Alpha 2-adrenergic receptors accelerate Na+/H+ exchange in neuroblastoma X glioma cells

The regulation of cytoplasmic pH (pHi) was examined in neuroblastoma X glioma hybrid cell-line cells (NG108-15 cells) using 2,7-biscarboxyethyl-5(6)-carboxyfluorescein. The pHi of NG108-15 cells suspended in nominally HCO-3-free, Na+-containing buffer could be reduced by the external application of acetate. The recovery of pHi to its resting value was blocked by the removal of extracellular Na+, by the addition of extra-cellular H+, and by the addition of analogs of amiloride selective for inhibition of Na+/H+ exchange. The rate of recovery of pHi from acid load exhibited an ionic selectivity of Na+ greater than Li+ much greater than K+, and no recovery was observed in N-methyl-D-glucamine+. Tetrodotoxin and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid had no effect on early pHi recovery. These data suggest that Na+/H+ exchange accounts primarily for the recovery of pHi in NG108-15 cells under our experimental conditions. Na+/H+ exchange in NG108-15 cells was accelerated by alpha 2-adrenergic receptors. Thus, (-)epinephrine, but not (+)epinephrine, elicited an intracellular alkalinization which was blocked by the alpha 2-adrenergic receptor selective antagonist yohimbine but not by the alpha 1-adrenergic receptor antagonist, prazosin, nor the beta-adrenergic antagonist, propranolol. Norepinephrine, clonidine, and the clonidine analog, UK-14304, also caused alkalinization of NG108-15 cells, whereas isoproterenol, a beta-adrenergic receptor agonist, and phenylephrine, a selective alpha 1-adrenergic receptor agonist, did not. Manipulations that blocked Na+/H+ exchange blocked the ability of alpha 2-adrenergic agonists to alkalinize the interior of NG108-15 cells without blocking the ability of these agonists to attenuate cAMP accumulation. These findings provide the first direct evidence of modulation of Na+/H+ exchange activity by a receptor linked to inhibition of adenylate cyclase and offer a possible mechanism whereby alpha 2-adrenergic receptors might influence cellular activity apart from changes in cyclic nucleotide metabolism.     

Late-Phase Accumulation of Inositol Phosphates Stimulated by Prostaglandins D2 and F2α in Neuroblastoma × Glioma Hybrid NG108-15 Cells

The accumulation of inositol phosphates (IPs) in response to prostaglandins (PGs) was studied in NG108-15 cells preincubated with myo-[3H]inositol. As a positive control, bradykinin caused accumulation of IPs transiently at an early phase (within 1 min) and continuously during a late phase (15-60 min) of incubation in the cells. PGD2 and PGF2 alpha did not significantly cause the accumulation of IPs at an early phase but significantly stimulated inositol bisphosphate (IP2) and inositol monophosphate (IP) formation at late phase of incubation. The maximum stimulation was obtained at greater than 10(-7) M concentrations of these PGs, the levels being three-and twofold for IP2 and IP1, respectively. 9 alpha, 11 beta-PGF2 has a slight effect but PGE2 and the metabolites of PGD2 and PGF2 alpha have no effect up to 10(-6)M. The effects of PGD2 and PGF2 alpha were not additive, but the effect of each PG was additive to that of bradykinin at a late phase of incubation. Inositol 1-monophosphate was mainly identified in the stimulation by 10(-5) M PGD2 and 10(-5) M PGF2 alpha, whereas both inositol 1-monophosphate and inositol 4-monophosphate were produced in the stimulation by 10(5) M bradykinin. Depletion of extracellular Ca2+ diminished the stimulatory effect of PGD2 and PGF2 alpha and late-phase effect of bradykinin, but simple Ca2+ influx into the cells by high K+, ionomycin, or A23187 failed to cause such late-phase effects. These results suggest that PGD2 and PGF2 alpha specifically stimulate hydrolysis of inositol phospholipids.     

