Inositol 1,4,5-triphosphate induces calcium release from a non- mitochondrial pool in amoebae of Dictyostelium.

Evidence is presented for two distinct CA2+ pools in amoebae of Dictyostelium discoideum. One pool, presumably mitochondrial, was sensitive to the mitochondrial inhibitors oligomycin and dinitrophenol and showed an affinity for Ca2+ in the micro M concentration range. The other Ca2+ pool, which was insensitive to these inhibitors, was of lower capacity but had higher affinity (in the nM range). Inositol 1,4,5-trisphosphate (5 micro M) added to saponin-permeabilized amoebae induced a rapid release of Ca2+ from the latter pool but had no effect on the presumed mitochondrial pool. Controls using addition of inositol 1,4-bisphosphate (the hydrolytic product of IP3) induced no such CA2+ release. The results provide strong support for the involvement of IP3 in signal transmission during chemotaxis of D. discoideum.

Preterm labour: a pharmacological challenge.

Preterm labour is a major cause of perinatal mortality and morbidity, but its prevention is difficult because most of the available drugs lack uterine selectivity and have potentially serious side-effects for the mother or the foetus. In this article, Andrés López Bernal and colleagues discuss new evidence that shows pregnancy is associated with changes in G protein signalling and second messenger formation in human myometrium. During gestation uterine relaxation is favoured by a pronounced increase in G alpha s levels, thereby facilitating the effect of agonists that increase cAMP formation. The change in G alpha s is reversed in spontaneous labour enabling the uterus to become responsive to contractile agents. Although it is not established that these changes in G protein function are causally related to the spontaneous onset of labour, nevertheless they provide a novel viewpoint towards increased understanding of the cellular mechanisms of uterine contractility, which may result in better drugs for the management of preterm labour.

Effects of normal and mutant ras genes on inositol lipid metabolism in Dictyostelium.

Dictyostelium cells transformed with multiple copies of a mutant Dictyostelium ras gene (ras-Thr12 that gave a Gly to Thr substitution at position 12 of the ras protein, showed 2 to 3 times greater incorporation of 32P into PtdInsP and PtdInsP2 (without changing the specific radioactivity) compared to the untransformed strain or a strain transformed with multiple copies of the normal ras-Gly12 gene. The ratio of labelled PtdInsP2/PtdInsP, however, was not affected by the ras-Thr12 gene. Stimulation with the chemoattractant, cyclic AMP, caused a rapid but transient decrease in the levels of labelled PtdInsP and PtdInsP2 in the normal and ras-Gly12-transformed strains but ras-Thr12-transformed strains failed to respond. In untransformed cells a small, very rapid rise in the level of labelled PtdInsP and PtdInsP2 was seen immediately after stimulation of the cells with cyclic AMP (before the transient decrease) and this rise was greatly accentuated in cells transformed with multiple copies of the normal ras-Gly12 gene. Agents that induce prolonged activation of phosphoinositidase C such as AlF4- or GTPYS gave a lowered steady-state level of incorporation of 32P into PtdInsP and PtdInsP2 in all strains. The results indicate that the enzyme in the inositol phosphate pathway that is affected by the ras gene is not phosphoinositidase C, but is an enzyme before PtdInsP kinase, possibly PtdIns kinase.

Identification and expression of G-proteins in human myometrium: up-regulation of G alpha s in pregnancy.

We report that human myometrium contains G alpha i1, G alpha i3, and G alpha q, and G alpha 11, which are expressed at similar levels in tissues from pregnant and nonpregnant women. G alpha i2 is also expressed, but at a slightly reduced level, in tissue taken from pregnant compared to nonpregnant donors. The major finding of this investigation is the substantial increase in G alpha s expression in pregnant myometrium. The increase in G alpha s levels may play a crucial role in maintaining relaxation of the uterus by favoring cAMP formation during pregnancy.

Prostaglandin E2 activates phospholipase C and elevates intracellular calcium in cultured myometrial cells: involvement of EP1 and EP3 receptor subtypes.

