Phosphoinositidase C Research Articles

Novel Ca 2+ signaling pathway for AVP mediated vasoconstriction

[Arg8]-vasopressin (AVP) is a peptide hormone which exerts vasoconstrictor effects by binding to V1a vasopressin receptors on vascular smooth muscle cells (VSMC). This effect is attributed to activation of phosphoinositidase C (PIC) and consequent release of Ca2+ from the sarcoplasmic reticulum. Interestingly, half-maximal activation of PIC requires nanomolar AVP concentrations, whereas circulating AVP concentrations rarely exceed 100pM. Using cultured VSMC, we previously identified a novel Ca2+ signaling pathway activated by 10–100pM AVP. This Ca2+ signaling pathway is distinguished from the PIC pathway by its dependence upon protein kinase C (PKC) activation and L-type voltage-sensitive Ca2+ channels (VSCC). In the present study, we tested the hypothesis that picomolar AVP concentrations would induce PKC- and VSCC-dependent vasoconstriction in isolated pressurized rat mesenteric arteries. AVP (10−14 to 10−6 M) induced a concentration-dependent constriction of arteries that was reversible with a V1a vasopressin receptor antagonist. 30pM AVP induced significant vasoconstriction (~45% of maximum), which was prevented by PKC inhibition with calphostin-C (250nM) or Ro-31–8220 (1μM) as well as by blockade of VSCC with verapamil (10μM). In contrast, maximal vasoconstriction induced by 10nM AVP was insensitive to either PKC inhibition or blockade of VSCC. These results suggest that two dose-dependent signaling pathways exist for AVP-induced vasoconstriction. Accordingly, we conclude that the vasoconstrictor actions of picomolar AVP within the systemic circulation may be dependent upon the novel Ca2+ signaling pathway involving PKC activation and L-type VSCC. NIH RO1-HL070670 and Falk Fellowship

Generation of Diacylglycerol during the Acrosome Reaction Induced by Ca2+ and Ca2+ Ionophore A23187 in Frozen-Thawed Spermatozoa from Fertile and Subfertile Japanese Black Bulls.

This study investigated the activation of phosphoinositidase C (PIC) and phosphatidylcholine-specific phospholipase C (PLC) during the acrosome reaction of spermatozoa induced by Ca2+ and Ca2+ ionophore A23187 in a subfertile Japanese black bull. Sperm motility, viability, morphology and penetration of cervical mucus by spermatozoa in a subfertile bull were comparable to those in a fertile bull. When frozen-thawed spermatozoa were stimulated with 3 mM Ca2+ and 1 μM A23187, both bulls' spermatozoa showed a time-dependent increase in percent acrosome reactions, but the increase was more rapid and higher in spermatozoa from the fertile bull. The amounts of 1,2-diacylglycerol (DAG) mass, a product of PIC and PLC, peaked at 1 min after a similar treatment with a gradual decline towards 10 min in both bulls' spermatozoa but were significantly higher throughout stimulation for the subfertile bull. These results suggest that active PIC/PLC may be present in spermatozoa of the subfertile bull, and that other mechanisms were possibly associated with the lowered induction of the acrosome reaction. However, it remains unclear whether the high levels of 1,2-DAG mass were responsible.

Prostaglandin E2-induced sensitization of bradykinin-evoked responses in rat dorsal root ganglion neurons is mediated by cAMP-dependent protein kinase A.