Differential effects of prostaglandin F2 alpha and of prostaglandins E1 and E2 on cyclic 3',5'-monophosphate production and intracellular calcium mobilization in avian uterine smooth muscle cells

The effects of prostaglandins (PGs) E1 (PGE1), E2 (PGE2) and F2 alpha (PGF2 alpha) on cyclic 3',5'-adenosine monophosphate (cAMP) production and intracellular Ca mobilization were examined in smooth muscle cells of chicken uterus grown in primary culture. At subnanomolar concentrations, both PGE1 and PGE2 significantly suppressed cAMP levels. However, at higher concentrations (0.1-100 microM), both agonists caused a dose-related increase in cAMP production. PGF2 alpha, on the other hand, had no effect on cAMP production. Forskolin (1-100 microM), which also stimulated cAMP production in a dose-dependent fashion, potentiated the effects of both PGE1 and PGE2. In digitonin-permeabilized uterine cells preloaded with 45Ca2+, the addition of PGF2 alpha caused a biphasic 45Ca2+ efflux. There was a small but significant 45Ca2+ release (10.0 +/- 1.5%) within 30 s (rapid phase), followed by a larger one (32.0 +/- 2.0%) within 5 min (slow phase). PGE2, at doses above 1 nM (which significantly increased cAMP accumulation), promoted 45Ca2+ sequestration. This action of PGE2 was observed as early as 1 min and was complete by 5 min. In addition, 0.001 nM PGE2 (a dose that was ineffective on 45Ca2+ mobilization) enhanced PGF2 alpha-induced 45Ca2+ mobilization from 22.5 +/- 5% to 57.0 +/- 3.5%. These results show that PGs of the E series have distinctly different effects on cAMP production and intracellular Ca mobilization. PGF2 alpha action may be linked directly to intracellular Ca mobilization, whereas the effects of PGE may be exerted at multiple sites depending on its local concentration. At low concentrations, its action may be mediated by the suppression of cAMP levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of prostaglandins on cyclic AMP levels in isolated cells from developing chick limbs

Effects of prostaglandins (PGs) on accumulation of cyclic AMP (cAMP) in the presence of a phosphodiesterase inhibitor were investigated in cells isolated from avian limb buds at various stages of development. Cells were responsive to PGE2 at the earliest stage investigated (stage 20-21) which was well in advance of specific cytodifferentiation of limb tissues. At three later stages (24-25; 26-28; 30-32), the responsiveness of cells isolated from the developing skeletal anlagen of the limb progressively increased coincident with the differentiation and maturation of the cartilage phenotype. Cells isolated from stage 26-28 cartilage rods were responsive also to prostacyclin (PGI2); however, the response produced was only about 50% of the response to an equivalent concentration of PGE2. Cells were not responsive to either PGF2 alpha or 6-keto PGF1 alpha, at concentrations of 30-33 micrograms/ml demonstrating a degree of specificity for PGE2 and PGI2. In the absence of the phosphodiesterase inhibitor, PGE2 increased cAMP accumulation two-fold over the controls and produced a concentration-dependent response between 0.3-30 micrograms/ml. The results demonstrate that PGs are capable of modulating cAMP levels of undifferentiated limb mesenchymal cells as well as embryonic cartilage cells and suggest a role for these compounds in limb chondrogenesis.     