PGE2 is a powerful modulator of uterine contractility, but there is uncertainty as to which receptor subtypes (EP1, EP2, EP3, or EP4), G proteins, and second messenger systems are activated by PGE2 in myometrium. Here we show that in cultured human myometrial cells, PGE2 (1-100 microM) activates phospholipase C (PLC) up to 500% over the control level and elevates intracellular calcium ([Ca2+]i) from the resting level of 60-90 nM up to 350 nM in a concentration-dependent manner. Stimulation by the receptor subtype-selective analogs GR63799X (EP3), sulprostone (EP3 > EP1), and misoprostol (EP3 > EP2 > EP1) indicates that these effects are transmitted through EP3 receptors. Both effects are resistant to pertussis toxin (PT). Lower concentrations of PGE2 (1-300 nM) increase [Ca2+]i via a PT-sensitive pathway, without PLC activation. This [Ca2+]i increase occurs after an inverse dose-related delay and is inhibited by the selective EP1 antagonist AH6809 and calcium channel blockers. By comparison, oxytocin stimulates PLC up to 1000% over the control level and elevates [Ca2+]i up to 800 nM in a concentration-dependent manner without any measurable delay; both effects are partly sensitive to PT. These data provide functional evidence for the presence of different stimulatory mechanisms for PGE2 in myometrium: 1) a low affinity receptor (probably EP3D) that activates PLC through a PT-insensitive pathway; and 2) a high affinity receptor (probably EP1), independent from PLC and involving a PT-sensitive G protein (G(i)?). Both pathways lead to elevation of [Ca2+]i.

Down-regulation of G alpha s in human myometrium in term and preterm labor: a mechanism for parturition.

We have previously reported that G alpha s is expressed at considerably higher levels in myometrium taken from pregnant than from nonpregnant women. In the present study we have determined adenylyl cyclase activity in myometrial membranes by measuring the conversion of [alpha-32P]ATP to [32P]cAMP and have measured guanosine triphosphate-binding protein expression by immunoblotting with specific antibodies. Here we report that the increase in G alpha s expression in pregnant myometrium is associated with a significant increase in G alpha s-coupled adenylyl cyclase activity, as estimated by incubating myometrial membranes in the presence of 5'-guanylyl-imidodiphosphate with and without prostaglandin E2. Moreover, in myometrium from women in spontaneous labor G alpha s levels and G alpha s-coupled adenylyl cyclase activity are reduced to the levels observed in nonpregnant tissue. There was no apparent change in forskolin-stimulated adenylyl cyclase activity in nonpregnant, pregnant, and laboring tissue. The increase in G alpha s expression in pregnant myometrium may facilitate agonist-induced cAMP formation, resulting in prolonged relaxation of the uterus during gestation. Down-regulation of G alpha s would decrease the relaxing effect exerted by cAMP and may be a triggering mechanism for the initiation of labor.

Human myometrial G alpha s-small (with serine) and Gs-large (with serine) messenger ribonucleic acid splice variants promote the increased expression of 46- and 54-kilodalton G alpha s protein isoforms in pregnancy and their down-regulation during labor.

In previous studies using specific G alpha s antibodies we have identified several human myometrial G alpha s protein isoforms with molecular masses of 45, 46, 47, 54, and 58 kDa, respectively. During pregnancy, levels of the 46- and 54-kDa proteins are significantly increased compared to those in nonpregnant myometrium and then decreased at the onset of labor. In this study we investigated the expression of G alpha s messenger ribonucleic acid (mRNA) splice variants, which are generated as a result of alternative splicing of a single mRNA precursor, in term pregnancy and parturition to determine whether there was any correlation with the observed changes in G alpha s protein isoforms. A myometrial G alpha s complementary DNA was synthesized using RT-PCR and cloned into pCRtmII suitable for preparation of riboprobes for use in ribonuclease protection assays. Using this technique, we identified at least three myometrial G alpha s mRNAs, including two forms of G alpha s-Large (with or without the serine at amino acid 87) and one form of G alpha s-Small (with the serine at amino acid 72). G alpha s Small (with the serine) and G alpha s-Large (with the serine) mRNAs encode for the 46- and 54-kDa G alpha s protein isoforms, respectively, and were increased in term pregnancy and then subsequently decreased after the onset of labor. Our data suggest that posttranscriptional regulation of G alpha s mRNAs may be important in the differential expression of G alpha s protein isoforms during pregnancy and labor.

