Formation of mineralized bone nodules by rat calvarial osteoblasts decreases with donor age due to a reduction in signaling through EP(1) subtype of prostaglandin E(2) receptor.

The effects of prostaglandin E(2) (PGE(2)) on the parameters for proliferation and differentiation were studied in calvarial osteoblast-like cells isolated from rats of various ages. In cells not treated with PGE(2), it was found that mineralized bone nodule (BN) formation, alkaline phosphatase (ALP) activity, and the incorporation rate of [(3)H]thymidine into the cells sharply decreased with the age of the cell donor at 6-50 weeks and then remained at a relatively constant level up to 120 weeks. Before studying the effects of PGE(2) on these parameters, we determined the change in the levels of PGE(2) produced by the untreated cells during the culture period and found that the endogenous PGE(2) reached a maximum on the 4th day of the culture, regardless of the cell donor age, followed by a sharp decrease. The endogenous production was blocked by pretreatment with a cyclooxygenase-2 (COX-2) inhibitor, NS-398, indicating the generation of PGE(2) through a COX-2 pathway. The area of BN was effectively suppressed by NS-398 in the cells from 10- to 35-week-old rats, whereas it was enhanced in the cells from 90- to 120-week-old rats. Treatment with PGE(2 )markedly increased the BN formation and the ALP activity in the cells from 4- to 35-week-old rats (defined as young rats). By contrast, PGE(2) decreased [(3)H]thymidine incorporation into the cells from young rats. The area of BN and the ALP activity decreased significantly, whereas [(3)H]thymidine incorporation into the cells increased by 60-80% in the cells of 80- to 120-week-old rats (defined as aged rats). The stimulatory effects on the cell differentiation and the inhibitory effect on the proliferation in the cells from young rats was mimicked by an EP(1) agonist, 17-phenyl-omega-trinor PGE(2), while an EP(2)/EP(4) agonist, 11-deoxy-PGE(1) and an adenylate cyclase activator, forskolin suppressed the differentiation and enhanced the proliferation regardless of the cell donor age. PGE(2), 11-deoxy-PGE(1) and forskolin, but not 17-phenyl-omega-trinor PGE(2) increased cyclic adenosine monophosphate (cAMP) production. Generation of inositol 1, 4,5-triphosphate (IP(3)) was stimulated by 17-phenyl-omega-trinor PGE(2) or PGE(2), but not by 11-deoxy-PGE(1) or forskolin increased cAMP production in the cells from young rats. By contrast, PGE(2 )had little effect on IP(3 )generation in aged rats. From the overall results, we concluded that PGE(2) exerts stimulatory and inhibitory effects on differentiation through the EP(1)-IP(3) pathway and EP(2)/EP(4)-cAMP pathway, respectively, in the cells from young rats. The EP(1)-IP(3) pathway seems to be inactive in the cells from aged rats.     

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.     

Stretch-induced morphological changes of human endothelial cells depend on the intracellular level of Ca2+ rather than of cAMP

When exposed to a uni-axial cyclic stretch, cultured human umbilical vein endothelial cells (HUVECs) align and elongate perpendicular to the stretch axis. Previous studies showed that forskolin inhibited stretch-induced orientation of endothelial cells, suggesting that adenosine 3:5-cyclic monophosphate (cAMP) plays an important role in the shape change. However, we have recently shown that stretch-induced shape changes in cultured HUVECs are due to increased [Ca2+]i. In the present study, we examined the possible role of cAMP in stretch-induced shape changes in cultured HUVECs. Application of uni-axial cyclic stretch induced a gradual rise in cAMP reaching a peak level at 60 min after the onset of stretch. The adenylate cyclase activator, forskolin, increased the basal level of cAMP but inhibited the rise in [Ca2+]i resulting in no cell shape changes. In contrast, N 6,2-dibutyryladenosine 3:5-cyclic monophosphate (dbcAMP) enhanced the stretch-induced increase in cAMP and [Ca2+]i and resulted in cell shape changes. On the other hand, 2'5'-dideoxyadenosine (DDA), an adenylate cyclase inhibitor, inhibited stretch-induced increases in cAMP and [Ca2+]i resulting in no cell shape changes. In summary, our data showed that cell shape changes were consistently dependent on [Ca2+]i rather than cAMP levels. We conclude that the primary second messenger in the stretch-induced shape changes in HUVECs is intracellular Ca2+ rather than cAMP.     

