Effect of atropine, PGF2 alpha and cimetidine on the beta-carotene induced cytoprotection in ethanol-treated rats

The aim of the study was to evaluate the influence of atropine, PGF2 alpha and cimetidine on the gastric cytoprotective effect of beta-carotene. Mucosal damage was produced by intragastric (i.g.) addition of 96% ethanol in CFY-strain rats of both sexes weighing 180-220 g. Gastric cytoprotection caused by i. g. pretreatment with 1.0 mg/kg beta-carotene 30 minutes before ethanol administration, was observed after 1 hour. Atropine (0.5 mg/kg), cimetidine (50 mg/kg) and PGF2 alpha (200 micrograms/kg) were given intraperitoneally (i.p.) 30 minutes before ethanol administration with and without beta-carotene and the changes in the number and severity of the gastric ulcers were detected. PGF2 alpha did not influence the gastric cytoprotective effect of beta-carotene meanwhile it was inhibited by atropine and markedly by cimetidine. Deleterious effect of cimetidine on the beta-carotene-induced cytoprotection may be explained perhaps by the adverse effect of the two compounds on ATP-cAMP transformation hereby counteracting one another, but more data are needed to the better understanding of drug interactions relating to mucosal cytoprotection.     

The effect of different prostaglandins on gastric mucosal intracellular second messenger system in the rat.

After an oral administration of 100 micrograms/kg dose, the investigated prostaglandins: PGF2 alpha, PGE2 and a synthetic PGE2 derivative: FCE-20700, exerted a significant effect on cAMP and cGMP content of both parts (antral and fundic) of gastric mucosa, resulting in an elevated cAMP/cGMP ratio, while 6-keto-PGF1 alpha, the stable break-down product of prostacyclin, was inactive. Since the above-mentioned phenomenon seems to be proportionate to the cytoprotective (anti-ulcerogenic) property of the investigated prostaglandins, this cAMP/cGMP ratio "shift" is interpreted as a probable (molecular) sign of the reparative, (anti-ulcerogenic) processes.     

The protective effects of a prostaglandin without antisecretory properties against ethanol-induced injury in the rat stomach: a histologic study

This study examined the effect of 2-acetyl-2-decarboxy-15(S)-15 methyl PGF2 alpha (PGF2 alpha) on ethanol (EtOH) induced injury in the rat stomach to determine if a PG analogue devoid of antisecretory properties could confer full or partial gastric mucosal protection. Rats were orally administered saline or PGF2 alpha in a dose of 0.5 or 5.0 mg/Kg. Thirty minutes later animals received varying concentrations (i.e. 25%, 50%, and 100%) of EtOH orally. Five minutes following EtOH exposure, they were killed and samples taken from identical regions of the glandular mucosa for microscopic evaluation. All concentrations of EtOH tested damaged the gastric epithelium. The injury induced by 25% EtOH was almost exclusively confined to the surface epithelium and was not altered by either dose of PGF2 alpha pretreatment. In contrast, both 50% and 100% EtOH elicited comparable damage to the gastric mucosa involving both the deep and superficial mucosa of virtually the entire epithelium. The deep injury induced by these two EtOH concentrations was prevented by both the low and high dose of PGF2 alpha. Of particular importance the 5.0 mg dose of PGF2 alpha provided complete protection (i.e. both superficial and deep) to as much as 50% of the mucosa exposed to 50% or 100% ethanol. These findings indicate that PGF2 alpha possesses "cytoprotective" properties involving both the superficial and deep epithelium that are dose related.     

Stimulatory effect of prostaglandins on the production of hexosamine-containing substances by cultured fibroblasts (4) adenosine 3':5'-cyclic monophosphate independent stimulation by prostaglandin F2alpha.

