin receptor (TSHR). Studies with Cos-7 cells transfected with human CG and TSH receptor cDNAs suggest the converse may be more likely. Thus, in cells with TSHR, about 3 x 10(-11) and 3 x 10(-10) M TSH cause half maximal increases in cAMP and inositol phosphate (IP) levels, respectively, whereas 10(-6) M hCG has no effect on either. In cells with CGR, about 10(-11) and 10(-9) M CG or lutropin (LH) significantly increase cAMP and IP levels. Surprisingly, however, 10(-11) and 10(-9) M TSH are similarly effective in the two assays, respectively, and TSH increases cAMP and IP levels to the same extent as CG and LH. LH contamination of TSH is unlikely given similar results with highly purified TSH preparations from different sources, including recombinant TSH, and the specificity of simultaneously measured binding data. Thus, TSH binds with high affinity (Kd = 7 x 10(-11) M) to the human TSHR; hCG (up to 10(-7) M) does not displace TSH binding. Similarly, hCG binds with high affinity (Kd = 5 x 10(-10) M) to the hCGR and TSH is only a weak inhibitor (Ki = 1 x 10(-8) M). Stimulating TSHR autoantibodies, with no epitopes on the CGR, do not duplicate TSH action. The unusual agonist action of TSH with recombinant CGR is consistent with TSHR models describing separate agonist and antagonist determinants; it may be a factor in the precocious puberty of juvenile hypothyroidism with high TSH levels

Animals↗
PubMed1989

Pretreatment of cloned helper T lymphocytes with IL-2 induces unresponsiveness to antigen and concanavalin A, associated with decreased inositol phosphate and diacylglycerol production.

IL-2 pretreatment of cloned Th lymphocytes has been demonstrated to render these cells unresponsive to subsequent stimulation through the TCR. These cells remain unresponsive for up to 7 days after removal from IL-2. Cells rendered unresponsive to Ag by pretreatment with IL-2 also demonstrated reduced increases in intracellular calcium ([Ca2+]i) after stimulation, hence this unresponsiveness is believed to result from absence of sufficient [Ca2+]i for activation of lymphokine genes. We have confirmed these observations, and demonstrate that only that portion of the [Ca2+]i increase derived from extracellular sources is inhibited in IL-2 pretreated cells. Further, inositol degradation and diacylglycerol production after stimulation are observed to be markedly reduced in cells rendered unresponsive by IL-2 pretreatment, suggesting that signal transduction leading to cleavage of phosphatidylinositol 4,5-bisphosphate after Ag receptor engagement is incomplete in these cells. However, treatment of IL-2 pretreated cells with AlF4- results in both production of inositol phosphates as well as increased intracellular calcium, suggesting that phospholipase C remains active in these cells. It appears that chronic IL-2 exposure regulates Th activation by inhibiting the signal transduction which follows engagement of the TCR.

Aluminum↗
PubMed1993

Cholera toxin and dibutyryl cyclic adenosine 3',5'-monophosphate sensitize gonadotropin-releasing hormone-stimulated inositol phosphate production to inhibition in protein kinase-C (PKC)-depleted cells: evidence for cross-talk between a cholera toxin-sensitive G-protein and PKC.

The present study assesses the relationship between G-proteins and protein kinase-C (PKC) in the gonadotrope. Cells were depleted of PKC with 1 microM phorbol 12-myristate 13-acetate for 12 h, followed by medium 199-BSA for 6 h before treatment with vehicle, pertussis toxin (PTX), cholera toxin (CTX), or (Bu)2cAMP (dBcAMP) for 18 h. PTX (10 ng/ml) significantly decreased GnRH-stimulated inositol phosphate (IP) production over a range of 10(-8)-10(-6) M GnRH. The degree of this inhibition was the same in control cells and PKC-depleted cells. Pretreatment with CTX (0.5 microgram/ml) significantly decreased GnRH-stimulated IP production over a range of 10(-9)-10(-6) M GnRH in PKC-depleted cells. This effect was mimicked by pretreatment with 3 mM dBcAMP. Although CTX and dBcAMP both decreased GnRH-stimulated IP production in control cells, this effect was enhanced in PKC-depleted cells. CTX (0.1 microgram/ml) and dBcAMP (3 mM) both enhanced GnRH-stimulated LH release, whereas PTX (100 ng/ml) had no effect. This was observed in control as well as PKC-depleted cells. Both PKA and PKC are capable of regulating IP turnover by phosphorylating phospholipase-C at distinct sites. CTX activates a G-protein that increases cAMP. cAMP can then activate PKA. In PKC-depleted cells, CTX inhibits GnRH-stimulated IP production. This effect is mimicked by dBcAMP, which suggests a role for PKA in the gonadotrope. The results of this study provide evidence for cross-talk between a CTX-sensitive G-protein and PKC.

