Specific and nonspecific effects of nucleotides on hormone-induced phosphoinositide turnover in permeabilized human pituitary tumour cells (Flow 9000)

Previous studies have shown that agonist-induced inositol phosphate formation in the human embryonic pituitary cell line Flow 9000 is regulated by guanine nucleotides, and it is likely that a guanine nucleotide-binding protein is involved in coupling receptors to phosphoinositidase C (PIC) [(1986) Biochem.Soc.Trans. 14, 1135-1136]. We have now tested the specificity of various nucleotides in regulating PIC activity in the absence or presence of the hormone cholecystokinin (CCK-8) in saponin-permeabilized [3H]inositol-labelled Flow 9000 cells. We found that all nucleotides tested (i.e. CTP, UTP, ITP, TTP, GTP, GppNHp, GTP[S], ATP, AppNHp and ATP[S]) stimulated total [3H]inositol phosphate ([3H]IP) formation in a dose-dependent manner with similar potency and efficacy. However, only guanine nucleotides significantly enhanced CCK-8 stimulation of [3H]IP production. These results indicate a physiological role for guanine nucleotides in regulating hormone-induced phosphoinositide turnover. In addition, the effects of nucleotides on calcium-dependent PIC activity are discussed.     

Pertussis toxin does not inhibit muscarinic-receptor-mediated phosphoinositide hydrolysis or calcium mobilization.

Pertussis toxin was used to examine the role of the inhibitory guanine nucleotide regulatory protein, Ni, in muscarinic-receptor-mediated stimulation of phosphoinositide turnover and calcium mobilization. In cultured chick heart cells, pertussis-toxin treatment inhibited muscarinic-receptor-mediated attenuation of isoprenaline-stimulated cyclic AMP accumulation. This finding is consistent with the proposal that pertussis toxin blocks the capacity of Ni to couple muscarinic receptors to adenylate cyclase. In contrast, treatment of chick heart cells or 1321N1 human astrocytoma cells with pertussis toxin did not block muscarinic-receptor-mediated stimulation of phosphoinositide hydrolysis, as measured by [3H]inositol phosphate accumulation in the presence of Li+. Pertussis-toxin treatment also had little effect on basal and muscarinic-receptor-stimulated phosphatidylinositol synthesis, as measured by the incorporation of [3H]inositol into phosphatidylinositol. Activation of muscarinic receptors also enhances the rate of unidirectional 45Ca2+ efflux in 1321N1 cells; this response, like phosphoinositide hydrolysis, was not prevented by pertussis-toxin treatment. Our data suggest that muscarinic receptors are not coupled to phosphoinositide hydrolysis or calcium mobilization through Ni.     

Differential effect of 1,25-dihydroxycholecalciferol on phosphoinositide turnover in the antipodal plasma membranes of colonic epithelial cells.

1,25-dihydroxycholecalciferol stimulates membrane phosphoinositide turnover in colonic epithelial and other cells, but the effects of this hormone on phosphoinositide metabolism in specific antipodal plasma membranes has not been examined. In the present studies, addition of 10(-8)M 1,25-dihydroxycholecalciferol to rat colonic crypts for 90 seconds decreased the phosphatidylinositol-4,5-bisphosphate content and increased the diacylglycerol content of the baso-lateral, but not the brush border plasma membrane. Using Caco-2 cells grown as tight polarized monolayers, 1,25-dihydroxycholecalciferol reduced cellular phosphatidylinositol-4,5-bisphosphate and increased cellular inositol-1,4,5-triphosphate and diacylglycerol when added to the buffer bathing the baso-lateral, but not the brush border membrane surface. These data indicate, therefore, that 1,25-dihydroxycholecalciferol activates the phosphoinositol signal transduction cascade specifically in the baso-lateral cell membrane of colonic cells.     