The mechanism of the stimulatory effect of prostaglandin PG) F2alpha on the production of hexosamine-containing substances by cultured fibroblasts was studied with special reference to adenosine 3':5'-cyclic monophosphate (cAMP). At the stationary phase, the cells were exposed for 6 hrs to PGF2alpha, E1, cAMP or dibutyryl-cAMP in a wide range of concentrations. cAMP itself showed a slight stimulation on the production of hexosamine-containing substances, and the effect was enhanced by using the dibutyryl derivative. PGF2alpha had much a greater capacity than either the exogeneous cAMP or the dibutyryl-cAMP for enhancing the production of hexosamine-containing substances. To know whether cAMP is involved in the stimulatory effect of PGF2alpha, intracellular cAMP level was concomitantly measured in both PGF2alpha and PGE1 treated cultures. Although the cellular cAMP level in PGE1 treated cultures was much higher than that in the PGF2alpha treated cultures, the stimulatory effect on the production of hexosamine-containing substances in PGE1 treated cultures was always much smaller than that in the PGF2alpha treated cultures. Moreover, PGF2alpha had a significant stimulatory effect on the production of hexosamine-containing substances even at a low concentration as 100 pg/ml, which is small enough not to increase any cellular cAMP level. From these results, it was concluded that the stimulatory effect of PGF2alpha on the production of hexosamine-containing substances by cultured fibroblasts is not mediated by cAMP and is caused by a mechanism different from that caused by cAMP.     

The effect of PGF2 alpha on parathyroid hormone-stimulated cyclic AMP production in mouse osteoblastic cell, MC3T3E1.

The effect of prostaglandin F2 alpha (PGF2 alpha) was investigated in MC3T3E1 cells on the succeeding cAMP response to parathyroid hormone (PTH). PGF2 alpha increased the membrane-associated protein kinase C (PKC) activity, indicating the activation of this enzyme. The effect of PTH to increase cAMP production was enhanced by pretreatment with PGF2 alpha. Phorbol 12-myristate 13-acetate also enhanced cAMP production stimulated by PTH, and PKC inhibitor H7 attenuated the enhancement of PGF2 alpha. A23187 did not reproduce the PGF2 alpha effect, and this effect was not antagonized by the calmodulin antagonist W7. PGF2 alpha did not change the ED50 nor the maximally responsive dose of PTH in stimulating cAMP production. The effect of PGF2 alpha was not affected by pertussis toxin, and PGF2 alpha also enhanced cholera toxin- or forskolin-stimulated cAMP production. In accordance with the response of cAMP to PTH, the resorption of mouse limb bones stimulated submaximally by PTH was enhanced by the concomitant presence of PGF2 alpha. These results indicate that PGF2 alpha modulates cAMP response through the activation of PKC, the target of which might be the catalytic unit of adenylate cyclase. Such interaction between signal transduction systems may have significance in modulating the effect of PTH on bone, i.e., bone resorption.     

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.     

Effect of proximal selective vagotomy on gastric prostaglandin content in the Shay-rat ulcer model

During Shay-ulcer formation damages to the barrier of the gastric mucosa develop even before the appearance of macroscopic ulceration. Proximal selective vagotomy prevents these damages. Following pyloric ligation the prostaglandin content of the mucosa changes in parallel with the injuries of the mucosal barrier: TXB2 content of the forestomach increases, while PGF2 alpha content of both the forestomach and the antrum decreases. Following PSV operation the 6-keto-PGF1 alpha content of the mucosa decreases, whereas PGF2 alpha and TXB2 contents exhibit no alteration. As a combined effect of proximal selective vagotomy pretreatment and pyloric ligation the 6-keto-PGF1 alpha and PGF2 alpha contents of the mucosa remain low and the TXB2 increase, otherwise detectable after pyloric ligation, does not take place.     

Pertussis toxin abolishes the inhibitory effects of prostaglandins E1, E2, I2 and F2 alpha on hormone-induced cAMP accumulation in cultured hepatocytes