Animals↗
PubMed1991

Alpha-adrenoceptor subtypes in dog saphenous vein that mediate contraction and inositol phosphate production.

1. Studies have been made of the contractile responses to the alpha-adrenoceptor agonists phenylephrine (Phen), cirazoline (Cir) or BHT-920 (BHT) in dog isolated saphenous vein (DSV) rings, using the antagonists yohimbine (Yoh), idazoxan (Idaz), prazosin (Praz), WB-4101 (WB) and nitrendipine or zero Ca2+ medium. 2. Contractile concentration-response curves to Phen or BHT were displaced to the right of controls by Yoh (0.01-3 microM) with mean apparent antagonist dissociation constants (pKBs) of 7.9 and 8.6 respectively. Yoh did not show simple competitive antagonism against either agonist, since the Schild plot slopes were significantly less than unity. Neither the antagonist affinity of Yoh against Phen, nor the slope of the Schild plot was modified in the presence of catecholamine uptake inhibitors, nor in the presence of alpha,beta-methylene ATP, which desensitizes P2-purinoceptors, suggesting that Phen does not release ATP, or noradrenaline to cause contraction in DSV. In the presence of Praz (0.3 microM) the antagonist potency of Yoh (mean pKB 7.4) against Phen was slightly decreased. Yoh had low potency against responses induced by Cir (pKB 6.3). 3. WB (0.001-1.0 microM) was a very potent antagonist of Phen-induced contractions, however, the biphasic Schild plot against Phen could be separated into two affinity sites, a high pKB of 9.3 (equivalent to that obtained using Cir as the agonist; pKB 9.6) and a lower affinity (pKB 8.6). WB showed an even lower antagonist affinity (pKB 7.4) against BHT-induced contractions, suggesting that these effects might be mediated by alpha 2A-adrenoceptors. Praz also appeared to identify two sites using Phen-induced contractions, a high pKB of 8.4 was equivalent to that obtained with Cir (pKB 8.2) and a lower affinity site (pKB 7.7; pA2 7.6; slope 1.1) at which Praz showed competitive antagonism. Higher concentrations of Praz were required to antagonize contractions to BHT (pKB 5.9). 4. Idaz was a weak partial agonist in this tissue with threshold contractile effects at concentrations in excess of 3 microM. Idaz (0.1-1 microM) competitively antagonized the contractile effects of BHT, but showed low antagonist affinity against Phen at these concentrations. 5. Contractions to Phen were slightly antagonized by nitrendipine (1 microM), with a 36% decrease in Emax. Contractions to Phen and Cir were also markedly attenuated in zero calcium medium (with EGTA), but maximum responses of 4.2 +/- 0.1 and 3.6 +/- 0.1 g, could be obtained with these agonists respectively. Only part of the contractile effects to Phen or Cir are therefore due to calcium influx (but L-type channels are not totally implicated), while the contractile effects of BHT were abolished in zero Ca2 + medium. Yoh (0.1 microm) retained its antagonist effects on Phen-induced responses in zero Ca2 + medium. 6. The formation of inositol phosphates (InsPs) in the presence of lithium (10mM) was measured after incubation of intact DSV strips with myo-2-[3H]-inositol. Phen (1-1OO0 microM) and Cir (O.O1-1O microm) induced concentration-dependent increases in total labelled InsP1_3, but BHT showed minimal InsP stimulation. InsPs were recovered after Phen (100,M) stimulation (10min) as labelled InsP1 (71%), InsP2 (25%) and InsP3 (4%). Phen (100 microM)-stimulated InsP1-3 formation was significantly antagonized by Praz (10nM), but was not fully inhibited even after Praz 1 microM. Yoh and Praz (0.1 and 1.0 microM) were equipotent inhibitors of this response, while Idaz (0.3 microM) showed no effects. 7. The receptors in DSV which are stimulated by Phen to cause contraction show characteristics of the alpha lA-adrenoceptor (high pM antagonist affinity for WB-4101 and extracellular calcium sensitivity) and the alpha lB-adrenoceptor (contraction in calcium-free medium, increase in InsP and low nm antagonist affinity of WB). The paradoxical results obtained with Yoh (potent antagonist effects on Phen-stimulated PI and pKB 7.9 on contraction) and Praz (low affinity competitive antagonist of Phen-induced contraction, pKB 7.7 and failure to inhibit completely the PI response at 1 microM), cannot fully exclude an alpha 2B-subtype characterization of these responses. These pharmacological differences suggest that the adrenoceptor involved in the contractile and in particular the second messenger effects of Phen in DSV is not typically an alpha lB-adrenoceptor.