Multiple G proteins and phospholipase C isoforms in human myometrial cells: implication for oxytocin action.

Although a physiological role for oxytocin during parturition is well accepted, the mechanisms by which it activates myometrial contractility during labor have not been completely elucidated. We have previously shown the presence of Gq and two pertussis toxin (PT) substrates of the Gi family in human myometrial cells. In the present study, we have identified by Western blotting the G protein and phospholipase C (PLC) isoforms present in these cells and investigated their implication in oxytocin signaling by measuring the formation of inositol phosphates (IPs) and mobilization of intracellular calcium. We found G protein subunits alpha(q), alpha(11), alpha(i1), alpha(i2), alpha(i3), alpha(z), and two splice variants of alpha(s)- and beta-subunits. We have also detected the presence of five PLC isoforms: beta 1, beta 2, beta 3, gamma 1, and gamma 2. Oxytocin-induced IPs formation and intracellular Ca2+ mobilization were inhibited to approximately 50% after pretreatment of the cells with PT, suggesting that oxytocin activates PLC beta by interacting with at least two types of G proteins: a member of the Gq family (PT resistant) and a member of the Gi family (PT sensitive). The tyrosine phosphatase inhibitor pervanadate stimulated IPs formation in myometrial cells. Using the protein kinase inhibitors staurosporine, phenylarsine oxide, and Ro 31-8220 and the protein kinase C activator phorbol dibutyrate, we have shown that pervanadate and oxytocin activate PLC by different mechanisms. Furthermore, oxytocin did not activate tyrosine phosphorylation in human myometrial cells, as measured with an antiphosphotyrosine antibody, indicating that it does not activate a PLC gamma isoform. We conclude that oxytocin activates human myometrium by interacting with at least two G proteins and possibly three PLC beta isoforms.

Spatio-temporal expression of the trans-acting splicing factors SF2/ASF and heterogeneous ribonuclear proteins A1/A1B in the myometrium of the pregnant human uterus: a molecular mechanism for regulating regional protein isoform expression in vivo.

Many of the human myometrial proteins associated with uterine quiescence and the switch to coordinated contractions at the onset of labor exist as alternatively spliced isoforms. There is now extensive evidence to indicate that the nuclear concentrations of the trans-acting splicing regulators SF2/ASF and hnRNP A1/A1B are fundamental in regulating the expression of specific protein isoforms derived from alternative splicing of single precursor messenger ribonucleic acid transcripts. The question thus arose as to whether these factors were also involved in regulating the expression of specific myometrial protein species within different uterine regions during human gestation and parturition. SF2/ASF and hnRNP A1/A1B expression was therefore determined in paired upper (corpus) and lower segment myometrial samples taken from individual women at term/during spontaneous labor and compared with nonpregnant control samples using specific monoclonal antibodies. We report that SF2/ASF levels were substantially increased in the lower uterine region, and this was associated with a parallel decrease in levels of hnRNP A1/A1B during gestation. Conversely, the opposite pattern was observed within the upper uterine region during pregnancy, where hnRNP A1/A1B was significantly up-regulated and SF2/ASF levels were much less than those found in the lower uterine segment. The differential expression of hnRNP A1/A1B and SF2/ASF in the upper and lower uterine segments may have a primary role in defining the formation of specific myometrial protein species associated with the known contractile and relaxatory properties of these regions before and during parturition.

The differential expression of myometrial connexin-43, cyclooxygenase-1 and -2, and Gs alpha proteins in the upper and lower segments of the human uterus during pregnancy and labor.

There is evidence from many studies indicating that a number of specific quiescent and contractile associated proteins are temporally regulated in the myometrium during pregnancy. In this present investigation we provide data that strongly suggest that myometrial connexin-43, cyclooxygenase-1 and -2 (COX-1 and -2), and Gs alpha proteins are also spatially expressed within the human uterus during pregnancy and labor. Using paired lower and upper segment myometrial samples taken from individual women at term and during spontaneous labor, we have measured the expression of these proteins by immunoblotting with specific antibodies. We report that the myometrial gap junction connexin-43 protein is expressed at much greater levels in the upper uterine compared to the lower uterine segment and that this difference is even more pronounced during the course of labor. Conversely, myometrial COX-1 and -2 proteins appear to be expressed at much greater levels in the lower compared to the upper uterine segment. Moreover, the level of expression of both proteins is unaffected by the onset of parturition. In contrast, myometrial Gs alpha protein appears to be uniformly expressed in both lower and upper segments and is similarly down-regulated during parturition, as previously reported. The differential expression of COX-1 and -2 and connexin-43 in the uterus may allow cervical ripening before and dilatation during labor and facilitate effective propagation of contractions from fundus to cervix, which may be further facilitated by the down-regulation of Gs alpha at the onset of parturition.