Several prostaglandins inhibit the cAMP response to glucagon and beta-adrenergic stimulation in hepatocytes. To probe the mechanism of this inhibition, we have examined in primary hepatocyte cultures how pretreatment with pertussis toxin (islet-activating protein) influences the ability of the cells to respond to hormones and prostaglandins. Pertussis toxin augmented the effects of glucagon, epinephrine and isoproterenol, and also markedly enhanced the cAMP response to prostaglandin E1 (PGE1). Furthermore, whereas PGE1, PGE2, PGI2 and PGF2 alpha attenuated the cAMP responses to glucagon in control cultures, this inhibition was abolished in cells pretreated with pertussis toxin. A more detailed comparison was made of the effects of PGE1 and PGF2 alpha. In cells not treated with pertussis toxin, both these prostaglandins at high concentrations reduced the cAMP response to glucagon and isoproterenol by approximately 50%, but dose-effect curves showed that PGE1 was about 100-fold more potent as an inhibitor than PGF2 alpha. Pertussis toxin abolished the inhibitory effects of PGE1 and PGF2 alpha with almost identical time and dose requirements. The results obtained with PGE1, PGE2, PGI2 and PGF2 alpha suggest that prostaglandins of different series attenuate hormone-activable adenylate cyclase in hepatocytes through a common mechanism, dependent on the inhibitory GTP-binding protein.     

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.     

Effect of phorbol ester on prostaglandin regulation of proliferation in rabbit endometrial cells

We have proposed that two of the endogenously synthesized endometrial prostaglandins, prostaglandin F2 alpha (PGF2 alpha) and prostaglandin E1 (PGE1), play a regulatory role in growth control of the endometrium. PGF2 alpha increases DNA synthesis and PGE1 inhibits that effect. Primary cultures of rabbit endometrial cells were used here to examine the effects of the tumor-promoting, diacylglycerol mimicking, phorbol ester, 12-O-tetradecanoyl phorbol-13-acetate (TPA), on the prostaglandin control of cell proliferation. TPA treatment of these cultures results in: a decrease in control levels of proliferation and complete inhibition by TPA of PGF2 alpha stimulated DNA synthesis; a reduction in [3H]PGF2 alpha binding with short term treatment but an increase to above control binding level with long term treatment; an inhibition of the normal PGF2 alpha stimulated inositol polyphosphate synthesis; and a small increase in accumulation of PGF2 alpha in the culture media. Furthermore, in this culture system, TPA does not down regulate [3H]PGE1 binding; it does not alter the normal PGE1 stimulation of cAMP synthesis; and it has no effect on the normal endogenous PGE1 synthesis by these cultures. The above results are consistent with our previous observations that PGF2 alpha works through the intracellular messengers inositol polyphosphate/diacylglycerol whereas PGE1 works through cAMP.     

Synergistic effect of prostaglandin F2alpha and cyclic AMP on glucose transport in 3T3-L1 adipocytes

The combined effect of prostaglandin F2alpha (PGF2alpha) and cAMP on glucose transport in 3T3-L1 adipocytes was examined. In cells pretreated with PGF2alpha and 8-bromo cAMP for 8 h, a synergy between these two agents on glucose uptake was found. Insulin-stimulated glucose transport, on the other hand, was only slightly affected. The synergistic effect of these two agents was suppressed in the presence of cycloheximide and actinomycin D. In concord, immunoblot and Northern blot analyses revealed that GLUT1 protein and mRNA levels were both increased in cells pretreated with both PGF2alpha and 8-bromo cAMP, greater than the additive effect of each agent alone. The synergistic action of PGF2alpha with 8-bromo cAMP to enhance glucose transport was inhibited by GF109203X, a selective protein kinase C (PKC) inhibitor. In addition, in cells depleted of diacylglycerol-sensitive PKC by prolonged treatment with 4beta-phorbol 12beta-myristate 13alpha-acetate, a PKC activator, the synergistic effects of PGF2alpha and 8-bromo cAMP on glucose transport and GLUT1 mRNA accumulation were both abolished. Taken together, these results indicate that PGF2alpha may act with cAMP in a synergistic way to increase glucose transport, probably through enhanced GLUT1 expression by a PKC-dependent mechanism.     

Cyclic AMP formation of isolated human corpora lutea in response to HCG - interference by PGF2 alpha.

Human corpora lutea of defined ages were excised at operation, cut into pieces and incubated in the presence of HCG, PGF2 alpha and PGE2 alone or in combination. Following incubation cAMP formation in tissue and medium was determined. HCG-stimulated tissue cAMP content was most pronounced at a corpus luteum age of 7-10 days after ovulation. This stimulation was antagonized by PGF2 alpha in corpora lutea older than 6 days. PGE2 stimulated cAMP formation per se and this effect was more pronounced when HCG and PGE2 were combined. A possible role for PGF2 alpha as a luteolytic substance in the human is suggested.     