Differential modulation of carbachol and trans-ACPD-stimulated phosphoinositide turnover following traumatic brain injury

In the fluid percussion model of traumatic brain injury (TBI), we examined muscarinic and metabotropic glutamate receptor-stimulated polyphosphoinositide (PPI) turnover in rat hippocampus. Moderate injury was obtained by displacement and deformation of the brain within the closed cranial cavity using a fluid percussion device. Carbachol and (±)-1-Aminocyclopentane-trans-1,3.-dicarboxylic acid (trans-ACPD)-stimulated PPI hydrolysis was assayed in hippocampus from injured and sham-injured controls at both 1 hour and 15 days following injury. At 1 hour after TBI, the response to carbachol was enhanced in injured rats by up to 200% but the response to trans-ACPD was diminished by as much as 28%. By contrast, at 15 days after TBI, the response to carbachol was enhanced by 25% and the response to trans-ACPD was enhanced by 73%. The ionotropic glutamate agonists N-methyl-D-aspartate (NMDA), and -amino-3 hydroxy-5-methyl-4-isoxazolepropionate (AMPA), did not increase PPI hydrolysis in either sham or injured rats and injury did not alter basal hydrolysis. Thus, hippocampal muscarinic and metabotropic receptors linked to phospholipase C are differentially altered by TBI.Abbreviations used TBI traumatic brain injury - EAA excitatory amino acids - PPI polyphosphoinositides - IP inositol phosphates - NMDA N-methyl-D-aspartate - AMPA -amino-3-hydroxy-5-methylisoxazole-4-propionate - trans-ACPD (±)-1-Aminocyclopentanetrans-1,3-dicarboxylic acid - LTP long term potentiation     

Guanine nucleotide-dependent, pertussis toxin-insensitive regulation of phosphoinositide turnover by bradykinin in bovine pulmonary artery endothelial cells

In this paper we examine the effect of the vasodilator peptide bradykinin on endothelial cell regulation of phosphoinositide (PI) turnover. The data show that the activation of PI turnover by bradykinin in bovine pulmonary artery endothelial cells is insensitive to pertussis toxin, which ADP ribosylates a membrane protein of mol wt 40,000. However, this effect of bradykinin can be potentiated by guanosine 5'-O-(3-thio)triphosphate (GTP gamma S), an activator of G proteins, and depressed by guanosine 5'-O-(2-thio)diphosphate (GDP beta S), an inhibitor of G proteins. After endothelial cells were preincubated for 1 h with GTP gamma S, there was a three- to fourfold increase in PI turnover. Preincubation of cells with GDP beta S did not affect the basal level of PI turnover, but completely prevented activation of PI turnover by bradykinin. 4 beta-Phorbol-12 beta-myristate-13 alpha-acetate can block the bradykinin-stimulated inositol monophosphate formation in cultured endothelial cells. The effects of bradykinin on PI turnover were blocked by B2 antagonists but not by B1 antagonists. Taken together, these results indicate that in endothelial cells the bradykinin B2 receptor is coupled to phospholipase C via a G protein (or proteins) that is not a substrate for pertussis toxin (neither Gi nor Go).     

Stimulatory effects of estrogen on gonadotropin-releasing hormone-induced phosphoinositide turnover in granulosa cells.

Gonadotropin-releasing hormone (Gn-RH) stimulates phosphoinositide metabolism in granulosa cells by binding to its specific receptor, and suppresses gonadotropin-induced steroidogenesis. Incubation of immature rat granulosa cells with Gn-RH stimulated time-sequential [32P]phosphate incorporation into phosphatidic acid (PA) and phosphatidylinositol (PI) in a dose-dependent manner; EC50 was at 10 nM. Concurrent exposure to estradiol-17 beta (E2) (100 nM) and Gn-RH (1 microM) augmented 32P-labeling of PI by 5-fold, while Gn-RH alone induced 3.5-fold increase in PI-labeling. In cells preincubated with E2 for 48 h, Gn-RH provoked a 7-fold [32P]phosphate incorporation into PI, suggesting the induction by E2 of Gn-RH-responsible phosphoinositide turnover. E2 alone provoked a low but significant increase in basal labeling rate of PA and PI. Progesterone failed to mimic the action of E2. Essentially similar results were also obtained in mature rat granulosa cells. These results indicate that E2 augments Gn-RH-stimulated phospholipid turnover in granulosa cells, and suggest that estrogens within the microenvironment of the ovary may exert a local autoregulatory effect on their own production pathway through accelerating Gn-RH action to attenuate steroidogenesis.     