Adrenergic alpha-Agonists↗
PubMed1987

Stimulation of cytosolic free calcium and inositol phosphates by prostaglandins in cultured rat mesangial cells.

We studied the effects of four products of arachidonate cyclo-oxygenation on a phospholipase C-dependent signal transduction system in cultured rat glomerular mesangial cells. PGF2 alpha, PGE2 and the thromboxane A2/endoperoxide analogue U-46619 rapidly increased cytosolic free Ca2+, measured in monolayers loaded with the fluorescent intracellular probe fura-2. Peak responses were dose-dependent and unaffected by chelation of extracellular Ca2+, indicating release from internal stores. The thromboxane A2-receptor antagonist SQ 27,427 selectively inhibited responses to U-46619. The PGI2 analogue Iloprost had no effect on cytosolic Ca2+. PGF2 alpha, PGE2 and U-46619 also stimulated accumulation of total inositol phosphates during 15 min incubations. We conclude that phospholipase C activation mediates the effects of certain eicosanoids on the glomerular mesangium.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed2005

Can intervention in inositol phosphate signalling pathways improve therapy for cystic fibrosis?

Airway epithelial cells from cystic fibrosis (CF) individuals cannot secrete adequate Cl- through cystic fibrosis transmembrane regulator, and their Na+ channel (ENaC) activity is increased so that excessive Na+ and water is absorbed from the lumen. These aberrant transport activities can, at least partly, be compensated by pharmacologically increasing the activities of Ca2+-activated Cl- channels (CaCCs). The therapeutic value of this approach is currently being examined in clinical trials of candidate CF drugs such as INS-37217 (Inspire Pharmaceuticals) and Moli1901 (Lantibio, Inc.). This review argues that these drug development programmes will be helped if one can fully understand how the CaCCs are inhibited by inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P4), so that there can be pharmacological intervention in this process. Furthermore, genes that encode enzymes controlling Ins(3,4,5,6)P4 metabolism should be viewed as impacting upon CaCC activity; this, in turn, may influence the severity of the CF condition. Expression profiling of genes that regulate inositol phosphate metabolism may also illuminate variability in patient response to treatment regimens that target CaCCs. Compounds have been developed that can activate CaCCs by antagonising their inhibition by Ins(3,4,5,6)P4. One member of this drug family (INO-4995; Inologic) was recently shown to inhibit ENaC, thereby reducing fluid absorbtion by airway epithelial cells.

Animals↗
PubMed1992

Effect of nucleotides on the cytosolic free calcium activity and inositol phosphate formation in human glomerular epithelial cells.

1. Glomerular epithelial cells (GEC) were cultured from human kidneys and immunologically characterized. 2. The effect of extracellular nucleotides on the cytosolic free calcium activity [Ca2+]i was investigated with the fura-2 microfluorescence method. Extracellular UTP, UDP, UMP, ATP, adenosine 5'-O-(3-thio)-trisphosphate (ATP-gamma-S), inosine-triphosphate (ITP), guanyltriphosphate (GTP), 2-methylthio-ATP, AMP, alpha,beta-methylene-ATP and adenosine led to a rapid, transient, concentration-dependent increase of [Ca2+]i, followed by a plateau above the baseline level. 3. In a calcium-free extracellular solution, the rapid increase of [Ca2+]i was still present, whereas the plateau level was abolished. 4. ATP and UTP (ED50 both: 10(-5) M) stimulated inositol trisphosphate (InsP3) formation in GEC. 5. The order of potency for the purine nucleotides in stimulating InsP3 formation was ATP = ATP-gamma-S greater than ADP greater than 2-methylthio-ATP greater than AMP = a,beta methylene-ATP = adenosine. 6. The increase of InsP3 induced by ATP (10(-5) M) could be inhibited by the P2 receptor blocker suramin (greater than 10(-4) M). Reactive blue 2 exhibited a weak stimulating effect on the InsP3 formation and only a weak inhibitory effect at a concentration of 10(-3) M was observed. 7. Protein kinase C activation by preincubation of GEC with phorbol 12-myristate 13-acetate (PMA, 100 ng ml-1, 15 min) abolished the effect of ATP (10(-5) M) on InsP3 formation. Downregulation of protein kinase C by long term incubation (18 h) with PMA had no significant effect on the phosphoinositol turnover induced by ATP.8. The results indicate that an increase of [Ca2+]i and inositol phosphate breakdown can be mediated via activation of a P2 receptor in human GEC.