IL-1 activates the Na+/H+ antiport in a murine T cell

One of the early events following growth factor exposure is elevation of intracellular pH, a process mediated by the Na+/H+ antiport. We studied the effects of human rIL-1 alpha (HrIL-1 alpha) on intracellular pH (pHi) and calcium ([Ca2+]i) in a murine T cell line (MD10 cells), which proliferates in response to IL-1 alone. By using the intracellularly trapped fluorescent dyes (2(1),7(1)-bis-2-carboxyethyl)-5(and -6) carboxyfluorescein) and indo-1, we monitored immediate to early changes of pHi and [Ca2+]i in response to HrIL-1 alpha. Exposure to HrIL-1 alpha (120 pM) leads to an early, sustained intracellular alkalinization (delta pH = + 0.09 +/- 0.03) that plateaus within 20 min. Lower concentrations of the monokine (12 pM, 1.2 pM) have a positive but not statistically significant effect on pHi. These effects parallel the degree of MD10 IL-1R saturation predicted by the KD (49 pM) as assessed by 125I-HrIL-1 alpha binding by MD10 cells (Bmax = approximately 1300). Both the MD10 IL-1 receptor KD and the HrIL-1 alpha concentration required to induce early measurable alkaline pH shifts, however, exceed by three orders of magnitude the HrIL-1 alpha ED50 (50 fM) required for MD10 proliferation. The IL-1-induced rise in pHi is both sodium dependent and amiloride sensitive, indicative of activation of the Na+/H+ antiport. Additionally, PMA (100 nM) and IL-2 (2 nM) alkalinize MD10 cells, with the rise in pHi as a result of PMA exceeding the maximal IL-1 effect (delta pH = + 0.13 +/- 0.04). Furthermore, although PMA alkalinizes cells previously exposed to HrIL-1 alpha, the monokine does not alter the pHi of PMA-treated MD10 cells. Importantly, intracellular alkalinization induced by either HrIL-1 alpha or PMA is inhibited by staurosporine (1 mu iM). Finally, HrIL-1 alpha does not change MD10 [Ca2+]i, in either an acute or sustained fashion. These results indicate that IL-1 activates the Na+/H+ antiport in T cells by a mechanism that is unrelated to changes in [Ca2+]i but may involve protein kinase C activation.     

Ca2+ mobilization can occur independent of acceleration of Na+/H+ exchange in thrombin-stimulated human platelets

Intracellular free Ca2+ [( Ca2+]i) and pH (pHi) were measured simultaneously by dual wavelength excitation in thrombin-stimulated human platelets double-labeled with the fluorescent probes fura2 and 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein to determine the relationship between changes in [Ca2+]i and pHi, respectively. At 37 degrees C, thrombin (0.5 or 0.1 units/ml) increased [Ca2+]i with no detectable lag period to maximum levels within 13 s followed by a slow return to resting levels. There was a transient decrease in pHi within 9 s that was immediately followed by an alkalinization response, attributable to activation of Na+/H+ exchange, that raised pHi above resting levels within 22 s. At 10-15 degrees C, thrombin-induced changes in [Ca2+]i and pHi were delayed and therefore better resolved, although no differences in the magnitude of changes in [Ca2+]i and pHi were observed. However, the increase in [Ca2+]i had peaked or was declining before the alkalinization response was detected, suggesting that Ca2+ mobilization occurs before activation of Na+/H+ exchange. In platelets preincubated with 5-(N-ethyl-N-isopropyl)amiloride or gel-filtered in Na+-free buffer (Na+ replaced with N-methyl-D-glutamine) to inhibit Na+/H+ exchange, thrombin stimulation caused a rapid, sustained decrease in pHi. Under these conditions there was complete inhibition of the alkalinization response, whereas Ca2+ mobilization was only partially inhibited. Nigericin (a K+/H+ ionophore) caused a rapid acidification of more than 0.3 pH unit that was sustained in the presence of 5-(N-ethyl-N-isopropyl)amiloride. Subsequent stimulation with thrombin resulted in slight inhibition of Ca2+ mobilization. These data show that, in human platelets stimulated with high or low concentrations of thrombin, Ca2+ mobilization can occur without a functional Na+/H+ exchanger and in an acidified cytoplasm. We conclude that Ca2+ mobilization does not require activation of Na+/H+ exchange or preliminary cytoplasmic alkalinization.     