Involvement of phosphoinositide turnover in tracheary element differentiation in Zinnia elegans L. cells

Mesophyll cells of Zinnia elegans L., cultured in the presence of phytohormones, will transdifferentiate and undergo programmed cell death to become tracheary elements, thick-walled cells of the xylem. This system is a model system for study of plant cell development and differentiation. We report that a high concentration of extracellular Ca(2+) is necessary during the first 6 h of culturing for tracheary elements to form. Extracellular Ca(2+) is still required at later times, but at a much lower concentration. When cells transdifferentiate in adequate Ca(2+), microsomal phospholipase C activity increases and levels of inositol 1,4,5-trisphosphate rise at about hour 4 of culturing. The production of inositol 1,4,5-trisphosphate appears to be important for tracheary element formation, since inhibitors of phospholipase C inhibit both inositol 1,4,5-trisphosphate production and tracheary element formation. Pertussis toxin, an inhibitor of GTP-binding proteins, inhibits transdifferentiation and eliminates inositol 1,4,5-trisphosphate production. Tracheary element formation was not completely abolished by inhibitors that eliminated inositol 1,4,5-trisphosphate production, suggesting the involvement of other pathways in regulating transdifferentiation.     

Differential regulation by phosphatidylinositol 4,5-bisphosphate of pituitary plasma-membrane and cytosolic phosphoinositide kinases.

Regulation of phosphatidylinositol kinase (EC 2.7.1.67) and phosphatidylinositol 4-phosphate (PtdIns4P) kinase (EC 2.7.1.68) was investigated in highly enriched plasma-membrane and cytosolic fractions derived from cloned rat pituitary (GH3) cells. In plasma membranes, phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] added exogenously enhanced incorporation of [32P]phosphate from [gamma-32P]MgATP2- into PtdIns(4,5)P2 and PtdIns4P to 150% of control; half-maximal effect occurred with 0.03 mM exogenous PtdIns(4,5)P2. Exogenous PtdIns4P and phosphatidylinositol (PtdIns) had no effect. When plasma membranes prepared from cells prelabelled to isotopic steady state with [3H]inositol were used, there was a MgATP2- dependent increase in the content of [3H]PtdIns(4,5)P2 and [3H]PtdIns4P that was enhanced specifically by exogenous PtdIns(4,5)P2 also. Degradation of 32P- and 3H-labelled PtdIns(4,5)P2 and PtdIns4P within the plasma-membrane fraction was not affected by exogenous PtdIns(4,5)P2. Phosphoinositide kinase activities in the cytosolic fraction were assayed by using exogenous substrates. Phosphoinositide kinase activities in cytosol were inhibited by exogenously added PtdIns(4,5)P2. These findings demonstrate that exogenously added PtdIns(4,5)P2 enhances phosphoinositide kinase activities (and formation of polyphosphoinositides) in plasma membranes, but decreases these kinase activities in cytosol derived from GH3 cells. These data suggest that flux of PtdIns to PtdIns4P to PtdIns(4,5)P2 in the plasma membrane cannot be increased simply by release of membrane-associated phosphoinositide kinases from product inhibition as PtdIns(4,5)P2 is hydrolysed.     

Agents that stimulate phosphoinositide turnover also elevate cAMP in SK-N-SH human neuroblastoma cells.