Primary cultures of neonatal rat dorsal root ganglion (DRG) neurons were used to examine the mechanisms underlying both the direct activation and the sensitization of sensory neurons by prostanoids. Prostaglandin E2 (PGE2) elevated cytosolic calcium concentration ([Ca2+]i) in a subpopulation of small (< 19 microm) diameter, capsaicin-sensitive DRG neurons. PGE2 also stimulated substance P (SP) release from DRG cultures. In contrast to bradykinin, PGE2 did not stimulate phosphoinositidase C (PIC) and the PGE2-evoked increase in [Ca2+]i was dependent on extracellular calcium. Pre-treatment with PGE2 potentiated bradykinin-evoked increases in [Ca2+]i in small diameter neurons and increased the number of cells that responded to low concentrations of bradykinin. A similar effect was seen with prostaglandin I2 (PGI2) but not prostaglandin F2alpha (PGF2alpha). PGE2 pretreatment also potentiated bradykinin-evoked release of SP, inducing a leftward shift in the bradykinin concentration-response curve and an increase in the maximum response. PGE2 stimulated adenylyl cyclase activity in DRG cultures, at concentrations and times consistent with those required to observe both the direct and sensitizing effects of the prostanoid on [Ca2+]i responses. Furthermore, the direct and sensitizing effects of PGE2, on both [Ca2+]i responses and SP release, were mimicked by the membrane permeant cAMP analogue dibutyryl cAMP and inhibited by H89, an inhibitor of cAMP-dependent protein kinase A (PKA). These observations are consistent with the hypothesis that both direct activation and sensitization of sensory neurons by prostanoids, such as PGE2, are mediated by PKA-dependent phosphorylation mechanisms.

Differences in the stimulation of the phosphoinositide cycle by amine neurotransmitters in cultured rat forebrain neurones and astrocytes

In this study, we compared the stimulation by carbachol (CCh), noradrenaline (NA), and histamine (HA) of phosphoinositide hydrolysis in rat forebrain neuronal and glial cultures. When Ca 2+ was omitted from the stimulation buffer (low μM extracellular Ca 2+ ), amine-induced [ 3H]inositol phosphate accumulation was reduced to a higher extent in astrocytes (70–80% for CCh and NA and 100% for HA) than in neurones (around 50–60% for all the amines). Furthermore, guanosine 5′-by-[γ-thio]trisphosphate (GTP[S]) stimulation of phosphoinositidase C (PIC) in membranes was 5-fold higher in neurones than in astrocytes. These results indicate differences in the mechanism of PIC stimulation in the two cell types. After 30 min stimulation in the presence of 10 mM Li +, a higher accumulation of [ 3H]inositol 4-monophosphate and [ 3H]inositol 1,4-bisphosphate than of [ 3H]inositol — monophosphate occurred for all agonists in neurones, whereas the opposite was observed in astrocytes. Moreover, in these cells stimulation for 5 min in the absence of Li + produced a 2–3-fold accumulation of all metabolites of the 3-kinase pathway of inositol-l,4,5-trisphosphate metabolism but not of those of the 5-phosphatase pathway. Thus, regardless of the amine receptor stimulated, the 3-kinase route appeared to prevail in astrocytes and the 5-phosphatase pathway in neurones. The histamine response in neurones differed from that of the other agonists in that it rapidly declined. Taken together these results indicate that the heterogeneity in amine stimulation of the phosphoinositide cycle previously observed in brain slices could arise to a great extent from the cellular diversity of this preparation and be related to the differential contribution of the amine receptors located in neurones and astrocytes.

Enhanced type 1alpha metabotropic glutamate receptor-stimulated phosphoinositide signaling after pertussis toxin treatment.