Adenosine Triphosphate↗
PubMed1999

Potentiation of glutamatergic agonist-induced inositol phosphate formation by basic fibroblast growth factor is related to developmental features in hippocampal cultures: neuronal survival and glial cell proliferation.

We investigated the modulation by growth factors of phospholipase C (PLC)-linked glutamate receptors during in vitro development of hippocampal cultures. In defined medium, glial cells represent between 3 and 14% of total cell number. When we added basic fibroblast growth factor (bFGF) 2 h after plating, we found: (i) a neuroprotection from naturally occurring death for up to 5 days; (ii) a proliferation of glial cells from day 3; and (iii) a potentiation of quisqualate (QA)-induced inositol phosphate (IP) formation from 1 to 10 days in vitro (DIV) and 1S, 3R-amino-cyclopentane-1,3-dicarboxylate (ACPD) response from 3 to 10 DIV. The antimitotic cytosine-beta,D-arabinofuranoside (AraC) blocked glial cell proliferation induced by bFGF, but not neuroprotection. Under these conditions, the early potentiation of the QA response (1-3 DIV) was not changed, while the ACPD and late QA response potentiations were prevented (5-10 DIV). Epidermal growth factor was not neuroprotective but it induced both glial cell proliferation and late QA or ACPD potentiation. Surprisingly, the early bFGF-potentiated QA-induced IP response was blocked by 6, 7-dinitro-quinoxaline-2,3-dione (DNQX), suggesting the participation of ionotropic (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate (KA) receptors. The delayed bFGF-potentiated ACPD-induced IP response is inhibited by (S)-alpha-methyl-4-carboxyphenylglycine (MCPG), indicating possible activation of glial metabotropic receptors. These results suggest that, in hippocampal cultures, bFGF modulates AMPA and metabotropic glutamate receptors linked to the IP cascade, possibly in relation to the regulation of neuronal survival and glial cell proliferation, respectively.

Animals↗
PubMed1990

Agents that raise cAMP in human T lymphocytes release an intracellular pool of calcium in the absence of inositol phosphate production.

Prostaglandins (PGs) of the E series are recognized by specific receptors on T lymphocytes which lead to an increase in cAMP. The role of cAMP in modulation of T lymphocyte function is unknown. Here, we demonstrate that agents which increase cAMP in human T cells raise the intracellular free calcium concentration ([Ca2+]i). This increase in [Ca2+]i occurred following receptor stimulation with PGEs or by bypassing the receptor with the cell-permeant analog 8-(4-chlorophenylthio)-cAMP or forskolin, a direct activator of adenylyl cyclase. The calcium response to a submaximally stimulatory concentration of PGE2 was potentiated by the cAMP phosphodiesterase inhibitor isobutylmethylxanthine. A time course of cAMP production in response to PGE2 stimulation closely resembled the calcium response and suggested that the two events were coincident. The PGE2 concentrations required to achieve 50% maximum effect of cAMP production and increases in [Ca2+]i were similar, 0.07 and 0.15 microM respectively. Chelation of extracellular Ca2+ did not abolish the PGE2-stimulated Ca2+ response, suggesting that an intracellular source of calcium was sensitive to cAMP. Significant inositol phosphate production was not detected in response to PGE2 over a wide concentration range. The PGE2-induced calcium response curves were of lesser magnitude with shorter times to peak than those of a known inositol 1,4,5 trisphosphate-producing agonist, anti-CD3, suggesting distinct Ca2+ release mechanisms. However, the cAMP-releasable store appeared to be contained within the inositol trisphosphate-releasable store since no response could be seen with cAMP-elevating agents following emptying of the inositol trisphosphate-sensitive pool of Ca2+.