Stimulation of m1 and of m3 muscarinic receptors has previously been shown to increase intracellular cAMP levels in a variety of cells. Although the mechanism underlying this response is not fully understood, it has been hypothesized to be secondary to the IP3-mediated rise in intracellular calcium. In order to determine whether other means of elevating intracellular calcium also raise cAMP levels, we stimulated SK-N-SH human neuroblastoma cells with bradykinin or with maitotoxin. Both of these agents stimulated phospholipase C, stimulated inositol phosphate release and elevated cAMP levels, thus demonstrating that this cAMP response is not unique to muscarinic receptor stimulation.     

Phospholipid turnover during cell-cycle traverse in synchronous Chinese-hamster ovary cells. Mitogenesis without phosphoinositide breakdown.

The turnover of phospholipids was investigated in quiescent serum-starved Chinese-hamster ovary (CHO-K1) cells stimulated to progress through the cell cycle by the addition of dialysed bovine serum. A variety of radiolabelling techniques were employed to study the rapid effects of serum on phospholipids and later events during G1 and S phases of the cell cycle. Pulse-labelling studies using [32P]Pi revealed that there was a stimulation of the synthesis rate of all phospholipids investigated during the initial few hours after serum addition. The greatest stimulation (20-fold) was observed in phosphatidylcholine, and the smallest in the polyphosphoinositides (PPIs). Mock stimulation with serum-free medium caused a similar increase in PPI turnover, but little or no effect on turnover of other phospholipids. This effect could be accounted for by a stimulation of the turnover of cellular ATP pools increasing [32P]ATP specific radioactivity. Late G1 and S phases were associated with a decrease in the rate of synthesis of all phospholipids. Phosphatidic acid was the only phospholipid whose labelling fell below that in mock-stimulated cells during the period of the cell cycle. Stimulation of serum-starved cells that had been prelabelled with myo-[2-3H]inositol caused no change in the amounts of inositol trisphosphate, but both serum-stimulated and mock-stimulated cells exhibited similar small decreases in both inositol bisphosphate and inositol monophosphate, of approx. 30% after 30 s. When cells were serum-stimulated in the presence of 10 mM-Li+, there was no increase in the size of the total inositol phosphate pool. We conclude that mitogenic stimulation and cell-cycle traverse cause profound and complex effects on phospholipid turnover in CHO-K1 cells, but there is no evidence for a role of inositol lipid turnover in the proliferative response to serum in this cell line.     

Platelet phosphoinositide turnover in streptozotocin-induced diabetes

Increased platelet aggregation and secretion in response to various agonists has been described in both diabetic humans and animals. Alterations in the platelet membrane fatty acid composition of phospholipids and changes in the prostacyclin and thromboxane formation could only partly explain the altered platelet function in diabetes. In the present study, we have examined the role of phosphoinositide turnover in the diabetic platelet function. We report alterations in 2-[³H] myo-inositol uptake, phosphoinositide turnover, inositol phosphate and diacylglycerol (DAG) formation, phosphoinositide mass, and phospholipase C activity in platelets obtained from streptozotocin (STZ)-induced diabetic rats. There was a significant increase in the 2-[³H) myo-inositol uptake in washed platelets from diabetic rats. Basal incorporation of 2-[³H] myo-inositol into phosphatidylinositol 4,5-bisphosphate (PIP₂), phosphatidylinositol 4-phosphate (PIP) or phosphatidylinositol (PI) in platelets obtained from diabetic rats was, however, not affected. Thrombin stimulation of platelets from diabetic rats induced an increase in the hydrolysis of [³²P]PIP₂ but indicated no change in the hydrolysis of [³²P]PIP and [³²P]PI as compared to their basal levels. Thrombin-induced formation of [³H]inositol phosphates was significantly increased in both diabetic as well as in control platelets as compared to their basal levels. This formation of [³H]inositol phosphates in diabetic platelets was greater than controls at all time intervals studied. Similarly, there was an increase in the release of DAG after thrombin stimulation in the diabetic platelets. Based on these results, we conclude that there is an increase in the transport of myoinositol across the diabetic platelet membrane and this feature, along with alterations in the hydrolysis of PIP₂, inositol phosphates and DAG in the diabetic platelets, may play a role in increased phosphoinositide turnover which could explain the altered platelet function in STZ-induced diabetes.     