The regulation of phosphoinositide hydrolysis by the type 1alpha metabotropic glutamate receptor (mGluR1alpha) was investigated in stably transfected baby hamster kidney (BHK) cells. Incubation of the cells with L-glutamate, quisqualate, and 1-aminocyclopentane-1S, 3R-dicarboxylic acid resulted in a marked accumulation of [3H]inositol monophosphate (InsP1) and inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] mass in a time- and concentration-dependent manner. Pretreatment of BHK-mGluR1alpha cells with pertussis toxin [ 100 ng/ml, 24 hr] led to a dramatic 12-16-fold increase in the accumulation of [3H]InsP1 and a 2-fold increase in Ins(1,4,5)P3 in the absence of added agonist. Although only very low levels (</=1 microM) of L-glutamate could be detected in medium taken from control and PTX-treated cell monolayers, the PTX-elicited effect on basal [3H]InsP1 was fully reversed by preincubation of cells in the presence of glutamic-pyruvic transaminase and pyruvate, suggesting that an increased sensitivity to endogenous glutamate was responsible for the apparent agonist-independent activation of phosphoinositidase C (PIC) after PTX treatment. Consistent with this hypothesis, in the presence of glutamic-pyruvic transaminase/pyruvate, the maximal [3H]InsP1 response to quisqualate was increased by >/=75%, and the EC50 shifted leftward by 65-fold [-log EC50 values (molar), 7.26 +/- 0.23 versus 5.45 +/- 0.07; n = 4) in PTX-treated compared with control cells. In contrast, antagonist effects on agonist-stimulated [3H]InsP1 responses were similar in control and PTX-treated BHK-mGluR1alpha cells. These changes in the concentration-effect curves for mGluR agonists are consistent with a model in which the receptor associates with PTX-sensitive inhibitory (Gi/o) and PTX-insensitive stimulatory (Gq/11) G proteins that can each influence PIC activity. The present observations are consistent with a dual regulation of mGluR1alpha-mediated PIC activity that could be fundamental in controlling the output of phosphoinositide-derived messengers.

Oxytocin receptor-mediated activation of phosphoinositidase C and elevation of cytosolic calcium in the gonadotrope-derived alphaT3-1 cell line.

Gonadotropes synthesize and secrete LH and FSH under the control of GnRH, which acts via phosphoinositidase C (PIC)-linked G protein coupled receptors. Additionally, gonadotropin released from the pituitary is influenced by oxytocin, a peptide that has been shown to play a role in generation of the preovulatory LH surge. Although oxytocin receptors are present in the pituitary, studies have identified their presence on lactotropes but not on gonadotropes, raising the question of which cells act as the direct target of oxytocin in gonadotrope regulation. In this study, we examined effects of oxytocin on alphaT3-1 cells, a gonadotrope-derived cell line. Oxytocin, vasopressin, and vasotocin each stimulated accumulation of [3H]inositol phosphates in cells prelabeled with [3H]inositol, indicating activation of PIC. The rank order of potency (oxytocin > vasotocin > vasopressin) and sensitivity to inhibition by oxytocin and vasopressin receptor antagonists, revealed the effect to be mediated by oxytocin-selective receptors. Like other PIC activators, these nonapeptides caused biphasic (spike-plateau) increases in the cytosolic Ca2+. The spike response to oxytocin and GnRH were both retained in Ca2+-free medium, reflecting mobilization of intracellular Ca2+, and were comparably reduced by thapsigargin, implying mobilization of Ca2+ from a shared thapsigargin-sensitive intracellular pool. Brief stimulation with oxytocin, vasopressin, or vasotocin prevented subsequent Ca2+ responses to oxytocin, but not to GnRH, suggesting that the oxytocin receptor undergoes rapid homologous desensitization and reinforcing the interpretation that the nonapeptides act via the same receptor type. Oxytocin did not increase Ca2+ in cells stimulated with GnRH, whereas GnRH caused a spike Ca2+ increase even in the presence of oxytocin, implying that different mechanisms of desensitization (Ca2+ pool depletion and receptor uncoupling) are operating for two distinct PIC-coupled receptors in these cells. The demonstration that oxytocin acts directly via PIC-linked, oxytocin-selective receptors to increase cytosolic Ca2+ in a gonadotrope-derived cell line is consistent with the possibility that oxytocin has a comparable effect on nonimmortalized gonadotropes.