Antigens, CD↗
PubMed1990

Inositol phosphate release and steroidogenesis in rat adrenal glomerulosa cells. Comparison of the effects of endothelin, angiotensin II and vasopressin.

Endothelin has steroidogenic activity in adrenal glomerulosa cells, as do two other vasoconstrictor peptides, angiotensin II and vasopressin. The steroidogenic activities of angiotensin II and vasopressin are probably mediated via the phosphatidylinositol-turnover pathway and associated changes in cytosolic Ca2+ concentration. Endothelin caused a steroidogenic response, which was small compared with that to angiotensin II and quantitatively similar to the vasopressin response. Cytosolic free Ca2+ responses were similarly higher to angiotensin II than to either of the other two peptides. However, total inositol phosphate responses to endothelin and angiotensin II were similar when these were measured over 20 min, and were quantitatively greater than the vasopressin response. A detailed study has been made of the phosphatidylinositol-turnover response to endothelin in comparison with responses to angiotensin II and vasopressin. Each of the three peptides produced a rapid and transient rise in Ins(1,4,5)P3 (max. 5-15 s), followed by a slow sustained rise. Ins(1,4,5)P3 was metabolized by both dephosphorylation and phosphorylation pathways, but the relative importance of the two metabolic pathways was different under stimulation by each of the three peptides. These findings show that adrenal glomerulosa cells can distinguish between the stimulation of phosphatidylinositol turnover by three different effectors. These differences in the pathway may be associated with the observed different steroidogenic and Ca2+ responses to the three peptides.

Adrenal Glands↗
PubMed1996

GnRH-induced calcium mobilisation and inositol phosphate production in immature and mature rat ovarian granulosa cells.

In rat ovarian granulosa cells the effects of GnRH are determined by the state of granulosa cell development with mainly inhibitory actions in immature cells and stimulatory actions in differentiated mature cells. These developmentally related effects of GnRH may arise from changes in either one or more of the signal transduction pathways activated by GnRH. The present study therefore measured downstream signalling events associated with the activation of the phospholipase C (PLC) signal transduction pathway in both mature and immature rat ovarian granulosa cells. Results showed that GnRH produced similar total inositol phosphate and intracellular calcium ([Ca2+]i) responses in both immature and mature granulosa cells. In contrast to the biphasic GnRH-induced [Ca2+]i response in pituitary gonadotropes, stimulation of the endogenously expressed GnRH receptor in both immature and mature granulosa cells produced a prompt monophasic rise in [Ca2+]i. This calcium transient was abolished by pretreating either cell type with a potent GnRH receptor antagonist or the PLC inhibitor U73122, demonstrating a GnRH receptor-specific activation of PLC. Similarly, pretreatment of cells with the [Ca2+]i antagonists thapsigargin or cyclopiazonic acid abolished the GnRH-induced calcium transient, whereas EGTA and nifedipine, a voltage-operated calcium channel (VOCC) antagonist, had no effect. These results suggest that in either immature or mature granulosa cells GnRH mobilises calcium from thapsigargin/cyclopiazonic acid-sensitive [Ca2+]i stores but does not involve the influx of extracellular calcium through VOCCs. We conclude that GnRH-induced stimulation of the PLC signal transduction pathway is independent of the stage of granulosa cell maturity and that alternative mechanisms account for the opposite effects of GnRH on gonadotrophin-induced steroidogenesis in mature and immature rat granulosa cells in vitro.

Animals↗
PubMed1996

The luteinizing hormone/chorionic gonadotropin receptor has distinct transmembrane conductors for cAMP and inositol phosphate signals.

The luteinizing hormone/chorionic gonadotropin receptor is a member of the seven-transmembrane receptor family. It is coupled, presumably via Gs and Gq, to two signal pathways involving adenylyl cyclase/cAMP and phospholipase C/inositol phosphate (IP). Little is known about the events prior to G-protein coupling: for example, whether these signals are generated from a single or multiple independent origins and mechanisms, when and where they diverge, and how they are transduced. We report novel observations that the cAMP signal and the IP signal originate and diverge upstream of G-protein coupling. The generation of these two signals independently involves Lys583 in exoloop 3 of the rat receptor. For this study, Lys583 of the receptor was substituted with a panel of amino acids, and mutant receptors were assayed for hormone binding and induction of cAMP, inositol monophosphate, inositol bisphosphate, and inositol trisphosphate. No substitutions for Lys583 were permissible for cAMP induction, despite successful surface expression and hormone binding. In contrast, several substitutions were permissible for IP induction. Our results suggest two distinct transmembrane signal conductors for cAMP and inositol phosphate signals and imply particular models of receptor activation not previously suggested.