Mechanism of action of aldosterone release by prostaglandin E in isolated rat adrenal glomerulosa cells

The effect of prostaglandin E (PGE) on aldosterone release and the mechanism of action of PGE in mediating the release of aldosterone were studied using isolated rat glomerulosa cells. PGE1 stimulated aldosterone release in a dose-dependent fashion at concentrations between 10(-8) and 10(-6) M and caused approximately a two-fold increase over the basal aldosterone level at 10(-6) M. A significant and dose-dependent increase in cAMP production was also produced by PGE1 at concentrations greater than 10(-8) M. Aldosterone release induced by 10(-7) M or 10(-6) M PGE2 was significantly reduced by a competitive receptor blocking PG-antagonist, SC 19220 (10(-7) M), but not affected by (Sar1, Ileu8)-angiotensin-II (A-II), a competitive inhibitor of A-II. PGE-stimulated aldosterone release was almost completely abolished by depleting the extracellular Ca2+ by EGTA, or by verapamil, a Ca2+-channel blocker or W-7, a calmodulin inhibitor. These findings suggest that PGE stimulates aldosterone release through the membrane receptor binding and activation of adenylate cyclase and that Ca2+-calmodulin system plays an essential role in mediating the steroidogenic action of PGE in the adrenal glomerulosa cells. However, the physiological significance of PGE in the regulation of aldosterone secretion remains to be elucidated.     

Effects of prostaglandin E2 and F2 alpha on cytoplasmic pH in a clonal osteoblast-like cell line, MOB 3-4.

Prostaglandin E2 (PGE2, 5 ng/ml to 5 micrograms/ml) induced a dose-dependent increase in cAMP accumulation, inositol phosphates (IPs) accumulation, and cytoplasmic free Ca2+ ([Ca2+]i) in a clonal osteoblast-like cell line, MOB 3-4. In contrast, prostaglandin F2 alpha (PGF2 alpha, 5 ng/ml to 5 micrograms/ml) stimulated increases in IPs accumulation and [Ca2+]i without stimulating an increase in cAMP accumulation. Both PGE2 (greater than 0.5 micrograms/ml) and PGF2 alpha (greater than or equal to 5 micrograms/ml) increased cytoplasmic pH (pHi) from approximately 7.15 to 7.35 in BCECF-loaded cells. A tumor promotor, phorbol 12-myristate 13-acetate (PMA, 0.1-100 nM) also increased pHi without effect on phosphoinositide hydrolysis. Both PGE2-(5 micrograms/ml) and PMA- (100 nM) induced cytoplasmic alkalinization was inhibited by removal of extracellular Na+, or by pretreatment of the cells with amiloride (0.5 mM), an inhibitor of Na+/H+ exchange, or H-7 (100 microM), a nonspecific inhibitor of protein kinase C. Thus, MOB 3-4 cells appeared to possess PGE2 receptors and PGF2 alpha receptors: the former are coupled to adenylate cyclase and phospholipase C, and the latter are predominantly coupled to phospholipase C. Also the cells appeared to possess an amiloride-sensitive Na+/H+ exchange activity, which increases pHi in response to PGE2 and PGF2 alpha, as well as to PMA. Long-term (48 hr) exposure of the cells to PGE2 at a high concentration (5 micrograms/ml), but not to PGF2 alpha and PMA, decreased DNA synthesis in the serum-deficient medium. Thus, cytoplasmic alkalinization appeared insufficient for cell replication. At least in MOB 3-4 cells, the inhibitory effect of PGE2 on DNA synthesis may be due to the cAMP messenger system.     