The effect of different prostaglandins on cysteamine-induced duodenal ulcer in the rat

Most of the experiments have verified the cytoprotective effect of prostaglandins on the gastric mucosa and only few observations are known to deal with "cytoprotection" exerted on the duodenal or intestinal mucosa. Duodenal ulcers were produced by cysteamine (CEA). The treatment with different prostaglandins (PGI2, PGF2 alpha, PGE2) was carried out every six hours during the 48-hour experimental period. The obtained results indicate that neither prostacyclin nor prostaglandin-F2 alpha has any significant effect on CEA-induced duodenal ulceration, while prostaglandin-E2 exerts a slight anti-ulcerogenic effect. It is possible that cytoprotection is not the result of the same process in the duodenal and gastric mucosa: alternatively, three prostaglandins are not involved in the pathomechanism of CEA-induced duodenal ulceration.     

Prostaglandins enhance parathyroid hormone-evoked increase in free cytosolic calcium concentration in osteoblast-like cells.

Prostaglandins (PGs) are autocrine or paracrine hormones that may interact with circulating hormones such as parathyroid hormone (PTH) in bone. We examined the interaction of the PGs, PGF2 alpha, PGE2, and 6-keto-PGF1 alpha with PTH to enhance the rapid, initial transient rise in free cytosolic calcium ([Ca2+]i) and cAMP levels stimulated by PTH. Pretreatment of UMR-106, MC3T3-E1, and neonatal rat calvarial osteoblast-like cells by PGs resulted in an enhancement of the early transient rise in [Ca2+]i stimulated by PTH. PGF2 alpha was approximately 100 times more potent than PGE2. PGE2 itself was more potent than 6-keto-PGF1 alpha in enhancing PTH-stimulated rise in [Ca2+]i. Near-maximal augmentation was achieved at PGF2 alpha doses of 10 nM and PGE2 of 1 microM. The degree of augmentation in [Ca2+]i by PGF2 alpha was independent of preincubation time. PGF2 alpha pretreatment did not alter the EC50 for the PTH-induced [Ca2+]i increase but only the extent of rise in [Ca2+]i at each dose of PTH. The augmented increase in [Ca2+]i was mostly due to enhanced PTH-mediated release of Ca2+ from intracellular stores. PGF2 alpha did not stimulate an increase in PTH receptor number as assessed by [125I]-PTH-related peptide binding. PG pretreatment partially reversed PTH inhibition of cell proliferation, suggesting that an increase in [Ca2+]i may play a role in tempering the anti-proliferative effect of PTH mediated by cAMP. These studies suggest a new mode by which PGs can affect cellular activity.     

Gastric anti-ulcerogenic drug effect. A possible mechanism of its molecular basis.

During experimental gastric ulceration in rats an elevation in the mucosal cAMP/cGMP ratio can be encountered. The cause of this significant elevation is mainly (but not entirely) the dramatic fall of the cGMP level. Similar observations were obtained with prostacyclin application (100 micrograms/kg, p.o.), too. This prostaglandin derivative is well known, among others, because of its pronounced anti-ulcerogenic (cytoprotective) effect, too. Other substances of different molecular structure and properties may also exert such effect. The exact mechanism of action of this above-mentioned cytoprotection is still not completely understood. H2-receptor blocker drug cimetidine, given in such small dose (5 mg/kg, p.o.) which does not interfere with gastric acid secretion, also exerts very significant cytoprotective effect in stress (restraint)- and drug (indomethacin)-induced gastric ulcer models. Under cimetidine effect--together with a noticeable endogenous prostacyclin mobilization--the gastric mucosal cAMP/cGMP ratio was also strongly elevated. We conclude that this elevation in the mucosal cAMP/cGMP ratio might be a possible molecular basis of the gastric cytoprotective (anti-ulcerogenic) drugs but it needs further investigations whether all substances exerting cytoprotective effect, e.g. atropine, somatostatin, sulfhydryl drugs, etc., have the same "shifting" property or not? Moreover the phenomenon of the so-called "adaptive cytoprotection" can not be ruled out completely either, therefore this problem needs attention, too.     

