The effect of granulocyte macrophage-colony stimulating factor on the prostaglandin E(2)-like activity of the small intestine of rat during total body irradiation

Radiation-induced gastrointestinal toxicity is important for subjects receiving radiation to the pelvis. Eicosanoids and free radicals may be involved in the mechanism. rHuGM-CSF is a subcutaneously administered drug which may reduce some side effects of radiation. This experimental study was undertaken to determine the effectiveness of rHuGM-CSF on PGE(2)-like activity of the small intestine in rats. Thirty-two adult male Wistar-Albino rats entered the study to be randomized to one of the four groups: Group I. Control; II. Drug administered; III. Irradiated; IV. Irradiated and drug administered. Radiation was by total body irradiation, 800 rads with Cobalt 60. On the 9th day the animals were killed and biopsies were taken from the terminal ileum. PGE(2)-like activity was evaluated. Animals were weighed on the day of irradiation and end of the experiment. A statistically significant difference was found according to pre- and post-treatment weights in the irradiated and nonirradiated drug administered groups (Groups II and IV) (P=0.035 and 0.018, respectively). PGE(2)-like activity in the intestinal tissue was statistically significant higher in the drug-treated animals, both in non-irradiated and irradiated groups. Surprisingly, irradiation was found to decrease the PGE(2)-like activity in the intestinal tissue (P=0.008). rHuGM-CSF was found to increase PGE(2)-like activity in the intestinal tissue. The cellular mechanisms underlying this must be clearly determined and weighed carefully in considering the drug for clinical usage.     

Des-Aspartate-Angiotensin I Attenuates Mortality of Mice Exposed to Gamma Radiation via a Novel Mechanism of Action

ACE inhibitors and ARBs (angiotensin receptor blockers) have been shown to attenuate radiation injuries in animal models of lethal gamma irradiation. These two classes of drug act by curtailing the actions of angiotensin II-linked inflammatory pathways that are up-regulated during gamma radiation in organ systems such as the brain, lung, kidney, and bone marrow. ACE inhibitors inhibit ACE and attenuate the formation of angiotensin II from angiotensin I; ARBs block the angiotensin AT₁ receptor and attenuate the actions of angiotensin II that are elicited through the receptor. DAA-I (des-aspartate-angiotensin I), an orally active angiotensin peptide, also attenuates the deleterious actions of angiotensin II. It acts as an agonist on the angiotensin AT₁ receptor and elicits responses that oppose those of angiotensn II. Thus, DAA-I was investigated for its anticipated radioprotection in gamma irradiated mice. DAA-I administered orally at 800 nmole/kg/day for 30 days post exposure (6.4 Gy) attenuated the death of mice during the 30-day period. The attenuation was blocked by losartan (50 nmole/kg/day, i.p.) that was administered sequential to DAA-I administration. This shows that the radioprotection was mediated via the angiotensin AT₁ receptor. Furthermore, the radioprotection correlated to an increase in circulating PGE₂ of surviving animals, and this suggests that PGE₂ is involved in the radioprotection in DAA-I-treated mice. At the hematopoietic level, DAA-I significantly improved two syndromes of myelosuppression (leucopenia and lymphocytopenia), and mice pre-treated with DAA-I prior to gamma irradiation showed significant improvement in the four myelodysplastic syndromes that were investigated, namely leucopenia, lymphocytopenia, monocytopenia and thrombocytopenia. Based on the known ability of PGE₂ to attenuate the loss of functional hematopoietic stem and progenitor cells in radiation injury, we hypothesize that PGE₂ mediated the action of DAA-I. DAA-I completely attenuated the increase in circulating level of two inflammatory cytokines, TNFα and IL-6, in irradiated mice; and this shows that DAA-I exerted additional anti-inflammatory actions, which could also have contributed to its radioprotection. These findings show that DAA-I acts via a novel mechanism of action on the angiotensin AT₁ receptor to specifically release PGE₂, which mediates radioprotection in the gamma irradiated mice.     

Prostaglandin stimulation of gastrointestinal transit in post-operative ileus rats

The prostaglandins PGF_2α, PGE₂ and 16,16-dimethyl PGE₂, when administered intravenously, orally, subcutaneously or intraduodenally to laparotomized rats, decreased gastric emptying, small intestinal transit and colonic transit as compared to unoperated controls. All three prostaglandins increased colonic transit above that found with unoperated controls. This activity was independent of small intestinal fluid accumulation (i.e., enteropooling) since ligating the ileal-cecal junction had no effect on colonic transit. Small intestinal transit was increased, but not normalized, by PGE₂ and 16,16-dimethyl PGE₂. 16,16-Dimethyl PGE₂ completely restored gastric emptying when given intravenously to laparotomized rats at doses greater than 5.0 μg/kg. This effect on gastric emptying lasted approximately 4 hrs. Thus, 16,16-dimethyl PGE₂, when given intravenously, normalized gastric emptying, significantly increased small intestinal transit, and made the colon hypermotile. Prostaglandins may be beneficial in the treatment of post-operative ileus and other conditions of sluggish gastrointestinal propulsion.     