Differential effects of parathyroid hormone and its analogues on cytosolic calcium ion and cAMP levels in cultured rat osteoblast-like cells

While the stimulatory effect of parathyroid hormone (PTH) on osteoblast-like cell adenylate cyclase is well known, the effect of PTH on cytosolic calcium ion ([Ca2+]i) mobilization is controversial, one group finding no effect but others reporting various increases. We investigated the effects on [Ca2+]i of synthetic rat PTH fragment 1-34 (rPTH(1-34)) and two bovine PTH analogues that inhibit PTH's stimulation of adenylate cyclase (bovine 8,18Nle, 34Tyr-PTH(3-34) and 34Tyr-PTH(7-34]. [Ca2+]i was measured before, during, and after exposure to PTH analogues in perifused, attached osteoblast-like rat osteosarcoma cells (ROS 17/2.8) that had been scrape-loaded with the luminescent photoprotein aequorin. Resting [Ca2+]i was 0.094 +/- 0.056 microM (mean +/- S.D., n = 103) and rose in a time- and dose-specific way after exposure to rPTH(1-34). At 10(-10) M rPTH(1-34), [Ca2+]i rose 100% within 30 s to a plateau; higher concentrations of PTH yielded increasing initial peaks of [Ca2+]i followed by lower plateaus. At 10(-6) M, the initial peak was 5-fold basal, or 0.64 +/- 0.07 microM. Both analogues of PTH were at least partial agonists for [Ca2+]i mobilization and did not reduce peak [Ca2+]i when co-perifused with rPTH(1-34). However, the analogues did reduce significantly rPTH(1-34)-induced cAMP accumulation and did not increase cAMP accumulation by themselves. Thus, rPTH(1-34) strongly mobilizes [Ca2+]i in ROS 17/2.8 cells, at near-physiologic concentrations. Failure of the PTH analogues to block the effect of PTH on [Ca2+]i while inhibiting the effect on cAMP accumulation suggests separate pathways for PTH activation of adenylate cyclase and mobilization of calcium.     

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.     

Chemoattractant-elicited alterations of cAMP levels in human polymorphonuclear leukocytes require a Ca2+-dependent mechanism which is independent of transmembrane activation of adenylate cyclase

The affinity of the chemoattractant receptor for N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) on human polymorphonuclear leukocytes (PMNs) is regulated by guanine nucleotides, and chemoattractants stimulate increased intracellular cAMP levels in PMNs. Our data, however, indicate that this receptor does not activate membrane-bound adenylate cyclase via direct nucleotide regulatory protein (N) coupling but instead raises cAMP levels indirectly via a mechanism which appears to require Ca2+ mobilization. This conclusion is based on the following data: 1) prostaglandin E1 (PGE1) activated and alpha 2-adrenergic treatment inhibited adenylate cyclase activation in PMN plasma membranes; fMet-Leu-Phe, however, neither activated nor inhibited adenylate cyclase in these membranes; 2) depletion of extracellular Ca2+ had no effect on isoproterenol and PGE1 elicited cAMP responses in intact PMNs while peak fMet-Leu-Phe and A23187-induced responses were reduced by approximately 50 and 80%, respectively; 3) 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate, a purported Ca2+ antagonist, caused almost complete inhibition of fMet-Leu-Phe and ionophore-induced cAMP responses in intact cells but had no effect on PGE1 and isoproterenol; 4) alpha 2-adrenergic agonists inhibited PGE1 but not chemoattractant- or A23187-elicited cAMP responses in intact PMNs; and 5) pretreatment of cells with a phosphodiesterase inhibitor (isobutylmethylxanthine) greatly potentiated the PGE1 and isoproterenol cAMP responses but nearly abolished the peak fMet-Leu-Phe response. Thus, chemoattractants appear to utilize a novel mechanism to raise cAMP levels which appear to require Ca2+ mobilization and could be mediated in part through a transient inhibition of phosphodiesterases. We suggest that stimulation of PMN functions by chemoattractants may utilize an N-coupled process to generate a Ca2+ signal which could in turn raise intracellular cAMP levels indirectly and thereby provide negative regulation.     