Inhibition of phosphoinositide turnover by praziquantel in Schistosoma mansoni.

The effect of praziquantel on phosphoinositide turnover was examined in Schistosoma mansoni to determine if this anthelminthic modulates signal transduction pathways in parasites. Adult worms were radiolabeled with [3H]myoinositol for 24 hr and total inositol phosphate levels determined in the presence of praziquantel. Praziquantel inhibited inositol phosphate turnover when activated with NaF plus AlCl3 or with the nonhydrolyzable guanine nucleotide-binding protein analogue GTP gamma S. Furthermore, praziquantel decreased basal turnover of inositol phosphates. Inhibition was seen in both male and female worms as well as in schistosomula. These data indicate that inhibition of phosphoinositide turnover may contribute to the effect of praziquantel on parasite survival within the definitive host.     

Differential regulation of the phosphoinositide 3-kinase and MAP kinase pathways by hepatocyte growth factor vs. insulin-like growth factor-I in myogenic cells

Hepatocyte growth factor (HGF) promotes the proliferation of adult myoblasts and inhibits their differentiation, whereas insulin-like growth factor I (IGF-I) enhances both processes. Recent studies indicate that activation of the phosphoinositide 3'-kinase (PI3K) pathway promotes myoblast differentiation, whereas activation of the mitogen-activated protein kinase/extracellular signal-regulated protein kinase (MAPK/ERK) promotes proliferation and inhibits their differentiation. This simple model is confounded by the fact that both HGF and IGF-I have been shown to activate both pathways. In this study, we have compared the ability of HGF and IGF-I to activate PI3K and MAPK/ERK in i28 myogenic cells. We find that, although the two stimuli result in comparable recruitment of the p85alpha subunit of PI3K into complexes with tyrosine-phosphorylated proteins, the p85beta regulatory subunit and p110alpha catalytic subunit of PI3K are preferentially recruited into these complexes in response to IGF-I. In agreement with this observation, IGF-I is much more potent than HGF in stimulating phosphorylation of Akt/PKB, a protein kinase downstream of PI3K. In contrast, MAPK/ERK phosphorylation was higher in response to HGF and lasted longer, relative to IGF-I. Moreover, the specific PI3K inhibitor, Wortmannin, abolished MAPK/ERK and Elk-1 phosphorylation in HGF-treated cells, suggesting the requirement of PI3K in mediating the HGF-induced MAPK pathway. UO126, a specific MAPK pathway inhibitor, had no effect on PI3K activity or Akt phosphorylation, implying that at least in muscle cells, the MAPK/ERK pathway is not required for HGF-induced PI3K activation. These results provide a biochemical rationale for the previous observations that HGF and IGF-I have opposite effects on myogenic cells, consistent with studies linking PI3K activation to differentiation and MAPK/ERK activation to proliferation in these cells. Moreover, the finding that PI3K activity is required for HGF-induced MAPK activation suggests its additional role in proliferation, rather than exclusively in the differentiation of adult myoblasts.     

Kinetic analysis of receptor-activated phosphoinositide turnover

We studied the bradykinin-induced changes in phosphoinositide composition of N1E-115 neuroblastoma cells using a combination of biochemistry, microscope imaging, and mathematical modeling. Phosphatidylinositol-4,5-bisphosphate (PIP2) decreased over the first 30 s, and then recovered over the following 2-3 min. However, the rate and amount of inositol-1,4,5-trisphosphate (InsP3) production were much greater than the rate or amount of PIP2 decline. A mathematical model of phosphoinositide turnover based on this data predicted that PIP2 synthesis is also stimulated by bradykinin, causing an early transient increase in its concentration. This was subsequently confirmed experimentally. Then, we used single-cell microscopy to further examine phosphoinositide turnover by following the translocation of the pleckstrin homology domain of PLCdelta1 fused to green fluorescent protein (PH-GFP). The observed time course could be simulated by incorporating binding of PIP2 and InsP3 to PH-GFP into the model that had been used to analyze the biochemistry. Furthermore, this analysis could help to resolve a controversy over whether the translocation of PH-GFP from membrane to cytosol is due to a decrease in PIP2 on the membrane or an increase in InsP3 in cytosol; by computationally clamping the concentrations of each of these compounds, the model shows how both contribute to the dynamics of probe translocation.     

