Cyclic nucleotides regulate the morphologic alterations required for chemotaxis in monocytes

The initial morphologic response of human monocytes to chemoattractants is a change in shape from round to a triangular "motile" configuration (polarization). At doses chemotactic in vitro, chemoattractants induced rapid (t 1/2 = 45 sec), sustained (greater than 40 min) polarization of monocytes in suspension. Extracellular Ca++ was not required for polarization induced by chemoattractants, but in the absence of Ca++ kinetics were slowed (t 1/2 = 6.5 min). Phenylephrine, carbamycholine, serotonin, and ascorbate also caused rapid polarization of monocytes. Unlike chemoattractants, polarization by the pharmacologic agents was unsustained (less than 15 min), absolutely required extracellular Ca++, and affected about 50% of the cells responsive to chemoattractants. Based on relative sensitivities to alpha 1- and alpha 2-adrenergic agonists and antagonists, polarization caused by adrenergic agents was mediated by alpha 2-receptors. Muscarinic and alpha 2-adrenergic agonists, serotonin, and ascorbate enhanced the rate and number of monocytes polarizing to suboptimal doses of chemoattractants. Thus, the initial morphologic changes induced by chemoattractants appear to utilize an activation pathway shared with a variety of agents that enhance cGMP levels and inhibit adenylate cyclase. In contrast, theophylline, histamine, and isoproterenol, all agents that activate adenylate cyclase and elevate cAMP levels, inhibited monocyte polarization to chemoattractants. As in PMN, pharmacologic agents that increase cAMP levels inhibited monocyte chemotaxis in vitro, whereas those that inhibit adenylate cyclase and increase cGMP enhanced monocyte chemotactic responses. Thus, the initial morphologic response of monocytes to chemoattractants as well as the processes required for sustained directional motility are modulated by cyclic nucleotides.     

Phosphatidic acid inhibition of PGE1-stimulated cAMP accumulation in WI-38 fibroblasts: similarities with carbachol inhibition

To test the hypothesis that phosphatidic acid (PhA) is involved in the carbachol inhibition of hormone stimulated accumulation of cAMP we observed the effects of PhA on PGE1-stimulation of cAMP in WI-38 fibroblasts. PhA inhibited PGE1-stimulated cAMP accumulation of WI-38 fibroblasts; maximum inhibition (approximately 50-80%) occurred at a PhA concentration of 1.0 microM and significant inhibition was observed with a concentration of 0.1 microM. The full effects of PhA were evident within 15 sec after the co-addition of PGE1 and PhA. Addition of PhA to cells which had been pre-stimulated with PGE1 resulted in the rapid decay of cAMP levels to a new steady state level with a t 1/2 of approximately 65 sec. The inhibition produced by PhA did not appear to be simply attributable to a depolarization or increased intracellular Ca2+, since addition of either KCl or the Ca2+ ionophore A23187 did not lower PGE1-stimulated cAMP accumulation. When intact cells were pretreated with PhA then lysed and adenylate cyclase immediately assayed, no detectable changes in broken cell adenylate cyclase activities were observed. Also, PhA added directly to adenylate cyclase assays at concentrations as high as 100 microM produced no detectable inhibition of the membrane fraction adenylate cyclase activities. Nonetheless, our results suggest that adenylate cyclase activity in intact cells may be directly affected by physiological levels of PhA . Further, the similarities of carbachol [Butcher, R. W., Journal of Cyclic Nucleotide Research, 4:411 (1978)] and PhA inhibition support the hypothesis that carbachol (acetylcholine) exerts its effect on adenylate cyclase through alterations of the plasma membrane phospholipid composition.     

cAMP and human neutrophil chemotaxis. Elevation of cAMP differentially affects chemotactic responsiveness.

Neutrophils (PMN) treated with cAMP elevating agents were evaluated for their chemotactic responsiveness to FMLP and leukotriene B4 (LTB4). PGE1 and isoproterenol, increased PMN cyclic AMP production and inhibited chemotaxis to both FMLP and LTB4. In contrast, forskolin, which activates adenylate cyclase directly, inhibited chemotaxis to FMLP but not to LTB4. The phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), was required for inhibition of PMN chemotaxis to FMLP by forskolin, PGE1, and isoproterenol. Isoproterenol and PGE1 inhibited PMN chemotaxis to LTB4 in the absence of IBMX and chemotaxis was further inhibited in the presence of IBMX. PMN cAMP levels were stimulated 2- to 3-fold with isoproterenol, 6- to 10-fold with PGE1, and 5- to 7-fold with forskolin over basal levels in the presence of IBMX. These observations demonstrate that total cellular cAMP concentration is not correlated with inhibition of PMN chemotaxis to all stimuli; forskolin, which increased cyclic AMP 5- to 7-fold over basal levels, did not inhibit chemotaxis to LTB4, whereas isoproterenol, which increased cyclic AMP only 2- to 3-fold over basal levels, inhibited chemotaxis to LTB4. PMN cAMP extrusion was determined under basal conditions and in the presence of PGE1, isoproterenol, or forskolin. PMN extruded cAMP under all conditions examined.     