Activation of protein kinase C modulates the adenylate cyclase effector system of B-lymphocytes

Antibodies to surface immunoglobulins activate inositol phospholipid hydrolysis in B-lymphocytes, but very little is known concerning their effects on cAMP levels. In other cells, products from the hydrolysis of phosphatidylinositol 4,5-bisphosphate can increase and/or potentiate cAMP accumulation. In this study we have examined whether goat anti-mouse IgM (mu-chain-specific) stimulates and/or potentiates increases in the cAMP levels of splenocytes from athymic nude mice. Goat anti-mouse IgM, by itself, stimulated a 60% increase in cAMP within 2 min. Pretreating the cell suspensions at 37 degrees C with anti-IgM produced opposite effects on the forskolin- and prostaglandin E1 (PGE1)-induced increase in cAMP. Anti-IgM (25 micrograms/ml) potentiated the rise in cAMP induced by 100 microM forskolin 76%, but it decreased the response to 50 nM PGE1 by 30%. Direct activation of protein kinase C (Ca2+/phospholipid-dependent enzyme) by 12-O-tetradecanoylphorbol 13-acetate and/or sn-1,2-dioctanoylglycerol resulted in a similar pattern of responses. A 3-min preincubation with 97 nM 12-O-tetradecanoylphorbol 13-acetate potentiated the forskolin-induced response from 1.7 +/- 0.1 to 4.3 +/- 0.6 pmol of cAMP/10(6) cells but reduced the PGE1 response from 0.98 +/- 0.06 to 0.51 +/- 0.03 pmol of cAMP/10(6) cells. Similarly, preincubating the cells for 3 min with 5 microM sn-1,2-dioctanoylglycerol increased the forskolin response from 1.7 +/- 0.1 to 5.1 +/- 0.2 pmol of cAMP/10(6) cells but reduced the response to PGE1 from 1.15 +/- 0.03 to 0.75 +/- 0.04 pmol of cAMP/10(6) cells. Thus, activation of protein kinase C by hydrolysis products of inositol phospholipids, 12-O-tetradecanoylphorbol 13-acetate, or exogenous diacylglycerols modified adenylate cyclase itself and sites upstream of adenylate cyclase such as the receptor or G proteins coupling the receptor to the cyclase. Furthermore, modification of the PGE1 response by anti-IgM provides a mechanism by which antigen can differentially regulate T- and B-cells responding to macrophage-produced prostaglandins during an immune response.     

Studies on the mechanism of inhibition of hepatic cAMP accumulation by vasopressin

Vasopressin elicited a dose-dependent inhibition of glucagon-induced cAMP accumulation in isolated hepatocytes. This response was not diminished by incubation of cells with the calmodulin antagonists trifluoperazine or chlorpromazine and was only slightly reduced in Ca2+-depleted hepatocytes. Half-maximal inhibition of cAMP accumulation occurred at 8 X 10(-11) M vasopressin, a dose which does not increase cytosolic Ca2+ in hepatocytes. Direct activation of adenylate cyclase by forskolin was significantly inhibited by vasopressin in Ca2+-depleted cells. It is concluded that inhibition of hormone-induced cAMP accumulation by vasopressin in liver is not dependent on cellular Ca2+ mobilisation but may involve direct inhibition of adenylate cyclase.     