The protective effect of thromboxane synthetase inhibitor UK 38485 against bile duct ligation induced liver injury

In order to elucidate the relation between tissue eicosanoids and liver injury due to bile duct obstruction, we have examined the effects of iloprost, a stable analogue of prostaglandin I₂ (PGI₂), and UK 38485 (UK), an inhibitor of thromboxane synthetase, on prostaglandin E₂ (PGE₂) and leukotriene C₄ (LTC₄) in guinea pig liver. 56 male guinea pigs were divided into the following groups: (i) sham operations (SHAM), (ii) bile duct ligated (BDL) group, (iii) guinea pigs given UK (5 μg/kg body wt intraperitoneally 10 min, 8 h and 16 h after bile duct ligation), and (iv) guinea pigs treated with iloprost (ILO) (2 μg/kg body wt intraperitoneally 10 min, 8 h and 16 h after bile duct ligation). Liver damage was assessed by blind quantitation of liver cell necrosis. Bile duct ligation caused an increase in tissue PGE₂-like activity and a decrease LTC₄-like activity. But the most pronounced elevation of PGE₂ was observed in ILO treated group. The LTC₄-like activity level improved significantly in the UK-treated BDL group compared with the BDL only and ILO treated animals. Also, UK was found to be beneficial in preventing the liver cell necrosis due to cholestasis. It is concluded that the ratio of PGE₂/LTC₄ in liver is a valuable marker for cholestatic injury.     

Radioprotection to small intestine of the mice against ionizing radiation by semiquinone glucoside derivative (SQGD) isolated from Bacillus sp. INM-1

Ionizing irradiation induces severe damage to the intestinal crypt cells which are responsible for renovation and maintenance of the intestinal cellular architecture. Therefore, protection of intestinal cells and tissue against lethal irradiation using a semiquinone glucoside derivative (SQGD) isolated from radioresistant bacterium Bacillus sp. INM-1 is the prime focus of the present investigation. BALB/c mice were administered by SQGD (50?mg/kg.b.wt. i.p.) 2?h before whole body irradiation (10?Gy), and histological analysis of the jejunum section was carried out and compared to the irradiated mice. Significant (p?<?0.0001) increase in villus length, number of cells per villus, crypts numbers per villus section, total cells counts and mitotic cell counts per crypt and low goblet cells per villus section, and low apoptotic index per crypt section were observed in the irradiated mice pre-treated by SQGD at 48–168?h. Significant induction in NF-kβ at 24?h and Bcl-2/Bax ratio was observed in irradiated mice pre-treated by SQGD compared to only irradiated animals. SQGD pre-treatment before irradiation was found instrumental to reverse the radiation-induced degenerative changes by replenishment of the damaged cells by enhancing mitotic, proliferating, pro-survival, and apoptosis inhibitory activities probably through modulation of cell cycle arrest in G₁/S phase in the intestinal cellular milieu.     

On the multiplicity of platelet prostaglandin receptors I. Evaluation of competitive antagonism by aggregometry

Methods for the evaluation of competitive interactions at receptors associated with platelet activation and inhibition using aggregometry of human PRP have been developed. The evidence supports the suggestion that PGE₁ and PGI₂ share a common receptor for inhibition of platelet reactivity, but only a portion (if any) of the aggregation stimulation associated with PGE₂ is the result of PGE₂ binding (without efficacy) to this receptor. PGE₂ (@.3–20 μ ) is an effective antagonist of PGE₁, PGI₂, producing a shift of about one order of magnitude in the IC₅₀-values obtained from complete aggregation inhibition dose response curves. The antagonism of PGD₂ inhibition is particularly notable, 80 n PGE₂ levels are detectable. This and other actions of PGE₂ indicate another platelet receptor for PGE₂. PGE₁ acts at both the PGE₂ and PGI₂ receptor. Other substances showing PGI₂-like actions only at high doses (1–30 μ ), display additive responses with PGI₂ indicative of decreased affinity for the I₂/E₁ receptor and the absence of PGE₂-like aggregation stimulation activity.PGI₂ methyl ester has intrinsic inhibitory action not associated with in situ ester hydrolysis. The methyl ester is dissaggregatory showing particular specificity for inhibition of release and second wave aggregation.     