Activation of Na/H exchange by thrombin in peritoneal mast cells]

Using the fluorescent probe, BCECF, the changes in intracellular pH (pHi) in rat peritoneal mast cells were studied. alpha-Thrombin (0.1 nM) induced biphasic changes in pHi which consisted in a temporary decrease in pH with its subsequent steady increase due to the Na/H exchange activation which was inhibited by EIPA and controlled by extracellular Na+. The biphasic changes in pHi induced by DIP-alpha-thrombin (0.1 pM-1 nM), a catalytically inactive form with an intact recognition site, were similar to those of alpha-thrombin, whereas beta/gamma-thrombin (10-1000 pM), a catalytically active form characterized by structural disturbances in the recognition site, was able to induce only the initial phase of acidification. The thrombin recognition site modulators, alpha 1-thymosin and heparin, blocked the ability of the enzyme to induce the alkalinization of pHi. Nigericin stimulated the Na/H-exchange in mast cells. The rate of the Na/H-exchange activation determined with nigericin, decreased with an increase in the alpha-thrombin dose from 0.1 pM up to 10 nM. Activation of protein kinase C (PKC) in mast cells by PMA used at 1 nM and 10 nM led to the alkalinization of the cytoplasm as a result of the Na/H-exchange activation blocked by EIPA. The PKC inhibitor, H-7, suppressed the pHi increase induced by both PMA and alpha-thrombin. The alpha-thrombin-induced acidification of the cytoplasm was completely blocked by SITS in Ca(2+)-free media, whereas in media with Ca2+ SITS inhibited the pHi decline. Acidification of the cytoplasm by thrombin seems to be due to both Ca2+ influx and activation of Cl- fluxes. It is concluded that the observed activation of the Na/H-exchange by thrombin is induced by a cascade of intracellular reactions involving PKC.     

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.     

Starvation of a clonal osteoblast-like cell line, MOB 3-4-F2, down-regulates prostaglandin E2 receptors but increases cAMP response to prostaglandin E2.

Prostaglandin E2 (PGE2) stimulated cAMP production in the MOB 3-4-F2 cell line, a subclone of the osteoblast-like MOB 3-4 cell line. After being cultured in alpha-minimum essential medium supplemented with 10% heat-inactivated foetal calf serum (HIFCS), cells responded to PGE2 (greater than or equal to 50 ng/ml) with a small, but significant, increase in cAMP production. This response did not vary with duration of culture. In 2% HIFCS-containing medium, despite their lower basal cAMP level, cells responded to PGE2 (greater than or equal to 5 ng/ml) with strikingly increased cAMP production. In addition, prolonged culture in this serum-deficient medium enhanced this response. On the other hand, culture of cells in 2% HIFCS-containing medium decreased the apparent number of PGE2 receptors, which was also enhanced by prolonged culture, without effect on their apparent affinity. Their number in 10% HIFCS-containing medium, more than that in 2% HIFCS-containing medium, was almost constant, independent of the culture period. Starvation of MOB 3-4-F2 cells in serum-deficient medium, therefore, appeared to down-regulate PGE2 receptors but increase the cAMP response to PGE2. Moreover, prolonged starvation of cells appeared to facilitate these phenomena. Our findings suggest that cAMP response to PGE2 does not always reflect the number of available PGE2 receptors in the cells.     

Synthesis of prostaglandin F2 alpha, E2 and prostacyclin in isolated corpora lutea of adult pseudopregnant rats throughout the luteal life-span.