Stimulation of beta-adrenoceptors inhibits calcium-dependent potassium-channels in mouse macrophages

K+ efflux in mouse macrophages exhibited a rate constant (kK) of 0.67 +/- 0.04 (h)-1 (mean +/- SEM of 16 experiments). This was strongly stimulated by increasing concentrations of the Ca2+ ionophore A23187 up to a maximal value of 4.01 +/- 0.25 (h)-1 with an IC50 of 7.6 +/- 1.9 microM (mean +/- SEM of 6 experiments). Similar results were obtained with the Ca2+ ionophore ionomycin. Binding experiments with 3H-dihydroalprenolol revealed a high density of beta-adrenergic receptors (97.5 +/- 5.2 fmol/mg protein) with apparent dissociation constant of 2.03 +/- 0.06 nM. Isoproterenol at a concentration of 10(-6)-10(-5) M induced a two- to threefold stimulation of endogenous levels of cyclic AMP (cAMP). A23187-stimulated K+ efflux was partially inhibited by stimulation of adenylate cyclase with isoproterenol, forskolin or, PGE1; exogenous cAMP; and inhibition of phosphodiesterase with MIX (1-methyl-3-isobutylxanthine). Maximal inhibition of K+ efflux was obtained by simultaneous addition of isoproterenol and MIX. In dose-response curves, the isoproterenol-sensitive K+ efflux was half-maximally inhibited (IC50) with 2-5 X 10(-10) M of isoproterenol concentration. Propranolol was able to completely block the effect of isoproterenol, with an IC50 of about 1-2 X 10(-7) M. Isoproterenol and MIX were also able to partially inhibit ionomycin-stimulated K+ efflux. Isoproterenol and MIX did not inhibit A23187-stimulated K+ efflux in an incubation medium where NaCl was replaced by sucrose (or choline), suggesting the involvement of an Na+:Ca2+ exchange mechanism. Our results show that stimulation of beta-adrenoceptors in mouse macrophages counterbalances the opening of K+ channels induced by the calcium ionophore A23187. This likely reflects a decrease in cytosolic free calcium content via a cAMP-mediated stimulation of Na+:Ca2+ exchange.     

Regulation of inositol phospholipid and inositol phosphate metabolism in chemoattractant-activated human polymorphonuclear leukocytes

Binding of chemoattractants to specific cell surface receptors on polymorphonuclear leukocytes (PMNs) initiates a series of biochemical responses leading to cellular activation. A critical early biochemical event in chemoattractant (CTX) receptor-mediated signal transduction is the phosphodiesteric cleavage of plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2), with concomitant production of the calcium mobilizing inositol-1,4,5-trisphosphate (IP3) isomer, and the protein kinase C activator, 1,2-diacylglycerol (DAG). The following lines of experimental evidence collectively suggest that CTX receptors are coupled to phospholipase C via a guanine nucleotide binding (G) protein. Receptor-mediated hydrolysis of PIP2 in PMN plasma membrane preparations requires both fMet-Leu-Phe and GTP, and incubation of intact PMNs with pertussis toxin (which ADP ribosylates and inactivates some G proteins) eliminates the ability of fMet-Leu-Phe plus GTP to promote PIP2 breakdown in isolated plasma membranes. Studies with both PMN particulate fractions and with partially purified fMet-Leu-Phe receptor preparations indicate that guanine nucleotides regulate CTX receptor affinity. Finally, fMet-Leu-Phe stimulates high-affinity binding of GTP gamma S to PMN membranes as well as GTPase activity. A G alpha subunit has been identified in phagocyte membranes which is different from other G alpha subunits on the basis of molecular weight and differential sensitivity to ribosylation by bacterial toxins. Thus, a novel G protein may be involved in coupling CTX receptors to phospholipase C. Studies in intact and sonicated PMNs demonstrate that metabolism of 1,4,5-IP3 proceeds via two distinct pathways: 1) sequential dephosphorylation to 1,4-IP2, 4-IP1 and inositol, or 2) ATP-dependent conversion to inositol 1,3,4,5-tetrakisphosphate (IP4) followed by sequential dephosphorylation to 1,3,4-IP3, 3,4-IP2, 3-IP1 and inositol. Receptor-mediated hydrolysis of PIP2 occurs at ambient intracellular Ca2+ levels; but metabolism of 1,4,5-IP3 via the IP4 pathway requires elevated cytosolic Ca2+ levels associated with cellular activation. Thus, the two pathways for 1,4,5-IP3 metabolism may serve different metabolic functions. Additionally, inositol phosphate production appears to be controlled by protein kinase C, as phorbol myristate acetate (PMA) abrogates PIP2 hydrolysis by interfering with the ability of the activated G protein to stimulate phospholipase C. This implies a physiologic mechanism for terminating biologic responses via protein kinase C mediated feedback inhibition of PIP2 hydrolysis.     