Oxytocin Receptor-Mediated Activation of Phosphoinositidase C and Elevation of Cytosolic Calcium in the Gonadotrope-Derived T3-1 Cell Line

Gonadotropes synthesize and secrete LH and FSH under the control of GnRH, which acts via phosphoinositidase C (PIC)-linked G protein coupled receptors. Additionally, gonadotropin released from the pituitary is influenced by oxytocin, a peptide that has been shown to play a role in generation of the preovulatory LH surge. Although oxytocin receptors are present in the pituitary, studies have identified their presence on lactotropes but not on gonadotropes, raising the question of which cells act as the direct target of oxytocin in gonadotrope regulation. In this study, we examined effects of oxytocin on αT3–1 cells, a gonadotrope-derived cell line. Oxytocin, vasopressin, and vasotocin each stimulated accumulation of[ 3H]inositol phosphates in cells prelabeled with[ 3H]inositol, indicating activation of PIC. The rank order of potency (oxytocin > vasotocin > vasopressin) and sensitivity to inhibition by oxytocin and vasopressin receptor antagonists, revealed the effect to be mediated by oxytocin-selective receptors. Like other PIC activators, these nonapeptides caused biphasic (spike-plateau) increases in the cytosolic Ca2+. The spike response to oxytocin and GnRH were both retained in Ca2+-free medium, reflecting mobilization of intracellular Ca2+, and were comparably reduced by thapsigargin, implying mobilization of Ca2+ from a shared thapsigargin-sensitive intracellular pool. Brief stimulation with oxytocin, vasopressin, or vasotocin prevented subsequent Ca2+ responses to oxytocin, but not to GnRH, suggesting that the oxytocin receptor undergoes rapid homologous desensitization and reinforcing the interpretation that the nonapeptides act via the same receptor type. Oxytocin did not increase Ca2+ in cells stimulated with GnRH, whereas GnRH caused a spike Ca2+ increase even in the presence of oxytocin, implying that different mechanisms of desensitization (Ca2+ pool depletion and receptor uncoupling) are operating for two distinct PIC-coupled receptors in these cells. The demonstration that oxytocin acts directly via PIC-linked, oxytocin-selective receptors to increase cytosolic Ca2+ in a gonadotrope-derived cell line is consistent with the possibility that oxytocin has a comparable effect on nonimmortalized gonadotropes.

Muscarinic M3 receptor coupling and regulation

Current concepts regarding the regulation and coupling of muscarinic m3 receptors to G-proteins and various effectors are discussed. The last few years have provided much evidence that although muscarinic m1, m3 and m5 subtypes couple predominantly via pertussis toxin-insensitive G-proteins (Gq/11) to activate phosphoinositidase C (PIC), interactions with other G-proteins (Gi, Go, Gs) can be readily observed in cells expressing recombinant muscarinic receptors even at relatively low levels. The significance of this diversity and the potential for agonist "trafficking" could open up opportunities for novel approaches to selective agonist action. Finally, mechanisms underlying the regulation of muscarinic m3 coupling through Gq/11 to PIC are discussed. In particular, our recent studies on precursor lipid depletion, whether regulation is receptor or cell specific and the identification and role of receptor kinases are briefly reviewed.

GnRH and PACAP action in gonadotropes: Cross-talk between phosphoinositidase C and adenylyl cyclase mediated signaling pathways

In order to respond appropriately to their environment, gonadotropes, like other cells, must integrate informational input from multiple ligands acting through multiple intracellular signaling pathways. In recent years, an increasing number of examples of functional interactions between the phosphoinositidase C (PIC) and adenylyl cyclase signaling pathways in gonadotropes have been described, and the discovery that these cells are targets for pituitary adenylyl cyclase activating peptide (PACAP) has provided a physiological context for earlier work on gonadotrope regulation by cAMP. It has become clear that gonadotropes possess multiple PIC-coupled receptor types, in addition to receptors activating adenylyl and guanylyl cyclases, so that the potential for both coincidence signaling and cross-talk in these cells is immense; examples of both are seen in the effects of PACAP and GnRH on Ca 2+ mobilization and adenylyl cyclase activation in αT3-1 cells. In these cells, GnRH, acting via PIC-coupled receptors, can dramatically inhibit adenylyl cyclase activated by PACAP, but can also alter cellular levels of protein kinase A subunits, providing a mechanism for coordinated regulation of both messenger and effector.

Cyclic GMP regulates activation of phosphoinositidase C by bradykinin in sensory neurons.