The ability of de novo biosynthesis of prostaglandins (PGs) in individual whole corpora lutea (CL) obtained from sterile-mated adult pseudopregnant rats on different days of the luteal phase and the post-luteolytic period was evaluated. Production of PGs, progesterone and 20 alpha-dihydroprogesterone were determined after in vitro incubation of CL extirpated from Day 2 to Day 19 after mating. A time-relationship with increased accumulation of PGs in the medium was demonstrated from 18 s to 5 h, with large increments during the first 30 min. Basal accumulation of PGs in the incubation medium was highest for 6-keto-PGF1 alpha (the stable metabolite of prostacyclin) greater than PGE2 greater than PGF2 alpha greater than thromboxane B2 (TXB2) and basal accumulation of PGF2 alpha and PGE2 measured in the medium was maximal on Day 10-11 of pseudopregnancy, concomitantly with a decline in secretion of progesterone. Addition of arachidonic acid (AA) dose-dependently increased synthesis of PGs, with absolute amounts of PGE2 greater than 6-keto-PGF1 alpha greater than PGF2 alpha greater than TXB2 and addition of 14 microM indomethacin markedly inhibited accumulation of all PGs measured. Luteinizing hormone (LH, 10 micrograms/ml) stimulated progesterone secretion on all days during pseudopregnancy, but not on the post-luteolytic Day 19. LH increased PGF2 alpha, PGE2 and 6-keto-PGF1 alpha secretion on Day 13 of pseudopregnancy by 76%, 91% and 28%, respectively, but not on the other days tested. Furthermore, stimulation of PG-synthesis by addition of AA abrogated the LH-induced progesterone accumulation markedly, but only on Day 13 of pseudopregnancy. Epinephrine (5 micrograms/ml) increased production of progesterone and also PGs, but only on Day 2 of pseudopregnancy, whereas oxytocin (100 mIU/ml) was found to be without effect on progesterone as well as PG secretion on all days tested. The results of the present study demonstrates the independent ability of the rat CL to synthesize PGG/PGH2-derived prostaglandins, including the putative luteolysin PGF2 alpha. Secondly, we demonstrate that LH and AA-induced increases in PGF2 alpha and PGE2 production during the luteolytic period, may be an autocrine or paracrine mechanism involved in luteolysis.     

The existence of distinct classes of prostaglandin E2 receptors mediating adenylate cyclase and phospholipase C pathways in osteoblastic clone MC3T3-E1.

We have reported previously that PGE2 evoked an increase in intracellular calcium level ([Ca2+]i) in mouse osteoblastic cells (1). Here, we investigated the effects of PGE1 and PGF2 alpha on cAMP production and [Ca2+]i in comparison with those of PGE2. In osteoblastic clone, MC3T3-E1 cells, PGE1 stimulated cAMP production, but had no effect on [Ca2+]i, whereas PGF2 alpha evoked only [Ca2+]i increase. In contrast, PGE2 not only stimulated cAMP production, but also increased [Ca2+]i. From the Scatchard plot analysis of PGE2 it was confirmed that there were two classes of PGE2 binding sites (Kd value, 9.2 nM; binding site, 29 fmole/mg protein, and Kd value, 134 nM; binding site, 148 fmole/mg protein). As the increase in [Ca2+]i was caused by PGF2 alpha and PGE2, but not by PGE1, we investigated the displacement of [3H]-PGF2 alpha binding. The displacement capacity of unlabeled PGE2 was about 110 of that of PGF2 alpha, while that of PGE1 was very low even at 500-fold excess. These data indicate the possibility that the dual action of PGE2 is mediated by distinct receptor systems.     

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.     

The regulation of intracellular pH in monkey kidney epithelial cells (BSC-1). Roles of Na+/H+ antiport, Na+-HCO3(-)-(NaCO3-) symport, and Cl-/HCO3- exchange

Using the pH-sensitive absorbance of 5 (and 6)-carboxy-4',5'- dimethylfluorescein, we investigated the regulation of cytoplasmic pH (pHi) in monkey kidney epithelial cells (BSC-1). In the absence of HCO3-, pHi is 7.15 +/- 0.1, which is not significantly different from pHi in 28 mM HCO3-, 5% CO2 (7.21 +/- 0.07). After an acid load, the cells regulate pHi in the absence of HCO3- by a Na+ (or Li+)-dependent, amiloride-inhibitable mechanism (indicative of Na+/H+ antiport). In 28 mM HCO3-, while still dependent on Na+, this regulation is only blocked in part by 1 mM amiloride. A partial block is also observed with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) (1 mM). With cells pretreated with DIDS, 1 mM amiloride nearly totally inhibits this regulation. Cl- had no effect on pHi regulation in the acidic range. In HCO3(-)-free saline, Na+ removal leads to an amiloride-insensitive acidification, which is dependent on Ca2+. In 28 mM HCO3-, Na+ (and Ca2+) removal led to a pronounced reversible and DIDS-sensitive acidification. When HCO3- was lowered from 46 to 10 mM at constant pCO2 (5%), pHi dropped by a DIDS-sensitive mechanism. Identical changes in pHo (7.6 to 6.9) in the nominal absence of HCO3- led to smaller changes of pHi. In the presence but not in the absence of HCO3-, removal of Cl- led to a DIDS-sensitive alkalinization. This was also observed in the nominal absence of Na+, which leads to a sustained acidification. It is concluded that in nominally bicarbonate-free saline, the amiloride-sensitive Na+/H+ antiport is the predominant mechanism of pHi regulation at acidic pHi, while being relatively inactive at physiological values of pHi. In bicarbonate saline, two other mechanisms effect pHi regulation: a DIDS-sensitive Na+-HCO3- symport, which contributes to cytoplasmic alkalinization, and a DIDS-sensitive Cl-/HCO3- exchange, which is apparently independent of Na+.     