Role of guanine nucleotide regulatory protein in polyphosphoinositide degradation and activation of phagocytic leukocytes by chemoattractants

Leukocyte activation by chemoattractants provides an important model to study the biochemical mechanisms of stimulus-response coupling in these cells. Well-defined chemotactic factors induce readily quantifiable responses in phagocytic leukocytes. These include directed migration and the production and release of toxic substances including oxygen radicals and lysosomal enzymes. The development of radiolabeled synthetic oligopeptides with potent chemotactic activity allowed the demonstration of chemoattractant receptors on polymorphonuclear leukocytes (PMNs) as well as macrophages. In membrane preparations from these cells, these receptors exist in high- and low-affinity states which are regulated by guanosine di- and triphosphates. This suggested that chemoattractant receptors interact with guanine nucleotide regulatory proteins (N or G proteins). Although chemoattractants elicit a rapid but transient increase in intracellular cAMP levels, they neither stimulate nor inhibit membrane-bound adenylate cyclase, suggesting a novel role for N proteins in certain receptor-transduction mechanisms. Stimulation of phagocytes by chemoattractants is also associated with a rapid increase in cytosolic Ca2+ concentrations ([ Ca2+]i) which appears to result from the production of inositol 1,4,5-triphosphate (IP3) as a consequence of the diesteric cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2). Treatment of phagocytes with pertussis toxin (PT), which ADP-ribosylates and thereby inactivates certain N proteins, abolishes the cells' responsiveness to chemoattractants. More direct evidence for a role of a PT-sensitive N protein in leukocyte activation was provided by the demonstration that chemoattractants stimulate the hydrolysis of PIP2 in PMN membranes only in the presence of GTP. This receptor-mediated hydrolysis of PIP2 is not observed in plasma membranes prepared from PT-treated PMNs. Therefore, these studies suggest that occupancy of chemoattractant receptors activates a PT-sensitive N protein. The activated N protein shifts the Ca2+ requirement for phospholipase C activity from supraphysiological levels to ambient cytosolic Ca2+ concentrations. Cleavage of PIP2 results in the formation of the second messenger molecules, IP3 and 1,2-diacylglycerol, which can initiate cellular activation. These messengers also seem to activate responses which feed back to attenuate receptor stimulation of phospholipase.     

Nucleotide regulatory protein-mediated activation of phospholipase C in human polymorphonuclear leukocytes is disrupted by phorbol esters

Polymorphonuclear leukocytes (PMNs) activate phospholipase C via a guanine nucleotide regulatory (G) protein. Pretreatment of the PMNs with pertussis toxin (PT) or 4-beta-phorbol 12-myristate 13-acetate (PMA) inhibited chemoattractant-induced inositol trisphosphate generation. To determine the loci of inhibition by PT and PMA, G protein-mediated reactions in PMN plasma membranes were examined. Plasma membranes prepared from untreated and PMA-treated PMNs demonstrated equivalent ability of a GTP analogue to suppress high affinity binding of the chemoattractant-N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) to its receptor. The rate, but not the extent, of high affinity binding of GTP gamma[35S] to untreated PMN membranes was stimulated up to 2-fold by preincubation with 1 microM fMet-Leu-Phe. The ability of fMet-Leu-Phe to stimulate the rate of GTP gamma S binding was absent in membranes prepared from PT-treated PMNs, but remained intact in membranes from PMA-treated cells. Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) via phospholipase C could be activated in untreated PMN membranes by either fMet-Leu-Phe plus GTP or GTP gamma S alone at low concentrations of Ca2+ (0.1-1 microM). Membranes prepared from PT-treated PMNs degraded PIP2 upon exposure to GTP gamma S, but not fMet-Leu-Phe plus GTP. In contrast, membranes prepared from phorbol ester-treated PMNs did not hydrolyze PIP2 when incubated with GTP gamma S. Treatment with PT or PMA did not affect the ability of 1 mM Ca2+ to activate PIP2 hydrolysis in PMN membranes, indicating that neither treatment directly inactivated phospholipase C. Therefore, PT appears to block coupling of the chemoattractant receptors to G protein activation, while phorbol esters disrupt coupling of the activated G protein to phospholipase C. The phorbol ester-mediated effect may mimic a negative feedback signal induced by protein kinase C activation by diacylglycerol generated upon activation of phospholipase C.     