Cyclic AMP Analogues Potentiate k-Opiate and Attenuate α2-Adrenoceptor Agonist Effects on Intrasynaptosomal Free Calcium

The intrasynaptosomal free calcium concentration ([Ca2+]i) was measured in quin2-loaded synaptosomes prepared from rat cerebral cortex. Membrane-permeant cyclic adenosine-3',5'-monophosphate (cAMP) analogues [8-bromo-cyclic adenosine-3',5'-monophosphate (8-Br-cAMP) and dibutyryl-cyclic adenosine-3',5'-monophosphate (db-cAMP)] increased [Ca2+]i in a dose-dependent manner; The maximal increases were approximately 50% for 8-Br-cAMP and 35% for db-cAMP and occurred at approximately 10 microM with both analogues. Clonidine (1 microM) alone reduced [Ca2+]i by 26.5%; db-cAMP and 8-Br-cAMP attenuated this reduction to 14.2 and 8.2%, respectively. In contrast, the reduction (19.9%) in [Ca2+]i induced by the preferential kappa-opiate agonist dynorphin A(1-13) was not attenuated by the cAMP analogues; in fact, db-cAMP and 8-Br-cAMP potentiated the effect of dynorphin A(1-13) (1 microM), producing decreases in [Ca2+]i of 33.6 and 29.6%, respectively. We conclude that although alpha 2-adrenergic and kappa-opiate receptors both reduce [Ca2+]i, the alpha 2-adrenoceptor-mediated response and the kappa-opiate receptor-mediated response involve different effector mechanisms. It appears that presynaptic alpha 2-adrenoceptor agonist effects are linked to reductions in adenylate cyclase activity and cAMP production and a resultant increase in Ca2+ sequestration, Ca2+-channel blockade, or both. On the other hand, the kappa-opiate-mediated effects possibly involve an increase in cAMP production and a blockade of Ca2+ entry.     

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.     

Cyclic AMP raises cytosolic Ca2+ and promotes Ca2+ influx in a clonal pancreatic beta-cell line (HIT T-15)

The effect on cytosolic Ca2+ concentration ([Ca2+]i) of cAMP analogues and the adenylate cyclase-stimulating agents forskolin, isoproterenol and glucagon has been examined in an insulin-secreting beta-cell line (HIT T-15) using fura 2. All these manipulations of the cAMP messenger system promoted a rise in [Ca2+]i which was blocked by the Ca2+ channel antagonists verapamil and nifedipine or by removal of extracellular Ca2+. The action of the adenylate cyclase activator forskolin was glucose-dependent. The results suggest that cAMP elevates [Ca2+]i in HIT cells by promoting Ca2+ entry through voltage-sensitive Ca2+ channels, not through mobilization of stored Ca2+. Activation of Ca2+ influx may be an important component of the mechanisms by which cAMP potentiates fuel-induced insulin release.     

Inhibition of prostaglandin E1-induced elevation of cytoplasmic free calcium ion by protein kinase C-activating phorbol esters and diacylglycerol in Swiss 3T3 fibroblasts

In quiescent cultures of Swiss 3T3 cells, prostaglandin E1 (PGE1) known to elevate cAMP increased rapidly cytoplasmic free Ca2+ concentration ([Ca2+]i) as measured with the fluorescent Ca2+ indicator quin2. The primary source of the PGE1-induced elevation of [Ca2+]i was extracellular. Pretreatment of the cells with various doses of 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent protein kinase C-activating phorbol ester, inhibited the PGE1-induced elevation of [Ca2+]i in a dose-dependent manner. Inversely, TPA enhanced slightly the PGE1-induced increase of cAMP. TPA alone did not affect the basal level of [Ca2+]i or cAMP in the absence of PGE1. The inhibitory action of TPA on the PGE1-induced elevation of [Ca2+]i was mimicked by other protein kinase C-activating agents such as phorbol 12,13-dibutyrate and 1-oleoyl-2-acetylglycerol. 4 alpha-Phorbol 12,13-didecanoate known to be inactive for protein kinase C was ineffective in this capacity. Prolonged treatment of the cells with phorbol 12,13-dibutyrate resulted in the down-regulation and disappearance of protein kinase C. In these protein kinase C-deficient cells, PGE1 still elevated [Ca2+]i to the same extent as that in the control cells, but TPA did not inhibit the PGE1-induced elevation of [Ca2+]i. These results strongly suggest that protein kinase C serves as an inhibitor for PGE1-induced Ca2+ influx in Swiss 3T3 cells.     