Thrombin induces a calcium transient that mediates an activation of the Na+/H+ exchanger in human fibroblasts

The calcium dependence of growth factor-induced cytoplasmic alkalinization was determined in serum-deprived human fibroblasts (WS-1 cells). Intracellular pH (pHi) and intracellular calcium (Ca2+i) were measured using the fluorescent dyes 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein and fura2, respectively. Thrombin (10 nM) induced an alkalinization (0.18 +/- 0.01 pH units, n = 23) that was Na+-dependent and amiloride-sensitive, suggesting that the alkalinization was mediated by the Na+/H+ exchanger. Thrombin treatment caused a transient increase in Ca2+i (325 +/- 39 nM, n = 12) that preceded the observed increase in pHi. The increases in Ca2+i and pHi were dependent on the concentration of thrombin. The thrombin-induced increase in Ca2+i occurred in the absence of external calcium indicating that thrombin released calcium from internal stores. Inhibition of the thrombin-induced increase in Ca2+i with 8-diethylaminooctyl 3,4,5-trimethoxybenzoate hydrochloride or bis-(o-aminophenoxy)ethane-N,N,N',N'- tetraacetic acid also inhibited the thrombin-stimulated increase in pHi. The calcium ionophore ionomycin was used to increase Ca2+i independent of growth factor stimulation. When Ca2+i was elevated with ionomycin, a concomitant increase in pHi was observed. The increase in pHi due to ionomycin was dependent on Na+ and sensitive to amiloride. The removal of external Ca2+i inhibited the ionomycin-induced elevation of both Ca2+i and pHi. The ionomycin-induced increases in Ca2+i and pHi were not inhibited by 8-diethylaminooctyl 3,4,5-trimethoxy-benzoate hydrochloride. The results suggest that thrombin treatment can activate the Na+/H+ exchanger, and this activation is mediated by an increase in Ca2+i.     

Thrombin-induced changes of intracellular [Ca2+] and pH in human platelets. Cytoplasmic alkalinization is not a prerequisite for calcium mobilization

We have studied the effects of thrombin (0.1 U/ml) on intracellular Ca2+ ([Ca2+]i) and pH (pHi) in human platelets loaded with fluorescent indicators. Thrombin produced a transient decrease of pHi which reached its maximum within 15-25 seconds (s) and was followed by a sustained alkalinization which brought pHi above the resting value. [Ca2+]i increased transiently peaking at 5-10 s. The late alkalinization induced by thrombin was antagonized by ethylisopropylamiloride, an inhibitor of Na+-H+ exchange, and by sphingosine, an inhibitor of protein kinase C, with little effect on the [Ca2+]i transient. The early acidification was not inhibited by these treatments. We conclude tha the thrombin-induced changes of [Ca2+]i and pHi are mediated by different mechanisms. The late alkalinization is due to activation of Na+/H+ exchange mediated by protein kinase C and, contrarily to previous proposals (Siffert, W. and Akkerman, J.W.N. (1987) Nature 325, 456-458), it is not necessary for calcium mobilization from intracellular stores.     

