Activation Of Phosphoinositide Turnover Research Articles

Differential regulation on human skin fibroblast by α 1 adrenergic receptor subtypes

Alpha 1 adrenoceptor (α 1-AR) regulation of DNA synthesis was studied in human neonatal foreskin fibroblast. Saturation assay with a specific radioligand for α 1 adrenergic [ 3H]-prazosin revealed two saturated and specific binding sites with high or low affinity. Competitive binding assay with different antagonist subtypes, defined pharmacologically three major types of α 1-AR. The α 1-AR agonists (from 1 × 10 −10 to 1 × 10 −4 M) triggered a biphasic action on DNA synthesis reaching maximal stimulation at 1 × 10 −9 M and maximal inhibition at 1 × 10 −6 M. Prazosin, abolished the stimulatory (pA 2: 9.24) and inhibitory (pA 2: 8.80) actions of α 1-AR agonists. The α 1-AR stimulation resulted in the activation of phosphoinositide turnover (InsP) via phospholipase C (PLC) involving calcium/calmodulin (CaM) and nitric oxide synthase (NOS) that correlates with the DNA synthesis increment; whereas the inhibition resulted in a decrease of cyclic AMP (cAMP) accumulation via adenylate cyclase inhibition. The potency displayed by the specific antagonists tested in binding, DNA synthesis, InsP and NOS at low agonist concentration suggests that they can be elicited by the activation of the same receptor (α 1B-AR subtype); while the decrement in DNA synthesis and cAMP at high concentration account by the activation of α 1D-AR coupled to G i protein. Non-functional α 1A-AR in neonatal human foreskin fibroblast was observed. Results suggest that the expression of α 1-AR subtypes on human skin fibroblast may differentially activate signaling pathways that modulate physiological response of the cells.

Mechanisms involved in the regulation of mRNA for M2 muscarinic acetylcholine receptors and endothelial and neuronal NO synthases in rat atria

Agonists of the M(2) muscarinic acetylcholine receptor (mAChR) increase mRNA for this receptor and mRNA for endothelial and neuronal isoforms of NO synthase (eNOS or nNOS). Here we examine the different signalling pathways involved in such events in rat cardiac atria. In isolated atria, the effects of carbachol on mRNA for M(2) receptors, eNOS and nNOS were measured along with changes in phosphoinositide (PI) turnover, translocation of protein kinase C (PKC), NOS activity and atrial contractility. Carbachol increased mRNA for M(2) receptors, activation of PI turnover, translocation of PKC and NOS activity and decreased atrial contractility. Inhibitors of phospholipase C (PLC), calcium/calmodulin (CaM), NOS and PKC prevented the carbachol-dependent increase in mRNA for M(2) receptors. These inhibitors also attenuated the carbachol induced increase in nNOS- and eNOS-mRNA levels. Inhibition of nNOS shifted the dose response curve of carbachol on contractility to the right, whereas inhibition of eNOS shifted it to the left. From our results, activation of M(2) receptors induced nNOS and eNOS expression and activation of NOS up-regulated M(2) receptor gene expression. The signalling pathways involved included stimulation of PI turnover via PLC activation, CaM and PKC. nNOS and eNOS mediated opposing effects on the negative inotropic effect in atria, induced by stimulation of M(2) receptors. These results may contribute to a better understanding of the effects and side effects of cholinomimetic treatment in patients with cardiac neuromyopathy.