Multiple signaling pathways of histamine H2 receptors. Identification of an H2 receptor-dependent Ca2+ mobilization pathway in human HL-60 promyelocytic leukemia cells

In order to analyze the complex activities of histamine H2 receptor activation on neutrophils, human HL-60 promyelocytic leukemia cells were differentiated into neutrophils by incubation with dimethyl sufoxide, loaded with the Ca2+-sensitive indicator dyes, indo-1 or fura-2, and the levels of intracellular Ca2+ ([Ca2+]i) measured in a fluorescent-activated cell sorter and fluorimeter, respectively. Histamine increased [Ca2+]i in a dose-dependent manner with a half-maximal concentration (EC50) of approximately 10(-6) to 10(-5) M, which exhibited H2 receptor specificity. Prostaglandin E2 and isoproterenol also induced [Ca2+]i mobilization in HL-60 cells, whereas the cell permeable form of cAMP and forskolin failed to increase [Ca2+]i. Since H2-receptor mediated [Ca2+]i mobilization was not inhibited by reducing the concentration of extracellular Ca2+ nor by the addition of Ca2+ channel antagonists, LaCl3 and nifedipine, [Ca2+]i mobilization is due to the release of Ca2+ from intracellular stores. Furthermore, both 10(-4) M histamine and 10(-6) M fMet-Leu-Phe increased the levels of 1,4,5-inositol trisphosphate. However, histamine-induced mobilization of [Ca2+]i was inhibited by cholera toxin but not by pertussis toxin, whereas the action of fMet-Leu-Phe was inhibited by pertussis toxin but not by cholera toxin. These data suggest that H2 receptors on HL-60 cells are coupled to two different cholera toxin-sensitive G-proteins and activate adenylate cyclase and phospholipase C simultaneously.     

Heterologous desensitization of adenylate cyclase with prostaglandin E1 alters sensitivity to inhibitory as well as stimulatory agonists

Adenylate cyclase in cultured human fibroblasts is activated by prostaglandin E1 (PGE1) or beta-adrenergic agonists, e.g., isoproterenol, and inhibited by muscarinic agonists. Incubation with PGE1 reduced adenylate cyclase responsiveness to both PGE1 and isoproterenol; this so-called heterologous desensitization is believed to result from impaired function of the stimulatory guanyl nucleotide-binding protein of the cyclase complex. The effect of heterologous desensitization by PGE1 on inhibition of adenylate cyclase by the muscarinic agonist oxotremorine was examined. Muscarinic inhibition of basal and isoproterenol-stimulated cAMP accumulation was attenuated following exposure to PGE1; the concentration of oxotremorine required for half-maximal inhibition of cAMP accumulation was increased. In both intact cells and membrane preparations the number of binding sites for [3H]scopolamine, a muscarinic antagonist, was unaltered by desensitization. Following exposure to PGE1, receptor affinity for oxotremorine, assessed by competition with [3H] scopolamine, and the guanyl nucleotide sensitivity of agonist binding were reduced. The amount of inhibitory guanyl nucleotide-binding regulatory protein available for [32P]ADP-ribosylation by pertussis toxin was unaltered by desensitization. Thus, heterologous desensitization of adenylate cyclase with the stimulatory agonist PGE1 alters sensitivity to inhibitory as well as stimulatory ligands.     

Endothelial cell adenylate cyclase: activation by catecholamines and prostaglandin I2+

Following incubation of intact vascular endothelial cells with 1 mM 3-isobutyl-1-methylxanthine, and isoproterenol or PGI₂, cyclic AMP levels increased 4- and 3-fold, respectively. Isoproterenol-stimulated adenylate cyclase activity of cell lysates was selectively inhibited by the β-adrenergic blocking agent propranolol. Catecholamines stimulated adenylate cyclase with the potency series: isoproterenol > epinephrine > norepinephrine. Prostaglandin did not stimulate adenylate cyclase activity in cell lysates, even in the presence of guanine nucleotides or following preincubation of the intact cells with prostaglandins prior to freeze-thaw lysis.     