Agents that raise cAMP in human T lymphocytes release an intracellular pool of calcium in the absence of inositol phosphate production

Prostaglandins (PGs) of the E series are recognized by specific receptors on T lymphocytes which lead to an increase in cAMP. The role of cAMP in modulation of T lymphocyte function is unknown. Here, we demonstrate that agents which increase cAMP in human T cells raise the intracellular free calcium concentration ([Ca2+]i). This increase in [Ca2+]i occurred following receptor stimulation with PGEs or by bypassing the receptor with the cell-permeant analog 8-(4-chlorophenylthio)-cAMP or forskolin, a direct activator of adenylyl cyclase. The calcium response to a submaximally stimulatory concentration of PGE2 was potentiated by the cAMP phosphodiesterase inhibitor isobutylmethylxanthine. A time course of cAMP production in response to PGE2 stimulation closely resembled the calcium response and suggested that the two events were coincident. The PGE2 concentrations required to achieve 50% maximum effect of cAMP production and increases in [Ca2+]i were similar, 0.07 and 0.15 microM respectively. Chelation of extracellular Ca2+ did not abolish the PGE2-stimulated Ca2+ response, suggesting that an intracellular source of calcium was sensitive to cAMP. Significant inositol phosphate production was not detected in response to PGE2 over a wide concentration range. The PGE2-induced calcium response curves were of lesser magnitude with shorter times to peak than those of a known inositol 1,4,5 trisphosphate-producing agonist, anti-CD3, suggesting distinct Ca2+ release mechanisms. However, the cAMP-releasable store appeared to be contained within the inositol trisphosphate-releasable store since no response could be seen with cAMP-elevating agents following emptying of the inositol trisphosphate-sensitive pool of Ca2+.     

Effects of cholera toxin on cyclic AMP accumulation and bone resorption in cultured mouse calvaria

We have utilized the adenylate cyclase stimulator, cholera toxin, as a tool to test the role of cyclic AMP as a mediator of the effects on bone resorption by the calcium-regulating hormones, parathyroid hormone (PTH) and calcitonin. The effects on bone resorption were studied in an organ culture system using calvarial bones from newborn mice. Cyclic AMP response was assayed in calvarial bone explants and isolated osteoblasts from neonatal mouse calvaria. Cholera toxin caused a dose-dependent cAMP response in calvarial bones, seen at and above approx. 1-3 ng/ml and calculated half-maximal stimulation (EC50) at 18 ng/ml. The stimulatory effect of cholera toxin could be potentiated by the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX, 0.2 mmol/l). Cyclic AMP accumulation in the bones was maximal after 4-6 h, and thereafter declined. However, activation of the adenylate cyclase was irreversible and the total amount (bone + medium) of cAMP produced, in the presence of IBMX (0.2 mmol/l), increased with time, for at least 48 h. In osteoblast-like cells cholera toxin (1 microgram/ml) stimulated the cellular levels of cAMP with a peak after 60-120 min, which could be potentiated with IBMX. The total cAMP accumulation indicated an irreversible response. In short-term bone organ cultures (at most, 24 h) cholera toxin, at and above 3 ng/ml, inhibited the stimulatory effect of PTH (10 nmol/l) on 45Ca release from prelabelled calvarial bones. The inhibitory effect of cholera toxin (0.1 microgram/ml) on 45Ca release was significant after 6 h and the calculated IC50 value at 24 h was 11.2 ng/ml. Cholera toxin (0.1 microgram/ml) also inhibited PTH-stimulated (10 nmol/l) release of Ca2+, inorganic phosphate (Pi), beta-glucuronidase, beta-N-acetylglucosaminidase and degradation of organic matrix (release of 3H from [3H]proline-labelled bones) in 24 h cultures. 45Ca release from bones stimulated by prostaglandin E2 (1 mumol/l) and 1 alpha-hydroxyvitamin D3 (0.1 mumol/l) was also inhibited by cholera toxin (0.3 microgram/ml) in 24-h cultures. The inhibitory effect of cholera toxin on bone resorption was transient, and in long-term cultures (120 h) cholera toxin caused a dose-dependent, delayed stimulation of mineral mobilization (Ca2+, 45Ca, Pi), degradation of matrix and release of the lysosomal enzymes beta-glucuronidase and beta-N-acetylglucosaminidase.(ABSTRACT TRUNCATED AT 400 WORDS)     