Gestational changes in the levels of transforming growth factor-beta1 (TGFbeta1) and TGFbeta receptor types I and II in the human myometrium.

As term approaches, a number of key proteins [contraction-associated proteins (CAPs)] are expressed within the human myometrium that are essential for the activation of powerful coordinated contractions during labor. The nature of the signals that switch on the synthesis of CAPs in vivo is not known. The ryanodine-sensitive intracellular Ca2+ release channel (RyR2) is a CAP whose expression in vitro is activated by transforming growth factor-beta (TGFbeta). The present experiments were performed to determine whether TGFbeta and TGFbeta receptors are present in the human myometrium at term and to explore the idea that they might form part of a signaling system in vivo. TGFbeta receptor types I and II, but not III, were demonstrated in myometrial smooth muscle in tissue taken from nonpregnant, pregnant nonlaboring, and spontaneous laboring women. Western blotting was used subsequently to determine the relative expression of TGFbeta receptor types I and II. Using nonpregnant myometrium as a baseline control the levels of expression of receptor types I and II were significantly increased by 168 +/- 19% (n = 6) and 162 +/- 22% (n = 7) in pregnant nonlaboring myometrium. In spontaneous laboring myometrium the levels of TGFbeta receptor type I and II expression were 93 +/- 12% (n = 6) and 85 +/- 11% (n = 7), respectively, compared to nonpregnant control values and were significantly lower than levels in pregnant nonlaboring tissues. The total TGFbeta1 levels in the myometrial tissues were 334 +/- 10, 534 +/- 73, and 674 +/- 106 pg/g tissue wet wt in nonpregnant, pregnant nonlaboring, and spontaneous laboring myometrium (n = 3 in each group), respectively. Thus, the TGFbeta signaling system appears to be up-regulated in the myometrium before the onset of parturition. The apparent loss of receptors in the spontaneous laboring samples in the presence of elevated total levels of TGFbeta may be indicative of agonist-induced receptor down-regulation. These observations support the idea that cytokines, in particular TGFbeta1, may play a role in the normal processes that prepare the myometrium for parturition at term.

Inhibition of cyclic GMP formation and aggregation in Dictyostelium by the intracellular Ca2+ antagonist TMB-8.

Aggregation in Dictyostelium discoideum was shown in previous studies employing EGTA to require Ca2+, but the intra- or extracellular site of action of this ion and its role in chemotaxis were not determined [1]. In this investigation we show that the intracellular Ca2+ immobilising agent TMB-8 does not affect binding of the signalling nucleotide, cAMP, to the cell surface receptors but abolishes the rapid accumulation of intracellular cGMP and subsequent chemotactic aggregation. We infer that movement of Ca2+ from membrane-bound stores is triggered by binding of cAMP to the cell-surface receptor and that this plays a primary role in stimulating cGMP formation and chemotaxis.

Calcium transport in the cellular slime mould Dictyostelium discoideum.

Transport of Ca2+ into amoebae of Dictyostelium discoideum was studied using 45Ca and a lanthanum stopping technique. Ca2 uptake was found to be rapid and showed saturation kinetics. No difference was found in Ca2+ uptake between vegetative and aggregation competent cells, the V(max) for unstimulated amoebae being approx. 10 nmol/10(7) cells per min. Ca2+ uptake had the characteristics of passive facilitated diffusion using a saturatable carrier and NaN3 and ouabain were not inhibitory. The chemoattractants cAMP and folate, previously reported to stimulate the uptake of Ca2+ into amoebae, did not stimulate the rate of Ca2+ uptake by this carrier but increased the extent of Ca2+ taken up over the period 10-30 s after chemotactic stimulation. The significance of these findings for the function of Ca2+ in chemotactic signalling is discussed.