Imaging of phosphoinositide turnover activity in post-myocardial infarction: Effect of angiotensin converting enzyme inhibition

Imaging of phosphoinositide turnover activity in post-myocardial infarction: Effect of angiotensin converting enzyme inhibition

Endogenous nitric oxide signalling system and the cardiac muscarinic acetylcholine receptor‐inotropic response

1. In this paper we have determined the different signalling pathways involved in muscarinic acetylcholine receptor (AChR)-dependent inhibition of contractility in rat isolated atria. 2. Carbachol stimulation of M2 muscarinic AChRs exerts a negative inotropic response, activation of phosphoinositide turnover, stimulation of nitric oxide synthase and increased production of cyclic GMP. 3. Inhibitors of phospholipase C, protein kinase C, calcium/calmodulin, nitric oxide synthase and guanylate cyclase, shifted the dose-response curve of carbachol on contractility to the right. These inhibitors also attenuated the muscarinic receptor-dependent increase in cyclic GMP and activation of nitric oxide synthase. In addition, sodium nitroprusside, isosorbide, or 8-bromo cyclic GMP, induced a negative inotropic effect, increased cyclic GMP and activated nitric oxide synthase. 4. These results suggest that carbachol activation of M2 AChRs, exerts a negative inotropic effect associated with increased production of nitric oxide and cyclic GMP. The mechanism appears to occur secondarily to stimulation of phosphoinositides turnover via phospholipase C activation. This in turn, triggers cascade reactions involving calcium/calmodulin and protein kinase C, leading to activation of nitric oxide synthase and soluble guanylate cyclase.

Some mechanical properties of skinned fibres of pregnant human myometrium

The properties of contractile elements and intracellular Ca 2+ storage sites of pregnant human myometrium were studied by recording the mechanical responses in skinned (saponin-treated and membrane-permeable) fibres. Calmodulin increased the amplitude of contractions induced by Ca 2+ and the Ca 2+ sensitivity for contractile elements in small myometrium strips, but PGF 2α, PGE 2, oxytocin, or cyclic AMP failed to produce similar effects. After accumulation of Ca 2+ in intracellular Ca 2+ storage sites, 10 μmol/l PGF 2α, 10 μmol/l PGE 2, 30 mmol/l caffeine, and 20 μmol/l InsP3 (inositol-trisphosphate) produced contractions by releasing Ca 2+ from storage sites. However, 20 nmol/l oxytocin had no effects under the same conditions. The InsP3 sensitive Ca 2+ store was much larger than those of PGs or caffeine. These results suggest that pregnant human myometrium contracts with low Ca 2+ by a calmodulin sensitive system. The data also indicate that direct application of PGF 2α or PGE 2 into the cells discharges Ca 2+ from Ca 2+ storage sites and that oxytocin extricates Ca 2+ via a pathway involving InsP3 by activation of phosphoinositide turnover. We suggest that these agents induce added contractile responses due to a Ca 2+ release mechanism from store sites in addition to the influx of Ca 2+ from the extracellular space.

Assessment of Neuronal Phosphoinositide Turnover and Its Disruption by Lithium

The ability of lithium to interfere with signal transduction pathways that involve neurotransmitter receptor activation of phosphoinositide turnover has been proposed as a potential mechanistic explanation of the therapeutic actions of lithium in manic-depressive illness. Noncompetitive inhibition of inositol monophosphatase by submillimolar concentrations of lithium deprives active neurons of endogenously generated myo-inositol. If this deficit cannot be compensated for by uptake of extracellular myo-inositol, then the ability of the cell to synthesize and maintain inositol phospholipid pools will be compromised. Here we describe methods for the investigation of the phosphoinositide cycle, with particular emphasis on methods that have been used to highlight the complex actions of lithium to disrupt activation of this important signal transduction pathway by neurotransmitters.

Concerted effects of lipoproteins and angiotensin II on signal transduction processes in vascular smooth muscle cells.