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.     

Adaptive cytoprotection against alcohol injury in the rat stomach is not due to increased prostanoid synthesis

This study evaluated the effects of 25% ethanol, a mild irritant, on endogenous prostanoid synthesis in the rat stomach before and after exposure to oral 100% ethanol. Rats received water or 25% ethanol orally. After 15 min, a portion of each group was sacrificed and the remaining animals treated with 100% ethanol prior to sacrifice one minute later. Microsomal membrane fractions were prepared from the glandular gastric mucosa in all groups and incubated with 14C arachidonic acid in the presence of cofactors. Endogenous mucosal prostanoid synthesis was analyzed by radiochromatography and results correlated with the presence or absence of gastric injury macroscopically. Prostanoids measured included PGI2, PGF2 alpha, PGE2, PGD2, PGA2, and thromboxane A2. Additional experiments were performed in like manner to those just described with the exception that indomethacin (5 mg/kg intraperitoneally) pretreatment was rendered. Stomachs exposed to water or 25% ethanol alone demonstrated a modest and equivalent level of synthesis of all prostanoids measured. Exposure to 100% ethanol (with and without mild irritant pretreatment) significantly increased prostanoid synthesis (especially PGI2, PGF2 alpha, and PGE2) compared with stomachs exposed to water or 25% ethanol alone; only mild irritant treated mucosa was protected from injury by 100% ethanol. Indomethacin pretreatment reversed the increased prostanoid synthesis in mucosa exposed to 100% ethanol, with or without mild irritant pretreatment, and partially reversed the protective effect of 25% ethanol. Other experiments using tissue slices in which perturbations in mucosal levels of prostanoids were measured by radioimmunoassay under identical experimental conditions exhibited similar results. These data dispute the notion that adaptive cytoprotection is mediated by increased endogenous prostanoid synthesis. The partial reversal of this process by indomethacin was most likely secondary to some other action of this agent, such as a reduction in gastric blood flow, rather than direct effects on prostanoid synthesis.     

Changes in endogenous prostaglandin levels during D-galactosamine-induced liver injury

The role of prostaglandins (PGs) in liver injury induced by D-galactosamine was investigated in the rat. The contents of PGD2 and PGF2 alpha in the liver were significantly increased from 3 h and 24 h after the D-galactosamine administration, respectively, but that of PGE2 was not significantly changed. Administration of 16,16-dimethyl PGE2, a long acting derivative of PGE2, or indomethacin, but not 16,16-dimethyl PGF2 alpha, a long acting derivative of PGF2 alpha, significantly depressed the increase in the serum transaminase activities induced by D-galactosamine. The protective effect of indomethacin was not disturbed by the 16, 16-dimethyl PGF2 alpha administration. These results indicate that PGE2 has a cytoprotective effect against the D-galactosamine induced liver injury and suggest that the protective effect of indomethacin is ascribable to its suppression of synthesis of PGs other than PGE2 or PGF2 alpha, e.g., PGD2.     

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.     

Effect of prostaglandins on cyclic nucleotide levels in cultured keratinocytes.

Ginea pig ear epidermal cells (keratinocytes) were established in primary cultures using trypsin, and treated in their proliferative phase of growth with prostaglandins E1, D1, F1 alpha, E2, D2, or F2 alpha. This phase is induced by the addition of retinoic acid during cell plating. Intracellular content of cAMP and cGMP was measured by radioimmunoassay at various times after treatment. Maximum stimulation of cAMP levels was observed with PGD2, smaller increases with PGE2 and relatively transient rises with PGF2 alpha which were of low significance, but confirm earlier data. Similar results were observed with PGD1, PGE1, and PGF1 alpha with smaller increases. The effects of D and E PGs were biphasic. Significant increases in cGMP were immediately observed with PGD2 and PGE2. With PGF2 alpha, maximum cGMP levels were noted after some delay. All PGs tested showed some effect in elevating cyclic nucleotides in keratinocytes. The most striking result was the increase in cAMP on PGD2 treatment.