Evidence of alpha 1-adrenergic----protein kinase C----Na+/H+ antiporter-dependent increase in pinealocyte intracellular pH. Role in the adrenergic stimulation of cGMP accumulation

The regulation of intracellular pH (pHi) in isolated rat pinealocytes was studied using the fluorescent pH indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. Resting pHi was 7.09 when the extracellular pH (pHe) was 7.2. Treatment of pinealocytes with the physiological regulator of pineal function, norepinephrine, resulted in a concentration-dependent increase in pHi. Further analysis indicated that norepinephrine is probably acting via an alpha 1-adrenergic----[Ca2+]i----Ca2+/phospholipid- dependent protein kinase (protein kinase C) mechanism to activate the Na+/H+ antiporter, thereby causing cytoplasmic alkalization. A potential influence of cytosolic alkalization on the responsiveness of cyclic nucleotides to adrenergic agonists was also studied. Five analogs of the antiporter inhibitor amiloride reduced norepinephrine stimulation of cGMP accumulation with the same relative potency as they act on the antiporter. In contrast, although inhibitory effects of these compounds on cAMP accumulation were detectable, they occurred at 10-100-fold higher concentrations, and the relative potency of these inhibitors did not indicate they were acting via the antiporter. These findings provide evidence that 1) alpha 1-adrenergic receptor activation increases pinealocyte pHi through Ca2+----protein kinase C-dependent activation of the Na+/H+ antiporter; and 2) norepinephrine stimulation of cGMP accumulation is pHi-dependent. It would appear that alpha 1-adrenergic regulation of pHi via the Na+/H+ antiporter may be of general importance in the control of cGMP accumulation.     

Phorbol ester-like action of staurosporine on the cAMP response to prostaglandin E2 in two macrophage-like cell lines at distinct differentiation stages.

The purpose of this study is to investigate the involvement of protein kinase C (PKC) in prostaglandin E2 (PGE2)-stimulated cAMP production of two macrophage-like cell lines (G3 and XC). XC cells are thought to be placed at a more differentiated stage than G3 cells [Orikasa et al. (1991) Cell Immunol. 132, 350-365]. In RPMI 1640 containing 10% (v/v) heat-inactivated foetal calf serum (FCS), in which the cAMP response of both cells to PGE2 increased with duration of culture, XC cells showed greater response than G3 cells at 2 days of culture. In alpha-minimum essential medium (alpha-MEM) containing 20% (v/v) heat-inactivated horse serum (HS), the cAMP response of both cells was apparently greater than that in RPMI 1640 containing 10% (v/v) FCS. These cells increased cAMP production also in response to PGE1 and PGF2 alpha, and the order of potency in increase was PGE1 > PGE2 > PGF2 alpha. Interestingly, a short-term (20 min) treatment with phorbol 12-myristate 13-acetate (PMA), a direct activator of PKC or staurosporine, a relatively specific inhibitor of PKC, augmented the PGE2-stimulated cAMP production in these cells cultured in alpha-MEM containing 20% (v/v) HS. However, a long-term (24 h) treatment with these compounds did not alter the cAMP response. In G3 cells, PMA appeared more potent than staurosporine in terms of augmentation, whereas in XC cells, the former appeared less potent than the latter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding and second messengers of prostaglandins F2 alpha and E1 in primary cultures of rabbit endometrial cells