The interaction of Ca2+ with the calmodulin-sensitive adenylate cyclase from Bordetella pertussis

Bordetella pertussis, the etiologic agent of whooping cough, produces a calmodulin-sensitive adenylate cyclase which elevates intracellular cAMP in a variety of eucaryotic cells. Exogenous calmodulin added to the partially purified adenylate cyclase has been shown to inhibit invasion of animal cells by this enzyme (Shattuck, R. L., and Storm, D. R. (1985) Biochemistry 24, 6323-6328). In this study, several properties of the calmodulin-sensitive adenylate cyclase are shown to be influenced by Ca2+ in the absence of calmodulin. The presence or absence of Ca2+ during QAE-Sephadex ion exchange chromatography produced two distinct chromatographic patterns of adenylate cyclase activity. Two different forms of the enzyme (Pk1 and Pk2EGTA) were isolated by this procedure. Pk1 adenylate cyclase readily elevated intracellular cAMP levels in mouse neuroblastoma cells (N1E-115) while Pk2EGTA adenylate cyclase had no effect on cAMP levels in these cells. Gel exclusion chromatography of Pk1 adenylate cyclase gave apparent Stokes radii (RS) of 43.5 A (+/- 1.3) in the presence of 2 mM CaCl2 and 33.8 A (+/- 0.94) in the presence of 2 mM EGTA [( ethylenebis (oxyethylenenitrilo)]tetraacetic acid). These Stokes radii are consistent with molecular weights of 104,000 (+/- 6,400) and 61,000 (+/- 3,600), respectively. Pk2EGTA adenylate cyclase had an apparent RS of 33.0 (+/- 1.2) (Mr = 60,600 (+/- 2,800] in the presence of Ca2+ or excess EGTA. At 60 degrees C, Pk1 adenylate cyclase exhibited a Ca2+-dependent heat stability with a half-life for loss of enzyme activity of 10.3 min in 5 mM CaCl2 and a half-life of 2.8 min in the presence of 0.1 microM CaCl2. The stability of Pk2EGTA adenylate cyclase was not affected by changes in free Ca2+. The adenylate cyclase preparations described above were submitted to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and enzyme activity was recovered from gel slices by extraction with detergent containing buffers. The catalytic subunit isolated from SDS-polyacrylamide gels was activated 7-fold in the presence of Ca2+ with maximum activity observed at 1 microM free Ca2+. With both preparations, the apparent molecular weight of the catalytic subunit on SDS gels was 51,000 in the presence of 2 mM CaCl2 and 45,000 in the presence of 2 mM EGTA. The catalytic subunit of the enzyme was purified to apparent homogeneity by preparative SDS-polyacrylamide gel electrophoresis and resubmitted to SDS gel electrophoresis in the presence or absence of free Ca2+. The purified catalytic subunit also exhibited a Ca2+-dependent shift in its mobility on SDS gels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Biochemical evidence for PGI2 and PGE2 receptors in the rabbit renal preglomerular microvasculature