Low-density (LDL) and high-density (HDL3) lipoproteins dose-dependently activate phosphoinositide turnover and elevate cytosolic free Ca2+ concentrations ([Ca2+]i) in cultured vascular smooth muscle cells (VSMCs) from either human (microarterioles and aorta) or rat (aorta) sources. High-performance liquid chromatography analysis of cell extracts revealed comparable spectra of inositol phosphate isomers generated in response to either LDL, HDL3, or angiotensin II (Ang II). Thus, lipoproteins and Ang II may use similar, if not identical, signal transduction pathways for the generation and metabolism of inositol phosphates and intracellular Ca2+ mobilization in VSMCs. When Ang II was added in combination with either LDL or HDL3, the phosphoinositide and [Ca2+]i responses of VSMCs were either equal to or even greater than the sum of the effects elicited by the agonists individually. This additivity/synergy between Ang II and the lipoproteins was not accompanied by alteration in the half-maximally effective dose requirements of VSMCs for either Ang II (approximately 2 nmol/L, with or without lipoproteins) or lipoproteins (approximately 50 micrograms/mL for LDL and HDL3, with or without Ang II). Neither short-term (up to 10 minutes) nor long-term (48 hours) exposure of VSMCs to lipoproteins caused desensitization of phospholipase C and intracellular Ca2+ mobilization responses to either Ang II or lipoproteins. Since constant exposure of VSMCs to lipoproteins is a physiological circumstance, and because elevation of [Ca2+]i and activation of phosphoinositide turnover are pivotal events for VSMC contraction and growth, we suggest that the low concentrations of lipoproteins in the vessel intima may play an important role in regulating the response of the vasculature to Ang II.

Paradoxical signal transduction mechanism of endothelins and sarafotoxins in cultured pituitary cells: Stimulation of phosphoinositide turnover and inhibition of prolactin release

Endothelins (ET-1, ET-2, ET-3 and vasoactive intestinal contractor, VIC) and sarafotoxins (SRTX-b and SRTX-c) appear to bind with high affinity to a homogeneous class of binding sites in cultured rat pituitary cells. All of these ligands seem to interact with the same receptor (ET A-R), except for SRTX-c which apparently binds to a separate receptor. Binding was followed by phosphodiesteric cleavage of phosphoinositides, resulting in the formation of inositol phosphates. No consistent effect on basal or gonadotropin-releasing hormone (GnRH)-induced release of luteinizing hormone (LH) was exerted by ET or SRTX during 2 h of static incubation. On the other hand, both groups of vasoactive peptides inhibited basal and thyrotropin-releasing hormone (TRH)-induced prolactin secretion. Surprisingly, activation of phosphoinositide turnover by TRH in pituitary mammotrophs led to stimulation of prolactin secretion, whereas activation of the same pathway by ET or SRTX resulted in inhibition of prolactin secretion. ET and SRTX stimulated inositol phosphate formation in GH3 cell line and in the gonadotroph-like cell line αT-3 (which is capable of producing the α subunit of the gonadotrophins), indicating that the peptides interact with both pituitary mammotrophs and gonadotrophs. The very low concentrations (nM range) needed to stimulate phosphoinositide turnover and to inhibit prolactin secretion, as well as the recent finding that ETs are present in the hypothalamo-pituitary axis suggest that ET might participate in the neuroendocrine modulation of pituitary functions. One such possibility is that ETs might be members of the prolactin inhibiting factors (PIFs) family.

Activation of phosphoinositide turnover and protein kinase C by neurotransmitters that modulate calcium channels in embryonic chick sensory neurons

Gamma aminobutyric acid (GABA) and norepinephrine modulate the excitability of primary chick sensory neurons by decreasing the voltage dependent Ca current. Although previous electro-physiological studies indicate that neurotransmitter modulation of the Ca current in these neurons involves protein kinase C, the biochemical aspects of this mechanism have not been examined directly. We find that both norepinephrine (via a unique a receptor subtype) and GABA (via GABAb receptors) linked to pertussis toxin sensitive pathways, stimulate the metabolism of membrane phosphatidylinositol phospholipids in primary chick sensory neurons. In addition, norepinephrine causes the rapid translocation of C kinase activity from cytosolic to membrane associated distribution, consistent with its rapid activation in response to applied neurotransmitter. The pharmacology, pertussis toxin sensitivity and time course of the biochemical changes due to neurotransmitter treatment parallel the effects of these transmitters on calcium current modulation. These biochemical studies confirm the hypothesis that activation of protein kinase C is critically involved in calcium channel modulation in embryonic chick sensory neurons.