Functional characterisation of the Drosophila 5-HTdro1 and 5-HTdro2B serotonin receptors in insect cells: activation of a G(alpha) s-like protein by 5-HTdro1 but lack of coupling to inhibitory G-proteins by 5-HTdro2B.

Insect cells are routinely used for the production of receptor proteins. Expression of the Drosophila 5-HTdro1 serotonin receptor resulted in positive coupling of the receptor to adenylyl cyclase via the G(alpha)s G-protein subtype. The Drosophila 5-HTdro2B receptor stimulated the metabolism of inositol phospholipid via a pertussis toxin-insensitive G-protein, but exhibited no detectable inhibition of adenylyl cyclase. Immunoblot analysis of the endogenous G-proteins revealed that Sf9 cells lack the G-protein subtypes G(alpha i 1-3) and G(alpha)o, but express the subtype G(alpha)s and G(alpha)q.

Expression of human myometrial G alpha s messenger ribonucleic acid transcript during pregnancy and labour: involvement of alternative splicing pathways.

We have shown previously that expression of 46 and 54 kDa human myometrial G alpha s protein isoforms is increased during gestation and then subsequently decreased during labour. These proteins appear to be coded for by G alpha s-Small (with a serine residue at position 72) and G alpha s-Large (with a serine residue at position 87) mRNA splice variants respectively. In the study presented here we have used a G alpha s cDNA template to generate [32P]cytidine cRNA riboprobes for use in RNase protection assays, so as to measure total myometrial G alpha s mRNA levels in relation to the pattern of expression of G alpha s mRNA splice variants during pregnancy and labour. We report that total levels of human myometrial G alpha s mRNA remain similar in non-pregnant and pregnant women but are substantially reduced during parturition. Our data also provide strong evidence that alternative splicing of G alpha s precursor mRNA has a primary role in regulating expression of G alpha s protein isoforms during pregnancy and labour. The inclusion of an additional serine codon in G alpha s mRNAs during pregnancy involves a switch in alternative splicing pathways. We speculate that this switch may be due to a change in specificity of splicing factors that are modulated during pregnancy and labour.

Identification of G alpha s messenger ribonucleic acid splice variants in human granulosa cells.

Granulosa cells are essential for follicular development and corpus luteum formation and their functions are regulated by gonadotrophins through G protein-coupled receptors. The dominant second messenger pathway involves the stimulation of cyclic AMP formation by G alpha s-linked receptors. In this paper we have investigated the expression of G alpha s mRNA splice variants in relation to expression of G alpha s protein isoforms in granulosa cells obtained from patients undergoing in vitro fertilization. We have carried out ribonuclease protection assays using cRNA riboprobes which are capable of detecting all G alpha s mRNA isoforms as well as quantifying total amounts of G alpha s mRNA. Granulosa cells express the message for G alpha s-Large and G alpha s-Small and the presence of two distinct protein products was confirmed by immunoblotting using the antibody RM/1. Moreover, the data show that a significant fraction of G alpha s-Large and G alpha s-Small mRNAs contain an extra CAG codon. This should generate proteins with an extra serine residue, resulting in G alpha s variants with the consensus sequence of a protein kinase C phosphorylation site. These results highlight the possible interaction between different signalling pathways in the control of cAMP production and the need to investigate the relationship between G alpha s variants and different adenylyl cyclase isozymes in patients with normal and abnormal ovarian function.

Oxytocin-stimulated phosphoinositide hydrolysis in human myometrial cells: involvement of pertussis toxin-sensitive and -insensitive G-proteins.