Prior exposure of cultured neonatal rat dorsal root ganglion (DRG) neurons to bradykinin resulted in marked attenuation of bradykinin-induced activation of phosphoinositidase C (PIC). The (logconcentration)-response curve for bradykinin-induced [3H]inositol trisphosphate ([3H]IP3) formation was shifted to the right and the maximum response was reduced. Bradykinin increases cyclic GMP (cGMP) in DRG neurons [Burgess, Mullaney, McNeill, Coote, Minhas and Wood (1989) J. Neurochem. 53, 1212-1218] and treatment of the neurons with dibutyryl cGMP (dbcGMP) had a similar, inhibitory, effect on bradykinin-induced [3H]IP3 formation. NG-Nitro-L-arginine (LNNA) blocked bradykinin-induced formation of cGMP. It prevented the functional uncoupling induced by pretreatment with bradykinin, but not the inhibitory effect of dbcGMP on [3H]IP3 formation. The ability of LNNA to prevent desensitization was reversed by excess L-arginine, indicating that its actions were mediated through inhibition of nitric oxide synthase. In addition to functional desensitization, exposure to bradykinin reduced the number of cell-surface receptors detected with [3H]bradykinin, without affecting its KD value for the remaining sites. In contrast to bradykinin, pretreatment with dbcGMP had no effect on either the KD or B(max) for [3H]bradykinin binding. This implies that the inhibitory effect of dbcGMP was down-stream from the binding of bradykinin to its receptor and upstream of IP3 formation. The lack of effect of dbcGMP on [3H]bradykinin binding suggests that the decrease in receptor number induced by bradykinin was mediated by a different mechanism and was not a key factor in the rapid phase of desensitization in these cells.

Thyroid-stimulating hormone rapidly stimulates inositol polyphosphate formation in FRTL-5 thyrocytes without activating phosphoinositidase C.

The thyroid-stimulating hormone (TSH) receptor is widely regarded as one of a limited number of G-protein-coupled receptors that activate both adenylate cyclase and phosphoinositidase C (PIC) via G-proteins, but the existing experimental evidence for TSH-stimulated PtdIns(4,5)P2 hydrolysis remains inconclusive. We have compared the effects of TSH and of ATP (acting via P2-purinergic receptors) on the inositol lipids and polyphosphates of [2-3H]inositol-labelled FRTL-5 rat thyroid cells. ATP initiated a rapid decrease in 3H-labelled PtdIns4P and PtdIns(4,5)P2, whereas TSH did not. Stimulation with ATP and, less consistently, with noradrenaline (acting via alpha-adrenergic receptors) provoked rapid formation of Ins(1,4,5)P3, Ins(1,3,4,5)P4, Ins(1,3,4)P3 and Ins(1,4)P2, confirming activation of PtdIns(4,5)P2 hydrolysis. No concentration of TSH provoked detectable accumulation of Ins(1,4,5)P3 or Ins(1,4)P2 during the first few minutes of stimulation. However, an InsP3 [with the chromatographic properties of Ins(1,3,4)P3] and two InsP4 isomers [neither of which was Ins(1,3,4,5)P4] accumulated quickly in TSH-stimulated cells. ATP immediately provoked a large increase in intracellular calcium concentration ([Ca2+]i) in Indo 1-AM-loaded cells. TSH provoked a small and delayed [Ca2+]i elevation in only some experiments. We therefore confirm that activation of P2-purinergic receptors and alpha 1-adrenergic receptors provokes PIC activation, an accumulation of Ins(1,4,5)P3 and its metabolites and rapid [Ca2+]i mobilization in FRTL-5 cells. By contrast, TSH provokes no rapid PIC-catalysed PtdIns(4,5)P2 hydrolysis or immediate [Ca2+]i mobilization. These results fail to support the widespread view that the TSH receptor of FRTL-5 cells signals, in part, through PIC activation. Our results suggest that TSH activates another, still undefined, mechanism that causes accumulation of an InsP3 and two isomers of InsP4.