Prolactin as an immunoregulatory hormone in mammals and birds

The immunoregulatory function of prolactin (PRL) and the mechanism of its action in mammals seem to be well documented. Reciprocal interdependence between PRL secretion and immune system function is essential for normal ontogeny, development and aging. PRL receptors in lymphocytes participate in the transduction of its regulatory signal into the intracellular enzymatic machinery including that of the nucleus, leading to the expression of some genes and to the synthesis of new proteins. Activation of phosphoinositide turnover and subsequent increase in protein kinase-C activity seems to be a possible mechanism acting in the regulatory influence of PRL on mammalian immune cells. These cells in turn, under mitogen or antigen stimulation, secrete a substance with PRL-like activity. The regulatory function of PRL within the avian immune system is less well known, but it seems to have some features in common with those in mammals. Direct mitogenic action on thymocytes and splenocytes in the chicken might indicate the existence of PRL receptors in these cells and could explain the immunostimulatory effect of PRL observed in vivo, which is dependent on the time of hormone administration. As the avian PRL stimulates mitogenesis of rat Nb2 lymphoma cells, the mechanism of direct PRL action on immune cells in mammals and birds seems to be similar. PRL in chickens also modifies the level and the diurnal rhythm of corticosterone which, in turn, influences the immunoregulatory effect exerted by PRL. Thus, PRL seems to be an important factor, influencing directly or indirectly the avian immune system.

Coordinate, biphasic activation of p44 mitogen-activated protein kinase and S6 kinase by growth factors in hamster fibroblasts. Evidence for thrombin-induced signals different from phosphoinositide turnover and adenylylcyclase inhibition.

In CCL39 cells transfected with m1-muscarinic receptors, carbachol stimulates phosphoinositide turnover and early events associated with mitogenesis as efficiently as thrombin but, in contrast to thrombin, fails to induce cell proliferation (Seuwen, K., Kahan, C., Hartmann, T., and Pouysségur, J. (1990) J. Biol. Chem. 265, 22292-22299). We show here that the action of the two agents can be dissociated at the level of S6 kinase and mitogen-activated protein kinase (MAP kinase) activation. Mitogenic concentrations of thrombin and basic FGF were found to stimulate S6 kinase activity, measured in whole cell lysates, with a biphasic time course; an early peak of activity is induced 10 min following stimulation and a sustained phase of activity can be measured over several hours. A very similar profile emerged for p44 MAP kinase (p44mapk), assayed in immunoprecipitates. In this case, the activity first peaks at 6-8 min, preceding S6 kinase. In contrast to thrombin and FGF, carbachol stimulates S6 kinase and MAP kinase only transiently, corresponding to the first peak of activity, but the sustained phase is not observed. Similarly, phorbol dibutyrate induces an early phase of activity only. Pertussis toxin (PTX), which is known to block thrombin mitogenicity efficiently, inhibited the first peak of thrombin-induced S6 kinase and MAP kinase activity only partially, but totally blocked the sustained phase. The toxin had no effect on FGF-induced kinase activities. The cAMP elevating hormone PGE1 did not inhibit p44mapk or S6 kinase activation by thrombin or FGF, demonstrating that the PTX-sensitive signal generated by thrombin does not depend on a Gi-mediated sustained inhibition of adenylylcyclase. Surprisingly, PGE1 was found to stimulate sustained phase S6 kinase activity both alone and in synergy with FGF or thrombin. This result, as well as the biphasic activation of S6 kinase by thrombin, could be qualitatively reproduced in immunocomplex kinase assays using an antiserum immunoprecipitating p70 S6 kinase (p70S6k). Our data show that activation of phosphoinositide turnover and PKC does not quantitatively explain thrombin action, in particular the sustained phase of kinase activities, which critically depends on a PTX-sensitive signal different from adenylylcyclase inhibition. We postulate that this signal does not exclusively originate from the recently identified G protein-coupled thrombin receptor.