Prostaglandin and prostaglandin synthetase in the cricket, Acheta domesticus

Prostaglandin (PG) synthetase was present in the testes, seminal vesicles, and spermatophores of the male house cricket, Acheta domesticus. The enzyme was not detected in bursa copulatrix, spermatheca, spermathecal canal, and oviducts from virgin females, while substantial activity was measured in the same tissue from mated females. The female appears to receive the enzyme from the spermatophore. A PGE₂-like material was detected by radioimmunoassay in A. domesticus testes and to a lesser extent in the remainder of the male reproductive tract. PG went undetected in virgin female reproductive tissues, while the same tissues from mated females contained an average of 589 pg of PGE₂-like material per female. In in vivo studies, injected PGE₁, PGE₂, and to a smaller degree PGF_2α stimulated oviposition by virgin females. Moreover, N-acetyl-p-aminophenol, a PG synthetase inhibitor, suppressed oviposition in mated females. Post-copulatory PG biosynthesis in the female reproductive tract might be partially responsible for triggering oviposition in A. domesticus. Since PG synthetase appears to be acquired from the male, it could be considered a primer pheromone.     

The synthesis and biological activities of some 12-Aza-prostaglandin analogues

12-Aza-prostaglandin (PG) analogues containing the pyrrolidine-2,4-dione ring system have been synthesized from ,2-disubstituted glycine esters via cyclisation of their -ethoxycarbonylacetyl derivatives. 5-(6-Carboxyhexyl)-1-octylpyrrolidine-2,4-dione (5) had little or no PG-like activity on superfused intestinal or vascular smooth muscle preparations but it selectively antagonised smooth muscle responses to PGE₂, PGE₁, PGF_2α and PGA₂in vitro. At a concentration of 10⁻⁵ g/ml it reduced responses of the rat stomach strip to PGE₂ by over 80% but did not affect responses of this tissue to acetylcholine, 5-hydroxytryptamine (5-HT) or bradykinin. Polyphloretin phosphate (PPP), the known PG antagonist, had a similar effect at the same concentration (10⁻⁵ g/ml).5-(6-Carboxyhexyl)-1-(3-hydroxyoctyl)pyrrolidine-2,4-dione (12) had the same profile of activity on superfused smooth muscle preparations as PGE₂ or PGA₂. On intravenous injection into anaesthetised rats it caused dose-dependent falls in arterial blood pressure with associated tachycardias, which is typical of the response to PGE₂. The smooth muscle activity of (12) was not reduced by passage through isolated perfused guinea-pig lungs nor was its potency as a vasodepressor increased when given intra-arterially to rats. These results suggest that, unlike PGE₂, this analogue is not removed by the pulmonary circulation.     

Chronobiological Studies on the Hypotensive Effect of Prostaglandin E2 and Arachidonic Acid in the Rat

The present study was designed to determine whether biological rhythm variations could be detected in the hypotensive action of prostaglandin E₂ (PGE₂) and arachidonic acid (AA) in normal rats. Doses of 1.0 μg kg^?1 of PGE₂ or 0.5 mg kg^?1 of AA were administered to pentobarbital-anesthetized rats at 6 times of the day. Maximal reduction of systolic and diastolic blood pressures was obtained when PGE₂ or AA were administered to rats between 0930 and 1200. The lowest falls in blood pressure were found when the same doses of the two substances were injected between 0300 and 0500. Mechanisms to explain these circadian variations are suggested.     

Prostacyclin production of rat aorta “in vitro” is increased by the combined action of dipyridamole plus pentoxifylline

The present study evaluates the effect of dipyridamole and pentoxifylline, individually and in combination, on PGI₂-like production and arachidonic acid metabolism of rat aorta “in vitro”. Pentoxifylline 100 μM and dipyridamole 92 and 184 μM increased PGI₂-like activity, as measured by the platelet aggregation inhibitory capacity of the aortic ring incubates, by 71%, 46% and 60% respectively; a greater increase in PGI₂-like activity was observed with the combination of the drugs than when they were used separately. This effect was observed even at the lowest doses assayed. In fact, dipyridamole 9.2 μM plus pentoxifylline 1 μM increased the PGI₂-like activity by 30% while the individual increase was 4.5% and 10.6% respectively. To obtain more information on the effect of the dipyridamole-pentoxifylline combination on arachidonic acid metabolism, arteries were incubated with (1-¹⁴C) arachidonic acid, and the 6-keto-PGF_1α and PGE₂ quantified. Dipyridamole 92 μM plus pentoxifulline 1 and 10 μM increased 6-keto-PGF_1α and PGE₂ production by about 30% and 48% respectively while combination with pentoxifylline 100 μM increased the 6-keto-PGF_1α 76.5% and the PGE₂ 50%. The possible biological effect and therapeutic implications of increased PGI₂ production by the arteries due to the dipyridamole-pentoxifylline combination remains to be ascertained.     