Relationship of cAMP and calcium messenger systems in prostaglandin-stimulated UMR-106 cells

The effect of prostaglandins (PG) on free cytosolic calcium concentrations [( Ca2+]i) and cAMP levels was studied in the osteosarcoma cell line UMR-106. PGF2 alpha and PGE2, but not 6-keto-PGF1 alpha, induced an increase in [Ca2+]i which was mainly due to Ca2+ release from intracellular stores. The EC50 for PGF2 alpha was approximately 7 nM, whereas that for PGE2 was approximately 1.8 microM. Maximal doses of PGF2 alpha increased [Ca2+]i to higher levels than PGE2. Both active PGs also stimulated phosphatidylinositol turnover in UMR-106 cells. The effects of the two PGs were independent of each other and appear to involve separate receptors for each PG. PGE2 was a very potent stimulator of cAMP production and increased cAMP by approximately 80-fold with an EC50 of 0.073 microM. PGF2 alpha was a very poor stimulator of cAMP production; 25 microM PGF2 alpha increased cAMP by 5-fold. The increase in cellular cAMP levels activated a plasma membrane Ca2+ channel which resulted in a secondary, slow increase in [Ca2+]i. High concentrations of both PGs (10-50 microM) inhibited this channel independent of their effect on cAMP levels. Pretreatment of the cells with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate inhibited the PG-mediated increase in phosphatidylinositol turnover and the increase in [Ca2+]i. However, pretreatment with 12-O-tetradecanoyl-13-acetate had no effect on the PGE2-mediated increase in cAMP. The latter finding, together with the dose responses for PGE2-mediated increases in [Ca2+]i and cAMP levels, suggests the presence of two subclasses of PGE2 receptors: one coupled to adenylate cyclase and the other to phospholipase C. With respect to osteoblast function, the cAMP signaling system is antiproliferative, whereas the Ca2+ messenger system, although having no proliferative effect by itself, tempers cAMP's antiproliferative effect.     

Potentiation of PGE1-induced increase in cyclic AMP by chemotactic peptide and Ca2+ ionophore through calmodulin-dependent processes

The interaction between prostaglandin E1 (PGE1) and chemotactic peptide formylmethionyl-leucyl-phenylalanine (fMLP) in cAMP production in guinea pig neutrophils was investigated. Both PGE1 and fMLP increased the cAMP content in neutrophils. At low concentrations of PGE1 (less than 10 nM), the effects of fMLP and PGE1 in stimulating cAMP accumulation were additive, but at high concentrations of PGE1, their effects were synergistic. The effects of PGE1 and Ca2+ ionophore A23187 instead of fMLP on cAMP accumulation were also synergistic. The synergy did not appear to be related to change in cyclic nucleotide phosphodiesterase activity, because it was still marked in the presence of isobutyl-3-methyl-1-xanthine, a phosphodiesterase inhibitor. Studies on the time course of PGE1-induced cAMP accumulation showed that cAMP production ceased within 5 min after the addition of high concentrations of PGE1. The period of cAMP production could not be prolonged by combined treatment with PGE1 and fMLP or Ca2+ ionophore A23187. The synergy was found to be caused through Ca2+-dependent processes, because depletion of the medium of Ca2+ and addition of the Ca2+ antagonist TMB-8 inhibited the synergistic increase in cAMP. Moreover, the calmodulin antagonist W-7 also effectively inhibited the synergistic increase in cAMP. These results suggest that the potentiation of PGE1-induced cAMP production by fMLP or Ca2+ ionophore A23187 is catalyzed by calmodulin-dependent processes. However, the synergistic increase in cAMP production was not inhibited by arachidonic acid cascade inhibitors such as indomethacin, BW755C, or nordihydroguiaretic acid, and a combination of PGE1 and a protein kinase C activator, tetradecanoyl phorbol acetate (TPA), did not cause synergistic increase in cAMP. Marked increase in cAMP was also induced by a combination of cholera toxin and fMLP or Ca2+ ionophore A23187, but not by a combination of forskolin and fMLP or Ca2+ ionophore A23187. The synergistic increase in cAMP was not sustained in isolated membranes. On the contrary, PGE1-induced cAMP production in isolated membranes was suppressed by their pretreatment with fMLP or Ca2+ ionophore A23187. These data suggest that the synergistic effects of PGE1 and fMLP or Ca2+ ionophore in increasing the cAMP level are due to potentiation of PGE1-induced cAMP production by Ca2+ and calmodulin-dependent processes.     