Regulation of ATP-dependent surfactant secretion and activation of second-messenger systems in alveolar type II cells

Several different classes of agonists are known to stimulate exocytosis in type II cells. These agonists cause increases in second messengers, such as adenosine 3',5'-cyclic monophosphate (cAMP) or cytosolic Ca2+, and/or stimulate protein kinase C. We studied generation of cAMP and phosphoinositide (PI) turnover in monolayer cultures of type II cells and measured [Ca2+]i in single cultured cells. ATP [10-4 M], which stimulates secretion of phosphatidylcholine (PC) and increases cellular cAMP, also stimulated PI turnover and increased [Ca2+]i. 12-O-tetradecanoylphorbol-13-acetate (TPA), which stimulates PC secretion and activates protein kinase C, did not increase [Ca2+]i. Pretreatment of type II cells with the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibited the PC secretion induced by ATP and TPA and blocked the increase in PI turnover caused by ATP. ATP-dependent surfactant secretion and stimulation of PI turnover could also be inhibited by pretreatment of the cells with pertussis toxin. We used the fluorescent probe indo-1 to measure [Ca2+]i in single cultured type II cells. ATP produced rapid transient increases in [Ca2+]i, which could be prevented by pretreatment of the cells with either TPA or W-7. Our data suggest that pertussis toxin-sensitive G protein(s) are involved in ATP-dependent activation of PI turnover and in secretion of surfactant in type II cells. Activation of protein kinase C blocks the ATP-stimulated increase in [Ca2+]i. Finally, calmodulin may be involved in the regulation of ATP-dependent increase in [Ca2+]i, the activation of PI turnover, and the secretion of surfactant in type II cells. lung; exocytosis; cell calcium; G proteins; phosphoinositide turnover

Regulation of ATP-dependent surfactant secretion and activation of second-messenger systems in alveolar type II cells

Several different classes of agonists are known to stimulate exocytosis in type II cells. These agonists cause increases in second messengers, such as adenosine 3',5'-cyclic monophosphate (cAMP) or cytosolic Ca2+, and/or stimulate protein kinase C. We studied generation of cAMP and phosphoinositide (PI) turnover in monolayer cultures of type II cells and measured [Ca2+]i in single cultured cells. ATP [10(-4) M], which stimulates secretion of phosphatidylcholine (PC) and increases cellular cAMP, also stimulated PI turnover and increased [Ca2+]i. 12-O-tetradecanoylphorbol-13-acetate (TPA), which stimulates PC secretion and activates protein kinase C, did not increase [Ca2+]i. Pretreatment of type II cells with the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibited the PC secretion induced by ATP and TPA and blocked the increase in PI turnover caused by ATP. ATP-dependent surfactant secretion and stimulation of PI turnover could also be inhibited by pretreatment of the cells with pertussis toxin. We used the fluorescent probe indo-1 to measure [Ca2+]i in single cultured type II cells. ATP produced rapid transient increases in [Ca2+]i, which could be prevented by pretreatment of the cells with either TPA or W-7. Our data suggest that pertussis toxin-sensitive G protein(s) are involved in ATP-dependent activation of PI turnover and in secretion of surfactant in type II cells. Activation of protein kinase C blocks the ATP-stimulated increase in [Ca2+]i. Finally, calmodulin may be involved in the regulation of ATP-dependent increase in [Ca2+]i, the activation of PI turnover, and the secretion of surfactant in type II cells.