Animals↗
PubMed1992

Agonist-induced production of inositol phosphates in human airway smooth muscle cells in culture.

Smooth muscle cells (SMC) from human bronchi were isolated by elastase treatment, subcultured, and characterized by their positive reaction with a monoclonal antibody against alpha-smooth muscle actin (alpha SMA). In each cell line tested, at least 95% of the cells were positively stained. The functional properties of these cells were examined by measuring the metabolism of inositol phosphates (IPs). For that purpose, cells were incubated for 3 days before reaching confluency in the presence of myo-[3H]inositol in order to label the phosphoinositide pool, and the various [3H]IPs were separated by HPLC on a SAX column with a phosphate gradient. IP1 isomers were separated in three peaks; IP2, IP3, IP4, IP5 and IP6 (phytic acid) were each eluted as single peaks. The identity of the [3H]peaks was verified with corresponding [3H]IP standards. The accumulation of [3H]IPs was measured by incubating cells up to 30 min in the presence of 10 mM LiCl, with or without a bronchoconstrictor agent (carbachol, histamine, PGF2 alpha). Histamine, 10(-4) M, elicited a four times larger IP accumulation than carbachol, 10(-4) M, and than PGF2 alpha, 5 10(-5) M. Dose-response curves were established for histamine and carbachol in the range 10(-7)-10(-4) M. At 10(-7) M, carbachol was more effective than histamine in stimulating the IP metabolism. Atropine blocked the response to carbachol, and diphenhydramine inhibited the effect of histamine, indicating the specificity of the response to the agonists. These results indicate that cultured human bronchial SMC are a suitable preparation for studying physiological aspects of membrane transduction in the airways.

Bronchi↗
PubMed1989

Vasopressin V1 receptors on the principal cells of the rabbit cortical collecting tubule. Stimulation of cytosolic free calcium and inositol phosphate production via coupling to a pertussis toxin substrate.

The effects of arginine vasopressin (AVP) on the cytosolic free calcium concentration ([Ca2+]f) were examined in freshly immunodissected rabbit cortical collecting tubule cells using fluorescent Ca2+ indicators fura-2 and indo-1. The addition of AVP to a cell suspension resulted in a rapid and transient increase in the [Ca2+]f. The 1-deamino-8-D-AVP (dDVP), a V2 receptor agonist of AVP that stimulated adenosine 3',5' cAMP production in these cells, had no effect on [Ca2+]f and did not affect AVP-induced increase in [Ca2+]f. The AVP-induced increase in [Ca2+]f but not cAMP production was blocked by the V1 receptor antagonist, [1-(beta-mercapto-beta-beta-cyclopentamethylene propionic acid), 2-(O-methyl)tyrosine] Arg8-vasopressin. The AVP-stimulated increase in [Ca2+]f appeared to be largely due to Ca2+ release from intracellular stores as reduction of extracellular Ca2+ with EGTA had little if any effect on the AVP-induced increase in [Ca2+]f. This AVP-induced increase in [Ca2+]f was associated with an increase in inositol-1,4,5-trisphosphate production and appeared to involve a guanine nucleotide-binding protein (G), since the pretreatment of cells with pertussis toxin for 4-6 h inhibited this effect. Finally, measurements of [Ca2+]f in single cells suggest that only the principal cells of the collecting tubules respond to AVP with an increase in [Ca2+]f. In summary, these results demonstrate that the principal cells of the cortical collecting tubule possess two distinct receptor systems for vasopressin, the well-known V2 receptor coupled to adenylate cyclase, and a V1 receptor system that leads to the mobilization of cytosolic calcium, coupled through a pertussis toxin substrate (G protein) to a production of inositol phosphates.

Adenylate Cyclase Toxin↗
PubMed1994

Anergic T-lymphocyte clones have altered inositol phosphate, calcium, and tyrosine kinase signaling pathways.