Signal transduction in cells following binding of chemoattractants to membrane receptors

Binding of chemoattractants to specific cell surface receptors on human polymorphonuclear leukocytes (PMNs) initiates a variety of biologic responses, including directed migration (chemotaxis), release of superoxide anions, and lysosomal enzyme secretion. Chemoattractant receptors belong to a large class of receptors which utilize the hydrolysis of polyphosphoinositides to initiate Ca2+ mobilization and cellular activation. Receptor occupancy leads to phospholipase C-mediated hydrolysis of polyphosphoinositol 4,5-bisphosphate (PIP2) yielding inositol 1,4,5-trisphosphate (IP3) and 1,2 sn-diacylglycerol (DAG). These products synergize to initiate cell activation via calcium mobilization (IP3) and protein kinase C activation (DAG). Pertussis toxin, which ADP-ribosylates and inactivates some GTP binding proteins (G proteins), abolishes all chemoattractant-induced responses, including Ca2+ mobilization, IP3 and DAG production, enzyme secretion, superoxide production and chemotaxis. Direct evidence for chemoattractant receptor: G protein coupling was obtained using PMN membrane preparations which contain a Ca2+-sensitive phospholipase C. Hydrolysis of polyphosphoinositides at resting intracellular Ca2+ levels (100 nm) was only observed when the membranes were stimulated with the chemoattractant N-formyl-methyl-leucyl-phenylalanine (fMet-Leu-Phe) in the presence of GTP. Myeloid cells contain two distinct pertussis toxin substrates of similar molecular weight (40 and 41 kD). The 41 kD substrate resembles Gi, whereas a 40 kD substrate is physically associated with a partially purified fMet-Leu-Phe receptor preparation and may therefore represent a novel G protein involved in chemoattractant-stimulated responses. Metabolism of 1,4,5-IP3 to inositol proceeds via two distinct pathways in PMNs: (1) degradation to 1,4-IP2 and 4-IP1 or (2) conversion to 1,3,4,5-IP4, 1,3,4-IP3, 3,4-IP2 and 3-IP1. Initial formation (0-30 s) of 1,4,5-IP3 and DAG occurs at ambient intracellular Ca2+ levels, whereas formation of 1,3,4-IP3 and a second sustained phase of DAG production (30 s-10 min) require elevated cytosolic Ca2+ influx. The later peak of DAG, which is not derived from phosphoinositides, appears to be required for stimulation of respiratory burst activity. Products formed during activation can feed back to attenuate chemoattractant receptor-mediated stimulation of phospholipase C by uncoupling receptor-G protein-phospholipase C interaction.     

Multifactorial regulation of prostaglandin synthesis in preovulatory goldfish ovarian follicles.

Goldfish preovulatory ovarian follicles (prior to germinal vesicle breakdown) were utilized for studies investigating the actions of activators of different signal transduction pathways on prostaglandin (PG) production. The protein kinase C (PKC) activators phorbol 12-myristate 13-acetate (PMA; 100-400 nM), 1-oleoyl-2-acetylglycerol (5 and 25 micrograms/ml), and 1,2-dioctanoylglycerol (10 and 50 micrograms/ml) stimulated PGE production; the inactive phorbol 4 alpha-phorbol didecanoate, which does not activate PKC, had no effect. Calcium ionophore A23187 (0.25-4.0 microM) stimulated PGE production and acted in a synergistic manner with activators of PKC. Although produced in lower amounts than PGE, PGF was stimulated by PMA and A23187. The direct activator of phospholipase A2, melittin (0.1-1.0 microM), stimulated a dose-related increase in PGE production, whereas chloroquine (100 microM), a putative inhibitor of phospholipase A2, blocked basal and PMA + A23187-stimulated PGE production. Several drugs known to elevate intracellular levels of cAMP including the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.1-1.0 mM), forskolin (10 microM), and dibutyryl cAMP (dbcAMP; 5 mM) attenuate PMA + A23187-stimulated PGE production. Melittin-stimulated production of PGE was inhibited by dbcAMP, suggesting that the action of cAMP was distal to the activation of phospholipase A2. In summary, these studies demonstrate that activation of PKC and elevation of intracellular calcium levels stimulate PG production, in part, through activation of phospholipase A2. The adenylate cyclase/cAMP signalling pathway is inhibitory to PG production by goldfish ovarian follicles.     

Biphasic effect of calcium on luteinizing hormone-stimulated cyclic adenosine 3',5'-monophosphate production in granulosa cells of the fowl (Gallus domesticus).