Calcium affects phosphoinositide turnover in human erythrocytes

Changes in extracellular Ca2+ concentration ([Ca2+]) were observed to affect 32Pi incorporation into polyphosphoinositides (PPI) and phosphatidic acid (PA) of human erythrocytes. A decrease of extracellular [Ca2+] from 1.5 mmol/l to 0.04 mumol/l increased the specific radioactivity (S.A.) of phosphatidylinositol 4,5-bisphosphate to 182% and that of phosphatidylinositol 4-phosphate to 120% of controls. Simultaneously S.A. and concentration of PA decreased. Further decrease of the extracellular [Ca2+] from 0.04 mumol/l to lower values as well as depletion of intracellular Ca2+ using ionophore A 23187 in Ca2(+)-free medium did not accelerate the PPI turnover rates any more. None of the above changes in extracellular [Ca2+] had any effect on the phosphorylation pattern of erythrocyte membrane proteins. Isolated erythrocyte membranes were incubated in the presence of [gamma-32P]ATP in media with various [Ca2+]. The decrease of [Ca2+] from 0.04 mumol/l (physiological concentration inside the cell) to lower values did not influence the turnover of PPI and PA monoester phosphates. Only after [Ca2+] was increased to 1-5 mumol/l an increase of PPI and PA turnover was observed. Our data suggest that the changes in extracellular [Ca2+] affect the metabolism of PPI and PA (despite the intracellular location of the latter) and may thus influence the properties of red cell plasma membrane.     

Fish oil affects phosphoinositide turnover and thromboxane A metabolism in cultured vascular muscle cells

Fish oil has been reported as having beneficial effects on cardiovascular diseases. Elevated serum lipoproteins, prostaglandins and intracellular free calcium concentrations [( Ca2+]i) of the vasculature and thus the phosphoinositide (PI) turnover may be involved in the pathogenesis of these disorders. Therefore, the effect of fish oil on the potency of both low-density lipoprotein (LDL) and angiotensin II (AII) to stimulate the PI turnover in cultured rat vascular smooth muscle cells (VSMC) has been studied. Furthermore, a possible link between PI turnover activity and thromboxane A2 (TXA2) metabolism in these cells has been investigated. In VSMC cultured for up to 7 weeks with either fish oil or n-3 eicosapentaenoic acid (EPA) a decrease to 5-48% of the LDL-induced inositol trisphosphate (IP3) formation (= 100%) was found. A similar range of decreased IP3 synthesis was observed, when AII was used instead of LDL. Both LDL- and AII-stimulated TXA2 synthesis was suppressed concomitantly within the range 34-60%. Blockade of VSMC TXA2 biosynthesis with either indomethacin or TXA2 synthetase blocker (SQ-80338) inhibited LDL-induced formation of IP3 in a dose-dependent manner. Similar results were obtained, when TXA2 receptor coupling antagonists (SQ-27427 or BM-13177) were used. However, blockers of TXA2 synthesis and of TXA2 receptor binding failed to affect AII-induced formation of IP3.     