Effects of oral administration of PGE2 on gastric secretion and experimental peptic ulcerations

The anti-secretory and anti-ulcer effects of prostaglandin E₂ (PGE₂) using iso-osmotic buffer as a vehicle have been investigated in several types of laboratory animals. Orally administered PGE₂ was found to be highly effective in preventing formation of ulcers in several experimental models -- pylorus ligated induced ulcers in rats, histamine induced ulcers in guinea pigs, reserpine induced ulcers in rats and pentagastrin induced ulcers in guinea pigs and cats. PGE₂ also suppressed acid secretion but not pepsin activity. It was concluded that the anti-ulcer effects of PGE₂ were due to its anti-secretory activity rather than antipepsin activity. In view of PGE₂'s activity in preventing ulceration induced by histamine and reserpine in addition to pentagastrin, it is suggested that the anti-pentagastrin activity of PGE₂ is not specific.     

The role of plasma arachidonic acid metabolites in the pathogenesis and the prognosis of Henoch-Schönlein Purpura

Henoch-Schönlein Purpura (HSP) involve small vessel inflammation. Arachidonate biochemical pathways play an important role in the pathogenesis of vascular inflammation. The aim of this study was to investigate the change in the ratio of plasma arachidonic acid metabolites in the patients with HSP and evaluate the association between clinical activity and prostanoid activity in the acute phase of HSP. Plasma prostaglandin E₂ (PGE₂)-like activities were found to be 7.2 ± 0.8 ng/ml in control group (n=12) while it was 5.3 ± 0.6 ng/ml in the patients with HSP (n=12). Plasma leukotriene C₄ (LTC₄)-like activities were found to be 16.0 ± 1.1 ng/ml in control while it was 30.9 ± 4.3 ng/ml in the patients. The differences of LTC₄-like activities and the ratios between the HSP patients and the controls were significant (p < 0.01, p < 0.001 respectively), but no significant difference was found in PGE₂-like activities. Plasma LTC₄-like activity and ratio were also significantly increased in the patients with high clinical score (p < 0.05, p < 0.02 respectively). These results suggested that not only cyclooxygenase products but also LTs may play an important role in vascular inflammation. Therefore ratio must be taken into consideration in the pathogenesis and the prognosis of HSP.     

Prostaglandin E2 and leukotriene C4 levels following different reperfusion periods in rat brain correlated with morphological changes

Prostaglandin E₂ (PGE₂) and leukotriene C₄ (LTC₄) are the metabolites of arachidonic acid (AA) that increase in forebrain following global ischemia and reperfusion. These mediators are highly potent vasoconstrictors of cerebral arteries leading to enhanced vascular permeability that induces the formation of vasogenic edema. In this study, after developing and experimental animal model simulating the concept of ischemic penumbra in the rat, the levels of PGE₂ and LTC₄ produced in the forebrain were measured and the effects of these mediators in short duration and prolonged reperfusion were investigated and then correlated with nueropathological findings. We found statistically significant reduction both in PGE₂ and LTC₄-like activities after just 10 min ischemia (p<0.05, p<0.05). PGE₂-like activity significantly increased in the 4th and 60th min of reperfusion (p<0.05, p<0.05). In the 15th min of reperfusion, PGE₂ was found to be significantly reduced (p<0.005) that may be due to the formation of free oxygen radicals by activation of PG hydroperoxidase reaction that inhibits PGE₂ production in the cylooxygenase pathway. LTs were not significantly increased in any reperfused group. Inhibition of the lipoxygenase pathway of AA metabolism may occur as a result of 15-HPETE (15-hydroperoxyeicosatetraenoic acid) production. Pathologically, edema and degeneration of brain tissue were seen beginning from the 4th min of reperfusion that reached a peak in the 60th min of reperfusion which is in accordance with biochemical changes in the damaged tissue. It is concluded that by preventing the formation of AA metabolites in the early hours of ischemia and reperfusion, it could be possible to increase blood flow in the ischemic penumbra that should limit the infarct area.     