Bradykinin Induces Phosphoinositide Turnover, 1,2-Diglyceride Formation, and Growth in Cultured Adult Human Keratinocytes

The effects of bradykinin on activation of phosphoinositide turnover, 1,2-diglyceride formation, and growth of cultured adult human keratinocytes were investigated. Keratinocytes specifically bound [3H]bradykinin with high affinity (kd = 3.4 nM) and displayed 1.5 X 10(5) binding sites/cell. Bradykinin caused a rapid dose-dependent increase in inositol trisphosphate (IP3) inositol bisphosphate, and inositol monophosphate. IP3 was maximally increased (fivefold) at 30 s and remained elevated for at least 10 min. Half maximal stimulation of IP3 formation was observed at 27 nM bradykinin. IP3 accumulation was equally elevated by bradykinin and lys-bradykinin but was not stimulated by des-Arg9-bradykinin, indicating that phospholipase C in cultured keratinocytes is coupled to B2 bradykinin receptors. Treatment of keratinocytes with active phorbol ester (TPA) caused a significant inhibition (50%) of bradykinin-induced IP3 accumulation, suggesting negative regulation of phospholipase C by protein kinase C. Bradykinin also caused a significant elevation in 1,2-diacylglycerol (DAG) content. DAG content was maximally elevated (twofold) at 1 min and remained elevated for at least 10 min. Bradykinin also caused a significant (twofold, p less than 0.02) increase in keratinocyte growth. These data demonstrate that bradykinin is a potent agonist of the phospholipase C/protein kinase C signal transduction system in cultured adult human keratinocytes and that activation of this pathway by bradykinin is associated with increased keratinocyte growth.

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.

Excitatory amino acids: The involvement of second messengers in the signal transduction process

1. Excitatory amino acids (EAA) can activate second messenger systems in addition to a direct gating of ion channels. A discrete coupling between novel EAA receptor subtypes and second messenger systems has been previously proposed. 2. EAAs have been suggested to activate both adenylate and guanylate cyclases and also to induce phosphoinositide (PI) turnover. The increased PI turnover was observed in both central neurons and glia, and a "quisqualate-type" receptor has been most frequently involved, which may differ from the quisqualate receptor previously defined by electrophysiological studies. 3. The roles of EAA-induced calcium influx into neurons and raised intracellular calcium levels are discussed regarding the activation of phosphoinositide turnover. 4. This review examines the data supporting a link between EAA receptors and second messengers and considers whether there is any need for adopting new EAA receptor subtypes. Also, the use of the Xenopus laevis oocyte for expressing EAA receptors and studying any putative links to second messenger systems is discussed.

Signal transduction in hamster fibroblasts overexpressing the human EGF receptor.

Human EGF receptors (HERs) were expressed in CCL39 hamster fibroblasts, a cell line responding only weakly to EGF despite the presence of 10-20,000 EGF receptors per cell. High expression of HERs (800,000 per cell) conferred EGF responsiveness. In these cells EGF is a potent mitogen, induces strong receptor autophosphorylation, phosphorylation of cellular substrates on tyrosine, activates the Na+/H+ exchanger, and weakly stimulates phosphoinositide (PI) turnover. Activation of PI turnover by a mitogen activating a receptor tyrosine kinase has not been observed previously in CCL39 cells. We present evidence, however, that the activation of this signaling pathway which is insensitive to pertussis toxin does not mediate the proliferative response. Unlike NIH 3T3 cells, CCL39 fibroblasts overexpressing HERs are not transformed nor can a transformed phenotype be observed in response to EGF or TGF alpha.

Serotonin Receptor Activation of Phosphoinositide Turnover in Uterine, Fundal, Vascular, and Tracheal Smooth Muscle