Full activation of TH1 helper T lymphocytes requires ligation of the specific T-cell antigen receptor (TCR) and a second signal provided by costimulator molecule(s) expressed on particular antigen-presenting cells. Stimulation via the TCR complex alone generates a subsequent unresponsive state characterized by an inability to produce interleukin 2. We report here that such anergic cells exhibit multiple alterations in TCR-associated signaling. The basal levels of intracellular free calcium and phosphatidylinositol 1,4,5-trisphosphate are elevated in anergic cells, and the levels fail to increase significantly upon subsequent restimulation. Examination of phospholipase C-gamma 1 reveals evidence for post-translational modification, correlating with increased tyrosine phosphorylation of the molecule. Tyrosine phosphorylation of additional substrates identified from whole-cell lysates also is altered compared to untreated cells, suggesting a modification in net tyrosine kinase activity. Although the level of kinase activity present in TCR/CD3 or Lck immunoprecipitates is modestly altered after induction of anergy, there is a dramatic increase in specific Fyn-associated tyrosine kinase activity in anergic cells and increased phosphorylation of a 110-kDa protein that is coimmunoprecipitated with Fyn. These results are consistent with a model in which anergic TH1 lymphocytes display a fundamental alteration in TCR-mediated tyrosine kinase activity, associated with changes in phospholipase C-gamma 1, inositol phosphates, and intracellular free calcium.

Animals↗
PubMed1994

Inhibition of inositol phosphate second messenger formation by intracellular loop one of a human calcitonin receptor. Expression and mutational analysis of synthetic receptor genes.

Receptors for calcitonin (CTRs) have been cloned from several species, and two isoforms have been found to be expressed in human tissue. One human CTR isoform (hCTR-1) contains a 16-amino acid insertion in its first intracellular (I1) loop that is not present in porcine CTR (pCTR), rat CTR, or the other human CTR (hCTR-2). To facilitate the study of CTRs by mutational analysis, we have constructed synthetic hCTR-1 and hCTR-2 genes. Activation of hCTR-1 expressed transiently in COS-1 cells stimulates the formation of cAMP but not of inositol phosphates (IPs) whereas pCTR, a chimeric CTR in which the I1 loop of pCTR was substituted for the I1 loop of hCTR-1, and hCTR-2 stimulate cAMP and IP formation. A series of chimeric CTRs in which intracellular loops 1, 2, and 3 and the carboxyl tail of pCTR were substituted individually or in combination for those of hCTR-1 were constructed. All chimeras stimulated cAMP formation whereas chimeras containing the I1 loop of hCTR-1 with its 16-amino acid insertion were incapable of stimulating IP formation. There was no correlation between maximal stimulation of cAMP and IP formation by these CTRs. Thus, an inserted sequence in the I1 loop of hCTR-1 abolishes stimulation of the IP signal transduction pathway while allowing stimulation of the cAMP pathway.

Amino Acid Sequence↗
PubMed1992

Epidermal growth factor potentiates the transmitter-induced stimulation of C-AMP and inositol phosphates in human pigment epithelial cells in culture.

Salbutamol, isoproterenol and dopamine stimulate C-AMP production in human retinal pigment epithelium (RPE) cells by activation of beta 2-type receptors. Epidermal growth factor (EGF) in contrast does not alter basal levels of C-AMP but elevates in an apparently dose-dependent manner the isoproterenol-induced stimulation of C-AMP. EGF also potentiates the forskolin-induced stimulation of C-AMP but has no effect on the elevation of C-AMP caused by NECA (5'-[N-ethyl]-carboxamido adenosine), an adenosine A2-receptor agonist. EGF, isoproterenol and NECA have no effect on basal levels of inositol phosphates (InsPs) in human RPE cells, but EGF specifically elevates the carbachol-induced stimulation of InsPs. The carbachol effect on InsPs is attenuated by the phorbol ester PMA (4 beta-phorbol 12 myrisate 13-acetate). PMA did not, however, affect the stimulation of C-AMP caused by isoproterenol. The interaction of EGF and C-AMP is further demonstrated in experiments where the incorporation of [3H]thymidine into RPE cells was studied, as an index for proliferation. EGF stimulates RPE cell proliferation while isoproterenol and dibutyryl C-AMP nullify the EGF effect. Dibutyryl C-AMP has a negative effect on RPE cell proliferation while isoproterenol is ineffective. The data presented here suggest that after stimulation of EGF receptors, tyrosine-kinase-activated products can influence secondary messenger products produced from activation of beta 2-type (linked with C-AMP formation) and muscarinic (linked with InsPs production) receptors in RPE cells. We could find no evidence of an interaction between receptors associated with C-AMP and InsPs/diacylglycerol production.

Adolescent↗