Transfected Saos-2 cells overexpressing phosphoinositidase C β1 isoform accumulate it within the nucleus

The subcellular partitioning of the phosphoinositidase C (PIC) isoforms involved in signal transduction, with the selective localization of the PIC β 1 isoform in the nucleus, represents a crucial aspect of the complex mechanism of cell response to agonists. In order to further elucidate this phenomenon, we utilized human osteosarcoma Saos-2 cells, transfected with the cDNA for rat PIC β 1. In the cells overexpressing this isoform, immunocytochemical analyses at the electron microscope level reveal an increased synthesis at the cytoplasm and a significant accumulation within the nucleus of the protein. Interestingly, the sites of intranuclear localization are, as in wild type cells, the interchromatin domains. These results indicate that the transfected cells maintain the capability of accumulating the enzyme within the nucleus and can be considered a model for functional studies on the nuclear signal transduction also in response to specific agonists.

Changes in the components of a nuclear inositide cycle during differentiation in murine erythroleukaemia cells.

Differentiation of murine erythroleukaemia cells with the chemical agent DMSO leads to a cessation of proliferation and the production of a number of erythrocyte markers such as haemoglobin. We have previously demonstrated that activation of proliferation leads to an increase in the production of nuclear diacylglycerol (DAG). Here we demonstrate that differentiation leads to a decrease in the levels of nuclear DAG and the activity of the nuclear-associated phosphoinositidase C (PIC). The change in activity appears to be due to a decrease in the mass levels of the beta 1 isoform, as demonstrated by the use of isoform-specific antibodies. Moreover, the changes correlate with the cessation of proliferation and an increase in the number of cells in G1 phase of the cell cycle, rather than with the number of cells which have differentiated. Indeed, although treatment of the cells with phorbol 12-myristate 13-acetate (PMA) inhibits the differentiation programme as assessed by haemoglobin staining, it does not inhibit the number of cells blocking in G1 of the cell cycle or the changes in nuclear DAG or PIC activity. The possible involvement of this nuclear inositide cycle during progression through the cell cycle is discussed.

Desensitization of gonadotropin-releasing hormone action in the gonadotrope-derived alpha T3-1 cell line

Sustained exposure of gonadotropes to GnRH causes a pronounced desensitization of GnRH-stimulated gonadotropin release, but the mechanisms involved are poorly understood. Recent studies have suggested, however, that GnRH-stimulated phosphoinositidase C (PIC) activity does not undergo rapid ( < 5 min) homologous desensitization in alpha T3-1 cells, and we have, therefore, used this cell line to address the question of whether desensitization occurs distal to PIC activity and/or in an intermediate time frame. We show that GnRH stimulates a rapid increase in inositol 1,4,5-trisphosphate [Ins(1,4,5)P3; maximum at 10-20 sec with a modest reduction thereafter] and that the GnRH-stimulated accumulation of [3H]IPs (in cells stimulated in the presence of LiCl) increases linearly over 5-300 sec. This clearly indicates that desensitization of PIC does not occur within this period and that the dramatic reduction in the rate of Ins(1,4,5)P3 accumulation (10-30 sec) is due to its metabolism, rather than to a reduction in Ins(1,4,5)P3 generation. Pretreatment for 60 min with 10(-7)M GnRH reduced cell surface GnRH receptor number by 48% (without measurably altering Kd). The pretreatment also reduced maximal GnRH-stimulated [3H]IP accumulation (to 66% of the control) and increased the EC50 for GnRH-stimulated [3H]IP accumulation approximately 3-fold, demonstrating that desensitization of GnRH-stimulated [3H]IP accumulation can, indeed, occur within 60 min, but that this may be attributable to receptor loss (without appreciable uncoupling of residual receptors from their immediate effector system). Pretreatment for 60 min with GnRH also caused a dose-dependent reduction in both spike and plateau phases of the GnRH effect on cytosolic Ca2+. This effect could not be overcome by stimulation with high concentrations of GnRH and appears, therefore, to reflect not only receptor loss, but, also, an additional inability of agonist-occupied GnRH receptors to elevate cytosolic Ca2+. The effect of KCl on cytosolic Ca2+ was similarly reduced by GnRH pretreatment, suggesting that desensitization of voltage-operated Ca2+ channels mediates desensitization of the plateau phase Ca2+ response to GnRH. Such a mechanism could not, however, explain desensitization of the spike phase of the Ca2+ response to GnRH seen in normal or Ca2+ -free medium. Accordingly, the data reveal a novel mechanism for homologous desensitization to GnRH in which agonist-occupied GnRH receptors are rendered unable to mobilize intracellular Ca2+ and imply that desensitization of GnRH-stimulated Ins(1,4,5)P3 production and/or action occurs.