Both cAMP and Ca2+ play important roles in the steroidogenic action of LH in hen granulosa cells. However, the interaction of these intracellular messengers is not fully understood. In the present study we used two calcium ionophores (ionomycin and A23187), as well as trifluoperazine (TFP), an inhibitor of calmodulin, to investigate LH- and forskolin-induced cAMP production in granulosa cells isolated from the largest (F1) preovulatory follicle of White Leghorn laying hens. Between 0.1 and 1.0 microM, both ionophores significantly potentiated cAMP responses to LH in the presence of 0.1 mM extracellular Ca2+. When calcium was omitted from the medium, ionophores had no effect. When either calcium was raised above 1 mM, or the concentration of ionophores was increased above 1 microM, LH-induced cAMP production was drastically inhibited. In the presence of 0.5-2.0 mM calcium, A23187 inhibited forskolin-promoted cAMP synthesis. TFP, while having no effect on basal cAMP, suppressed LH-induced responses and the potentiating effect of ionomycin. It is concluded that for full activation of the adenylate cyclase/cAMP system by LH, Ca-calmodulin is required at a site upstream from the catalytic component of the enzyme. However, high intracellular Ca2+ and/or other effects of ionophores (such as uncoupling of oxidative phosphorylation) inhibit LH-induced cAMP production.     

Muscarinic receptor-linked elevation of cAMP in SH-SY5Y neuroblastoma cells is mediated by Ca2+ and protein kinase C.

The mechanisms of muscarinic receptor-linked increase in cAMP accumulation in SH-SY5Y human neuroblastoma cells has been investigated. The dose-response relations of carbachol-induced cAMP synthesis and carbachol-induced rise in intracellular free Ca2+ were similar. The stimulated cAMP synthesis was inhibited by about 50% when cells were entrapped with the Ca2+ chelator BAPTA or in the presence of the protein kinase C (PKC) inhibitor staurosporine. Production of cAMP could be induced also by the Ca2+ ionophore, ionomycin and by TPA, an activator of PKC. When added together TPA and ionomycin had a synergistic effect. When cAMP synthesis was activated with cholera toxin, PGE1 or PGE1 + pertussis toxin carbachol stimulated cAMP production to the same extent as in control cells. Ca2+ and protein kinase C thus seem to be the mediators of muscarinic-receptor linked cAMP synthesis by a direct action on adenylate cyclase.     

Anthrax toxin components stimulate chemotaxis of human polymorphonuclear neutrophils

Effects of the three-component toxin of Bacillus anthracis on chemotaxis of human polymorphonuclear leukocytes (PMN) were investigated in an effort to determine the basis of the reported antiphagocytic effect of the toxin. The three toxin components, edema factor (EF), protective antigen (PA), and lethal factor (LF), were tested alone and in various combinations for their effect on PMN chemotaxis under agarose to formyl peptides and zymosan-activated serum. No component was active alone; combinations of EF + PA, LF + PA, and EF + LF + PA markedly stimulated chemotaxis (directed migration), but had little or no effect on unstimulated random migration. The toxin components were not themselves chemoattractants. EF in combination with PA had previously been identified as an adenylate cyclase in Chinese hamster ovary (CHO) cells. We found that EF + PA produced detectable cyclic adenosine 3'-5'monophosphate (cAMP) in PMN, but the level of cAMP was less than 1% of that produced in CHO cells by EF + PA, and in PMN by other bacterial adenylate cyclases. LF + PA (which stimulated chemotaxis to an equivalent extent) had no effect on cAMP levels. Thus, the enhancement of chemotaxis by anthrax toxin (at least by LF + PA) does not seem to be related to adenylate cyclase activity.     

Regulation of cyclic AMP metabolism in rabbit cortical collecting tubule cells by prostaglandins