It has been proposed that a portion of the biologic actions of vasodilator prostaglandins occurs via an interaction with specific adenylate cyclase-linked receptors. This hypothesis was explored further in the renal microvasculature by examining the effects of PGI2, PGE1, and PGE2 on rabbit preglomerular microvascular adenylate cyclase. A membrane preparation derived from freshly isolated rabbit renal preglomerular microvessels was used in these studies. NaF, forskolin, or 5'-guanylyl imidodiphosphate were found to be effective in increasing adenylate cyclase activity in the absence of exogenous guanosine-5'-triphosphate. A dose-dependent stimulation of adenylate cyclase was also observed with guanosine-5'-triphosphate. PGE1, PGE2, and PGI2 produced a dose-dependent stimulation of adenylate cyclase activity only in the presence of guanosine-5'-triphosphate suggesting that this nucleotide is essential for prostaglandin-induced stimulation of the enzyme. PGI2 exhibited a time-dependent increase in adenylate cyclase activity and this increased activity reached a plateau at 20-25 min. When PGE1 and PGE2 were added together, no additive effect on adenylate cyclase stimulation was noted whereas PGI2 and PGE2 when added together produced an additive stimulatory effect. When viewed together, these data suggest the presence of separate PGI2 and PGE adenylate cyclase-linked receptors in rabbit renal preglomerular microvessels. These findings also suggest that in the renal microvasculature, cyclic AMP may be a second messenger mediating the vasodilatory effects of both PGI2 and PGE2.     

Possible involvement of p38 MAP kinase in prostaglandin E1-induced ALP activity in osteoblast-like cells

Prostaglandins are now recognized to be important regulators for both bone formation and resorption. Among them, prostaglandin E(1) (PGE(1)) has been reported to stimulate cAMP accumulation and to induce alkaline phosphatase (ALP) activity, a marker of differentiation, in osteoblast-like cells. Recently, we have shown that p38 mitogen-activated protein (MAP) kinase pathway regulates ALP activity in response to activation of Gi protein-coupled receptors in mouse osteoblast-like MC3T3-E1 cells (Suzuki et al., Endocrinology 140 (1999) 3177). In the present study, we investigated whether p38 MAP kinase is involved in ALP activation by PGE(1) in MC3T3-E1 osteoblast-like cells. PGE(1) dose-dependently enhanced ALP activities in the concentration range between 1 nM and 1 microM in MC3T3-E1 cells. SB203580, a specific inhibitor of p38 MAP kinase, blocked the increase in ALP activity induced by PGE(1). Further analysis with western blotting suggested that PGE(1) induced an increase in tyrosine (Tyr) phosphorylation of p38 MAP kinase. Both Bt(2)cAMP, a permeable analogue of cAMP, and forskolin, which directly activates adenylate cyclase, also induced an increase in Tyr phosphorylation of p38 MAP kinase. H-89, a potent inhibitor of protein kinase A (PKA), significantly suppressed PGE(1)-induced Tyr phosphorylation of p38 MAP kinase. The results of this study suggest that PGE(1) stimulates p38 MAP kinase through the activation of PKA, resulting in the enhancement of ALP activity.     

Effect of prostaglandin E1 on the cyclic adenosine-3',5'--monophosphate system and radiosensitivity of B-82 cells cultured in vitro

The cyclic AMP (cAMP) system of mice fibroblasts L, clone B-82 has been studied. B-82 cells possess receptors to prostaglandin E1 (PGE1) and are deficient in beta-adrenoreceptors. It has been shown that 10 mM NaF, 1.10(-4) M GTP and 1.10(-5) M PGE1 stimulate adenylate cyclase of B-82 cells. PGE1 causes increase in the intracellular content of cAMP and protects B-82 cells against irradiation. Specifict beta-agonist isoproterenol fails to modulate adenylate cyclase activity and does not change cAMP content in B-82 cells. Isoproterenol has no effect on the B-82 cells radiosensitivity, although it is known to be a potent radioprotector. It is presumed that the stimulation of cAMP system by radioprotector is necessary for the performance of radioprotective effect.