Phosphoinositidase C beta 1 isoform expression is modulated by interferon alpha in burkitt lymphoma cells

The expression of phosphoinositidase C (PIC) at nuclear and cytoplasmic level has been revealed in control and interferon treated Burkitt lymphoma cells by means of western blotting and immunocytochemical analysis employing specific monoclonal antibodies against beta 1, gamma 1 and delta 1 isozymes. Results have indicated that PIC isoform beta 1, mainly detectable in the nucleus, undergoes transient modifications early after interferon treatment. PIC delta 1 has been found only at cytoplasmic level, apparently insensitive to interferon treatment, while PIC gamma 1 was scarcely or not detected either in the cytoplasmic or in the nuclear compartment. These results suggest that interferon may exert its antiproliferative effect activating at least two distinct pathways of signal transduction, at cytoplasmic and nuclear level, involving inositol lipid cycle mainly in the nucleus by modulation of PIC beta 1 expression.

Phosphoinositide signalling enzymes in human T lymphocytes: modulation of phosphoinositidase C isoform gamma 1 upon interferon treatment.

In human T lymphocytes immunoblotting analysis of phosphoinositidase C (PIC) isoforms has shown that gamma 1 is the most represented isoform both at the cytoplasmic and nuclear level, and that interferon beta produces, within 90 min of treatment, an increase of its expression. These results, also supported by immunoelectronmicroscopic investigation, strongly suggest the involvement of PIC gamma 1 in the cascade of molecular events generated by the interaction between interferon and its cell surface receptors.

Effect of temperature on muscarinic cholinoceptor-mediated phosphoinositide metabolism and tension generation in bovine tracheal smooth muscle

The effect of decreased temperature on phosphoinositide metabolism was studied in flurbiprofen pretreated bovine tracheal smooth muscle (BTSM) by investigating the consequences of cooling on muscarinic-cholinoceptor-mediated [3H]inositol phosphate ([3H]InsP) and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) accumulation, basal phosphoinositidase C (PIC) activity and airways smooth muscle (ASM) tone. Cooling of [3H]Ins labelled BTSM slices from 37 degrees C to 27 degrees C for 20 min prior to the addition of agonist caused a substantial (73.0 +/- 2.5%) inhibition of carbachol (100 microM, 30 min)-stimulated [3H]InsP accumulation compared to values measured at 37 degrees C. The degree of inhibition of [3H]InsP accumulation was similar at all agonist time points (2-30 min) studied. In parallel experiments, cooling of unlabelled BTSM slices from 37 degrees C to 27 degrees C resulted in a 34% reduction in basal Ins(1,4,5)P3 mass (37 degrees C, 13.1 +/- 0.6 pmol mg-1 protein; 27 degrees C, 8.9 +/- 0.9 pmol mg-1 protein; P < 0.02) and markedly attenuated carbachol (100 microM)-stimulated increases in Ins(1,4,5)P3 accumulation. Basal PIC activity in the soluble fraction of BTSM homogenates, measured using a [3H]phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) /deoxycholate assay system, was also significantly lower at 27 degrees C compared to 37 degrees C (initial velocities of PtdIns(4,5)P2 hydrolysis of 853 +/- 167 (37 degrees C) and 418 +/- 119 (27 degrees C) pmol min-1 ml-1 (1/400 diluted) BTSM cytosol; p < 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)

Guanosine-5'-(3-O-thio)triphosphate-mediated stimulation of phosphoinositidase C in solubilized rat peripheral nerve myelin and its alteration in streptozotocin-induced diabetes.