Phosphoinositide hydrolysis is important in mediating the actions of oxytocin and prostaglandin (PG) F2 alpha on uterine contractions during labour. We have measured the effect of oxytocin, PGF2 alpha and other agents on the formation of inositol phosphates (IPs) in cultured human myometrial cells labelled with [3H]inositol and on changes in intracellular free Ca2+ concentration ([Ca2+]i) in cells loaded with Fura-2. Oxytocin induced the formation of [3H]IPs in a concentration-dependent manner with an EC50 (concentration of agonist producing 50% of the maximal response) of 1.4 +/- 0.5 nmol/l (mean +/- S.E.M.). The maximal response was obtained with 1 mumol oxytocin/l and represented a stimulation of 670% over basal. PGF2 alpha also stimulated the formation of [3H]IPs and the response at 1 mumol/l was a 204% stimulation over basal. The effects of PGF2 alpha were independent of extracellular Ca2+ but the effect of oxytocin was reduced with low extracellular Ca2+. Cyclic AMP formation, induced by forskolin or PGE2, had no effect on the stimulated levels of [3H]IPs. Pertussis toxin (PT) reduced the oxytocin-stimulated formation of [3H]IPs in a concentration-dependent manner. The maximal effect of PT resulted in an 80% reduction in the formation of [3H]IPs. However, PGF2 alpha stimulation was not affected by PT treatment. To analyse the action of PT further, we studied its effect on oxytocin-induced changes in [Ca2+]i. The basal [Ca2+]i was 112 +/- 4 nmol/l (n = 225 cells) and was not affected by PT treatment (109 +/- 3 nmol/l; n = 200 cells). In the absence of PT, 1 mumol oxytocin/l increased [Ca2+]i to a peak of 522 +/- 26 nmol/l, and in PT-treated cells, the [Ca2+]i peak was reduced to 348 +/- 16 nmol/l. Similar inhibitory effects of PT were obtained at oxytocin concentrations ranging from 1 to 100 nmol/l. Our data suggest that in human myometrial cells, the oxytocin-induced production of [3H]IPs and increase in [Ca2+]i are mediated by a PT-sensitive G-protein. However, a significant fraction of the oxytocin response appears to be mediated by a PT-insensitive G-protein, possibly a member of the Gq family.

The desensitization of oxytocin receptors in human myometrial cells is accompanied by down-regulation of oxytocin receptor messenger RNA.

We have recently provided evidence for the desensitization of oxytocin receptors in human myometrial cells. In the present study, we have investigated the possible mechanisms by which oxytocin (OT) might regulate OT receptor density. The steady state level of OT binding in cultured myometrial cells was 220 x 10(3) binding sites/ cell, but this was time-dependently reduced to 27 x 10(3) sites/cell by exposure to OT for up to 20 h. Similarly, OT exposure decreased the binding of OT to cell membranes. In contrast, Western blotting data showed that the total amount of OT receptor protein was not affected by OT treatment for up to 48 h. Flow cytometry experiments demonstrated that OT receptors are not internalized during prolonged exposure of the cells to OT. However, RNase protection assays and Northern analysis showed that OT receptor mRNA was reduced by OT treatment to reach a new low steady state level with a time course similar to that of the disappearance of cell surface OT binding sites. Possible mechanisms involved in mRNA down-regulation include transcriptional suppression and destabilization of mRNA by RNA binding proteins.

Inhibition of aggregation in Dictyostelium by EGTA-induced depletion of calcium.

During aggregation of Dictyostelium discoideum, amoebae move towards collecting centres emitting cyclic AMP. Previous work has indicated that Ca2+ can affect the operation of various parts of the chemotactic mechanism but there are contradictory reports about the role or requirement for Ca2+ during aggregation of intact amoebae. In this study we show that there is a requirement for Ca2+ during this aggregation process. Addition of EGTA (7 mM) and in some cases extensive washing with EGTA is needed to see inhibition of aggregation. Considerable stores of Ca2+ present within cells may explain the failure of some previous attempts to see a Ca2+ requirement during aggregation.

Hepatic hexose transport and the effect of N2-induced anoxia and KCN on this process.

Hepatic hexose transport was characterized using 3-O-methyl-D-glucose, which is not metabolized by the liver. The kinetic parameters determined in the starved state were taken as basal values for the transport system which showed saturation kinetics with high Vmax and Km values of 161 nmol/mg dry wt./min and 39 mM respectively. In the fed state, the Vmax was found to be increased nearly two-fold; this may be due to a phenomenon known as trans-stimulation. The effects of N2-induced anoxia and of KCN were investigated. In the fasted state, anoxia caused the transport characteristics Vmax and Km to decrease nearly two-fold whereas KCN had the opposite effect as the Vmax and Km were increased by three- and two-fold respectively. In the fed state, anoxia and KCN caused a marked decrease in the transport characteristics.