Hydrolysis of phosphatidylinositol is becoming recognized as the second messenger system for a number of hormones and neurotransmitters, including serotonin. The present study was designed to explore the effects of serotonin to enhance phosphoinositide turnover in several smooth muscle preparations, in an effort (a) to determine which smooth muscle preparation might provide a useful system for further study of phosphoinositide turnover and (b) to examine the role of 5-HT2 receptors in such responses. Basal-[3H]inositol monophosphate ([3H]IP) formation was 10-fold higher in the uterus than in the rat jugular vein, aorta, stomach fundus, or guinea pig trachea. Serotonin produced significant elevations in [3H]IP in the rat aorta, uterus, and jugular vein. Maximal elevation in [3H]IP was greatest in the jugular vein (eightfold increase) with an ED50 for serotonin of 0.4 microM. Serotonin (10(-7)-10(-4) M), although a potent contractile agonist in both the guinea pig trachea and rat stomach fundus, did not increase [3H]IP levels in these tissues. The selective 5-HT2 receptor blocker LY53857 (10(-8) M) antagonized the increase in [3H]IP produced by serotonin in the jugular vein, aorta, and uterus. Pargyline (10(-5) M) added to the trachea and fundus did not unmask an effect of serotonin or enhance the response to serotonin in the rat aorta. Thus, the jugular vein was the tissue most sensitive to activation of [3H]IP by serotonin. Increases in [3H]IP produced by serotonin may be linked to activation of 5-HT2 receptors in the jugular vein, aorta, and uterus since the selective 5-HT2 antagonist LY53857 could block this biochemical effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Complete dissociation between the activation of phosphoinositide turnover and of NADPH oxidase by formyl-methionyl-leucyl-phenylalanine in human neutrophils depleted of Ca 2+ and primed by subthreshold doses of phorbol 12,myristate 13,acetate

Evidences have been provided by many laboratories that the activation of the NADPH oxidase in neutrophils by forayl-methionyl-leucyl-phenylalanine (FMLP) is strictly linked to a transduction pathway that involves the stimulation, via GTP binding protein, of the phosphoinositide turnover and the increase in [Ca 2+]i. The results presented in this paper demonstrate that FMLP can activate the NADPH oxidase by triggering a transduction pathway completely independent of phosphoinositide turnover and Ca 2+ changes. In fact: i) Ca 2+-depleted neutrophils do not respond to FMLP with the activation of phosphoinositide hydrolysis and NADPH oxidase. Both the responses are restored by the addition of exogenous Ca 2+. ii) In Ca 2+-depleted neutrophils phorbolmyristate-acetate (PMA) activates the NADPH oxidase. iii) The pretreatement of Ca 2+-depleted neutrophils with non stimulatory doses of PMA restores the activation of the NADPH oxidase but not of the turnover of phosphoinositides by FMLP. This priming effect of PMA and the role of this phosphoinositide and Ca 2+-independent pathway for the stimulation of the NADPH oxidase by receptors mediated stimuli are discussed.

Relationships between phosphoinositide metabolism, Ca 2+ changes and respiratory burst in formyl-methionyl-leucyl- phenylalanine-stimulated human neutrophils : The breakdown ofphosphoinositides is not involved in the rise of cytosolic free Ca 2+

The relationships between the changes of cellular Ca 2+ the activation of phosphoinositide turnover and the functional responses induced by the stimulus-receptor interactions in neutrophils are matter of controversy. By measuring the concentration dependency of different formyl-leucyl-methionyl-phenylalanine (FMLP)-induced changes, the following values of ED 50 were found: 1.6 and 0.8 nM for the rise in [Ca 2+] i monitored with Quin-2, in the presence and absence of exogenous Ca 2+ respectively; 20 nM for the activation of phosphoinositide metabolism, monitored as change in the 32P i of phosphatidate; 14 nM for membrane-bound Ca 2+ mobilization, monitored with chlorotetracycline (CTC); 34 nM for 45Ca 2+ influx and 32 nM for the respiratory burst. Furthermore, low dose of FMLP causes an increase in [Ca 2+] i in absence of activation of breakdown of phosphatidylinositol, phosphatidylinositol 4-monophosphate and phosphatidylinositol 4,5-biphosphate monitored as changes in [ 3H]glycerol radioactivity. The results clearly demonstrate that the increase in [Ca 2+] i, due to the release from intracellular stores, is not caused by the breakdown of phosphatidylinositides. On the other hand, the data of the similarity of ED 50 are compatible with an involvement of phosphoinositide response in the release of membrane bound Ca 2+, monitored with CTC, and in the 45Ca influx and in the respiratory burst.