Involvement of protein kinase A in the regulation of intracellular free calcium and phosphoinositide turnover in rat myometrium

Preincubation of Fura 2-loaded rat myometrial cells with H-8, an inhibitor of protein kinase A, for 1 h reversed the inhibitory effects of 8-(4-chlorophenylthio)-cAMP (CPTcAMP) on the oxytocin-stimulated increase in (Ca2+)i (intracellular free calcium), with an EC50 of 47 microM. H-8 also prevented the inhibition by relaxin and isoproterenol of the oxytocin-induced increase in (Ca2+)i. The EC50 of H-8 in reversing the relaxin effect was 42 microM. H-8 reversal of the effect of relaxin on (Ca2+)i was evident both in the absence of extracellular calcium and in cells pretreated with pertussis toxin. H-8 also reversed the inhibitory effects of relaxin and CPTcAMP on the oxytocin-induced increase in [3H]inositol phosphate formation and [3H]phosphoinositide hydrolysis. Preincubation of myometrial cells for 1 h with H-7, another protein kinase inhibitor, only partially attenuated the inhibition by relaxin and CPTcAMP of the oxytocin-induced increase in (Ca2+)i and [3H]inositol phosphate formation at concentrations 4-5 times greater than those of H-8. Acute (15-min) exposure to phorbol myristate acetate (1.0 microM) did not affect basal (Ca2+)i or the oxytocin-stimulated increases in (Ca2+)i or inositol phosphate formation. These results imply a regulatory role for protein kinase A in the inhibition of the oxytocin-induced increase in (Ca2+)i and inositol phosphate formation by relaxants.     

Differential regulation of lipid and protein kinase activities of phosphoinositide 3-kinase gamma in vitro

G protein sensitive phosphoinositide 3-kinase gamma (PI3Kgamma) has been characterised as a pleiotropic signalling protein expressing lipid kinase and protein kinase activities. Whereas the regulation of the lipid kinase activity has been investigated in detail, the regulatory features of PI3Kgamma protein kinase activity are unknown. Here we report that Gbetagamma subunits of heterotrimeric G proteins induce a biphasic response of PI3Kgamma autophosphorylation in vitro, which contrasts the regulatory effects of the G proteins on PI3Kgamma lipid kinase activity. In addition to autophosphorylation PI3Kgamma is able to catalyse transphosphorylation of the adapter protein p101 and the protein kinase MEK-1. In the presence of the p101, Gbetagamma affects PI3Kgamma protein kinase activities in a complex manner. In summary, the differential regulatory effects of heterotrimeric G proteins on PI3Kgamma lipid and protein kinase activities in vitro reflect the functional diversity of the enzyme observed in vivo.     

Effects of hypoxia on phosphoinositide turnover and adenylate cyclase system in cultured endothelial cells]

By studying the effects of oxygen deficiency upon signal-transducing system it has been shown that in long hypobaric hypoxia activates PI-turnover in cultured human endothelial cells. The sensitivity of cells to histamine was decreased as well as the adenylate cyclase activity in membranes of this cells. The amount of beta-adrenoreceptors was not influenced significantly. Incubation of endothelial cells with histamine (10(-5) M) and phorbol ester (10(-9) M) = activator of protein kinase C within 1-2 h resulted in desensitization of cellular responses which can be seen not only as a disappearance of histamine-induced activation of PI-turnover but also as a decrease of beta-adrenoreceptor amount and adenylate cyclase activity. It seems that hypoxia may change the action of Ca-mobilizing hormones on PI-turnover and suppress adenylate cyclase in human endothelial cells. However the effects of hypoxia on signal-transducing systems in this cells are developed slower than those of Ca-mobilizing hormones.     

Odorant- and guanine nucleotide-stimulated phosphoinositide turnover in olfactory cilia

Isolated olfactory cilia from the channel catfish (Ictalurus punctatus) exhibited phosphatidylinositol-4,5-bisphosphate phosphodiesterase (E.C.3.1.4.11) activity. The phosphodiesterase activity was stimulated in the presence of an odorant for the catfish, namely the amino acid L-alanine. The enzyme activity was also stimulated in the presence of GTP and its nonhydrolyzable analogues. The activation of the phosphodiesterase by guanine nucleotides, in combination with the identification of guanine nucleotide-binding protein(s) in the isolated cilia, indicate the probable participation of a guanine nucleotide-binding protein in stimulation of phosphoinositide turnover in the olfactory receptor neuron.