The regulation of phosphoinositidase C (PIC) activity by guanosine-5'-(3-O-thio)triphosphate (GTP gamma S) was characterized in a cholate-solubilized peripheral myelin-enriched fraction from rat sciatic nerve. The GTP analog maximally enhanced PIC-catalyzed hydrolysis of exogenous phosphatidylinositol-4,5-bisphosphate (PIP2) in a dose-dependent manner only within a narrow range of cholate concentrations. Maximal stimulation was attained at 0.6 microM GTP gamma S and could be completely prevented by 1 microM guanosine-5'-(2-O-thio)diphosphate. Neither adenylyl-imidodiphosphate nor adenosine triphosphate (ATP) enhanced PIC activity. Carbamoylcholine (1 mM) added together with GTP gamma S increased the extent of PIP2 hydrolysis over that elicited by GTP gamma S alone and this stimulation was blocked by the muscarinic receptor antagonist, atropine (50 microM). In detergent-solubilized myelin preparations from streptozotocin-induced diabetic rats, a higher concentration of the guanine nucleotide analog was required to achieve stimulation comparable to that obtained with corresponding preparations from normal animals. These results suggest that sciatic nerve myelin possesses muscarinic receptors coupled via a GTP-binding protein to PIC and that this system can be reconstituted in detergent-solubilized extracts. It is possible that the function of G proteins in cell signaling is impaired in experimental diabetic neuropathy.

Nuclear phosphoinositide signalling enzyme in human B lymphoid cells.

The modulation of phosphoinositidase C (PIC) beta activity upon interferon treatment in Burkitt lymphoma cells (Daudi) and its localization and expression have been analyzed by Western blotting, immunocytochemical and immunoelectronmicroscopy analysis. Results have disclosed an early increase of phosphatidyl-inositol-bisphosphate (PIP2) hydrolysis at nuclear level upon interferon (IFN) treatment paralleled by the evidence of an increase of PIC beta 1 expression. PIC beta 1 expression has been detected in the nuclear compartment also in a clone of Daudi cells selected for the resistance to the antiproliferative action of interferon alpha but no modulation of the enzyme has been detected upon interferon treatment. Since no changes in terms of PIP2 hydrolysis have been found at nuclear level in this selected line, we suggest that the antiproliferative action of interferon on Burkitt lymphoma cells is mediated by a possible recruitment of nuclear PIC beta 1 expression.

Phosphoinositidase C Isoforms Are Specifically Localized in the Nuclear Matrix and Cytoskeleton of Swiss 3T3 Cells

The fine subcellular localization of different phosphoinositidase C (PIC) isoforms has been determined by both electron microscope immunocytochemistry and immunoblotting in whole Swiss 3T3 cells as well as in subcellular fractions. PIC-β, whose signaling activity has been recently demonstrated at the nuclear level (M. A. Martelli, R. S. Gilmour, V. Bertagnolo, L. M. Neri, L. Manzoli, and L. Cocco, Nature, 358, 242-244, 1992), is mainly localized in the interchromatin domains, while it is almost absent from the cytoplasm. PIC-γ is almost absent from the nucleus and resides in the cytosol, while PIC-δ is undetectable in these cells. PIC-β is retained in the inner nuclear matrix and lacks the nuclear pore-lamina complex, whereas PIC-γ is preferentially associated with cytoskeletal filaments. Moreover, PIC-β is present at the same sites of the nuclear matrix where phospholipids and protein kinase C can be identified. This indicates that some elements of the phosphoinositide signal transduction system are located inside the nucleus. Moreover, PIC-γ association with the cytoskeleton filaments suggests a possible involvement of this enzyme in cytoskeleton-mediated changes of cell shape.