Several factors and hormones are thought to play a role in the growth control of endometrial cells. We have shown that prostaglandin F2 alpha (PGF2 alpha) is a growth factor for primary cultures of rabbit endometrial cells grown in serum-free, chemically defined medium and that prostaglandin E1 (PGE1) antagonizes the PGF2 alpha induction of growth (Orlicky et al., 1986). [3H]PGF2 alpha binds to whole cells in a time (optimal approximately 30 min)- and temperature-dependent (optimal 37 degrees C), disassociable (90% disassociable within 30 min), saturable (Kd1 = 4.9 X 10(-8) M, n1 = 1.2 X 10(5) molecules/cell; Kd2 = 2.6 X 10(-7) M, n2 = 3.0 X 10(5) molecules/cell), and specific manner. [3H]PGE1 binds in a time-dependent (optimal 25 min), disassociable (90% disassociable within 10 min), saturable (Kd = 6.4 X 10(-8) M, n = 1.2 X 10(5) molecules/cell), and specific manner. This specific binding of [3H]PGF2 alpha and [3H]PGE1 is down-regulatable by prior treatment of the cultures with unlabeled ligand, and up-regulatable by prior treatment of the cultures with indomethacin to inhibit endogenous PG synthesis. Proteolytic enzyme treatment for 2 min reduces the specific binding of PGF2 alpha by 75%. PGE1 stimulates intracellular cAMP synthesis and accumulation in a time (optimal 10 min)- and concentration (half-maximal stimulation at 10(-6) M)-dependent manner but has no effect on intracellular cGMP. PGF2 alpha has no effect on either intracellular cAMP or cGMP in this system. We describe here for the first time the analysis at a biochemical level of the interaction between two prostaglandins, antagonistic to each other in terms of growth regulation.     

Cytoplasmic pH is differently regulated in the monoblastic U-937 and erythroleukemic K-562 cell lines

Regulation of cytoplasmic pH (pHi) of the human monoblastic U-937 and erythroleukemic K-562 cell lines was investigated. The apparent resting pHi, as assessed by the fluorescent pH probe quenel, were 6.61 and 6.75 for the U-937 and K-562 cells, respectively. When extracellular Na+ was substituted by equimolar choline+, pHi decreased by about 0.2 units. The protein kinase C activating beta-form of the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 10(-10) and 10(-7) M) induced a dose-dependent alkalinization in both cell types of 0.03-0.12 units, whereas the alpha-form was inactive. The response was detectable after about 2 min and reached steady-state 10-15 min later. In the K-562 cells the alkalinization was mediated by Na+/H+ exchange as it was accompanied by stimulation of H+ extrusion and abolished by Na+ removal. The TPA response in the U-937 cells, however, was unaffected by Na+ removal, not accompanied by H+-efflux, and thus unrelated to Na+/H+ exchange. Since electron microscopy indicated development of multivesicular bodies with an acidic interior, the alkalinization can probably be accounted for by an intracellular mechanism. Ionomycin (10(-5) M) induced a rapid increase in the cytoplasmic Ca2+ concentration of both cell types and this response was accompanied by acidification followed by a Na+-dependent recovery. In the U-937, but not in the K-562, cells this recovery was followed by a net alkalinization. It is concluded that both cell types possess a Na+/H+ exchange of importance for pHi but that this mechanism is regulated differently in the U-937 and K-562 cells.     

Effects of dibutyryl cAMP, LHRH, and aromatase inhibitor on simultaneous outputs of prostaglandin F2 alpha, and 13,14-dihydro-15-keto-prostaglandin F2 alpha by term placental explants

Explants from term human placentas were maintained in culture with daily changes of medium. Daily output of PGF2 alpha and PGFM1 decreased during the course of the incubation. Addition of 4 micrograms/ml DHEAS or 67 micrograms/ml LDL cholesterol had no effect on output of PGF2 alpha or PGFM. Addition of 1.6, 3.2, or 6.4 micrograms/ml of LHRH to the culture plates had no effect on output of PGFM or PGF2 alpha, but LHRH increased hCG output. Dibutyryl cAMP (1mM, 2mM, and 4 mM) increased output of PGF2 alpha, PGFM, and hCG. Aromatase inhibitor decreased hCG output, but it was without effect on output of PGF2 alpha, or PGFM, Significant correlations were demonstrated between progesterone, PGFM, PGF2 alpha, and hCG, suggesting that PGF2 alpha originates in the syncytiotrophoblast cell. The ability of LHRH to stimulate output of hCG but not PGF2 alpha while dbcAMP stimulated both suggests that either PGF2 alpha and hCG arise in different cells or that LHRH does not act through cAMP.