Prostaglandin E1 (PGE1) at 1 nM inhibits arginine-vasopressin (AVP)-induced water reabsorption in the rabbit cortical collecting tubule (RCCT), while 100 nM PGE1, by itself, stimulates water reabsorption (Grantham, J. J., and Orloff, J. (1968) J. Clin. Invest. 47, 1154-1161). To investigate the basis for these two responses, we measured the effects of prostaglandins on cAMP metabolism in purified RCCT cells. In freshly isolated cells, PGE2, PGE1, and 16,16-dimethyl-PGE2 acting at high concentrations (0.1-10 microM) stimulated cAMP accumulation; however, one PGE2 analog, sulprostone (16-phenoxy-17,18,19,20-tetranor-PGE2 methylsulfonilamide), failed to stimulate cAMP accumulation or to antagonize PGE2-induced cAMP formation; PGD2, PGF2 alpha, and a PGI2 analog, carbacyclin (6-carbaprostaglandin I2), also failed to stimulate cAMP synthesis. These results suggest that there is a PGE-specific stimulatory receptor in RCCT cells which mediates activation of adenylate cyclase. Occupancy of this receptor would be anticipated to cause water reabsorption by the collecting tubule. At lower concentrations (0.1-100 nM) PGE2, PGE1, 16,16-dimethyl-PGE2, and, in addition, sulprostone inhibited AVP-induced cAMP accumulation by fresh RCCT cells in the presence of cAMP phosphodiesterase inhibitors. Pertussis toxin pretreatment of RCCT cells blocked the ability of both PGE2 and sulprostone to inhibit AVP-induced cAMP accumulation. In membranes prepared from RCCT cells, sulprostone prevented stimulation of adenylate cyclase by AVP. These results suggest that E-series prostaglandins (including sulprostone) can act through an inhibitory PGE receptor(s) coupled to the inhibitory guanine nucleotide regulatory protein, Gi, to block AVP-induced cAMP synthesis by RCCT cells. Occupancy of this receptor would be expected to cause inhibition of AVP-induced water reabsorption in the intact tubule. Curiously, after RCCT cells were cultured for 5-7 days, PGE2 no longer inhibited AVP-induced cAMP accumulation, but PGE2 by itself could still stimulate cAMP accumulation. In contrast to PGE2, epinephrine acting via an alpha 2-adrenergic, Gi-linked mechanism did block AVP-induced cAMP formation by cultured RCCT cells. This implies that some component of the inhibitory PGE response other than Gi is lost when RCCT cells are cultured.     

Mechanism of regulation of adenylate cyclase activity in human polymorphonuclear leukocytes by calcium, guanosyl nucleotides, and positive effectors.

This study presents the results of a kinetic investigation of adenylate cyclase in human polymorphonuclear leukocytes. In the presence of a saturating concentration of substrate (1 mM), the basal activity was increased severalfold by increasing Mg2+ from 1 to 25 mM. A Hill coefficient of 1.9 was obtained for Mg2+ or ATP. The data suggest cooperative interactions between the substrate binding sites in the neutrophil adenylate cyclase complex. It has been observed that guanyl-5'-yl imidodiphosphate (Gpp(NH)p) (S0.5 = 10 MUM) significantly increased and Ca2+ (S0.5 = 0.5 MM) significantly decreased only the Vmax without affecting the Hill coefficient or S0.5 for ATP. The Hill coefficients for Ca2+ or Gpp(NH)p were 0.9 and 0.8, respectively. The Hill coefficient for Ca2+ was not changed by the increased Gpp(NH)p concentrations. It appears that neutrophil adenylate cyclase has distinct binding sites for Gpp(NH)p and Ca2+, one for each compond. The binding of ligands is not changed by the other effectors and the action is directed only toward the Vmax of the enzyme. The stimulatory action of positive effectors (prostaglandin E1, isoproterenol, histamine) was enhanced by Gpp(NH)p and depressed by Ca2+. No preferential stimulation by Gpp(NH)p nor inhibition by Ca2+ of the action of the positive effectors has been found. The data suggests that only one type of catalytic subunit responds to the action of several positive effectors. Extracellular Gpp(NH)p or Ca2+ do not affect the cyclic adenosine 3':5'-monophosphate (cAMP) level in whole neutrophils and the effect of positive effectors on cAMP production is also not significantly changed by 5 mM Ca2+ or 0.1 mM Gpp(NH)p. Ionophore A23187 in the presence of 5 mM Ca2+ enhances Ca2+ entry into cells and decreases the basal cAMP formation. It appears that Gpp(NH)p or Ca2+ act only at the intracellular site of the adenylate cyclase complex.     

A guanine nucleotide regulatory protein controls polyphosphoinositide metabolism, Ca2+ mobilization, and cellular responses to chemoattractants in human monocytes

Previous studies demonstrated that oligopeptide chemoattractant receptors on PMN and macrophages exist in high and low affinity states which are interconvertible by guanosine di- and triphosphates. These observations suggest that guanine nucleotide regulatory (N) proteins play a role in phagocyte activation by chemotactic factors. The data presented here indicate that chemotactic factor receptors on monocytes utilize an N protein to activate phospholipase C and subsequent biologic responses by the cells. This conclusion is based on the findings that inactivation of an N protein of 41,000 m.w. by Bordetella pertussis toxin (PT) treatment abolishes monocyte responsiveness to chemoattractants but not to lectins, PMA, or the Ca2+ ionophore A23187. Treatment with PT inhibited IP3 production, Ca2+ mobilization, and cellular activation as assessed by chemotaxis and changes in forward light scattering in response to the chemoattractants by at least 80%. Therefore, a PT-sensitive N protein plays an important role in the activation of monocytes by chemoattractants.