The effect of intravenously administered prostaglandin E2 on rat uterine 3′, 5′-adenosine monophosphate

PGE₂ administered intravenously increased levels of cyclic AMP in uterine tissue of rats ovariectomized 12 days before treatment. This action of PGE₂ on uterine tissue was dose-dependent, with a dose response curve from 50 to 600 μ/Kg and the maximum effect was seen 10 minutes after PGE₂ administration. Delay of prostaglandin treatment until 25 days post-ovariectomy prevented this response. Administration of estradiol benzoate to such animals however, allowed the rat uterus to respond with elevated cyclic AMP levels at 3 minutes but not at 10 or 45 minutes after PGE₂ treatment.     

The effects of PGF1α, PGE2 and 16,16 dimethyl PGE2 on gastric emptying and small intestinal transit in rat

Prostaglandins are well known for their ability to stimulate contraction in gastrointestinal smooth muscle, yet very little information is available on how their activity affects propulsion . Thus, studies were undertaken to determine the effect of various prostaglandins on qastric emptying (GE) and small intestinal transit (SIT) in unanesthetized fasted rats. Rats were treated with intravenous, subcutaneous, or oral PGF_2α, PGE₂, or 16,16 dimethyl PGE₂ at various doses, followed 1 (intravenous), 20 (subcutaneous) or 10 (oral) mins later by intragastric ⁵¹Cr oxide in black ink. Forty-five mins later, rats were sacrificed by CO₂ asphyxiation, the pylorus clamped, and the gut excised. SIT was expressed as the percent of intestinal length traveled by the most distal portion of ink. GE was expressed as the percent of the ⁵¹Cr emptied into the intestines. If GE was affected by prostaglandin treatment, the experiments were repeated with rats pre-implanted with duodenal cannula. This preparation allowed the visual transit marker to be deposited directly into the dueodenum, thus avoiding acceleration or delay of SIT caused by fluctuations in GE. The results of these studies show that: (1) intravenous 16,16 dimethyl PGE₂ (5–50 μg/kg), but not PGF_2α or PGE₂, accelerates GE and delays SIT; (2) oral prostaglandin administration increases SIT; (3) oral 16,16 dimethyl PGE₂ delays GE; (4) subcutaneous 16,16 dimethyl PGE₂ accelerates, has no effect upon, or delays GE depending upon dose, but accelerates SIT at all doses tested; and (5) subcutaneous PGE₂ accelerates SIT while PGF_2α does not. Thus, the effect of prostaglandins on GE and SIT depends upon the dosage and route of administration as well as type of prostaglandin used.     

Release and inhibitory effects of prostaglandin D2 in guinea pig urinary bladder and the role of urothelium

BackgroundWhile studying a urothelium-derived inhibitory factor in guinea pig urinary bladders we observed considerable release of prostanoids, including PGD₂-like activity. The present study was carried out to identify the prostanoids and to study their roles in modulating guinea pig urinary bladder motility.MethodsRelease of PGE₂ and PGD₂ in isolated guinea pig urinary bladder preparations was analyzed by high performance liquid chromatography (HPLC) combined with bioassay on bladder strips. Isolated urothelium-intact (UI) or -denuded (UD) bladder strips were subjected to electrical field stimulation (EFS) and applications of PGE₂ and PGD₂.ResultsA resting release of 95 ± 9 (n = 5) ng g tissue^− 1 h^− 1 PGE₂-like activity and 210 ± 34 (n = 4) ng g tissue^− 1 h^− 1 PGD₂-like activity was found, where PGD₂-like was subject to marked spontaneous inactivation during isolation. Prostanoids release was decreased by 70–90% by the cyclo-oxygenase inhibitor diclofenac in UI preparations. Urothelium removal decreased prostanoids release by more than 90%.PGE₂ increased basal tone and spontaneous contractions, whereas PGD₂ had little or no effect on these. Exogenous PGE₂ enhanced and PGD₂ inhibited contractile responses to EFS, exogenous acetylcholine- and ATP, whereas PGD₂ caused marked dose-dependent inhibition. PGE₂ and PGD₂ effects were more pronounced in diclofenac-treated UD tissues.ConclusionsPGD₂ and PGE₂ are released from guinea pig bladder urothelium and PGD₂ has inhibitory effects on bladder motility, mainly through a postjunctional action on smooth muscle responsiveness.General significanceThe release and inhibitory effects merit further studies in relation to normal biological function as well as overactive bladder syndrome.     

Rat osteogenic sarcoma cells: Effects of some prostaglandins, their metabolites and analogues on cyclic AMP production

Cyclic AMP production by freshly isolated cells, from a ³²P-induced transplantable rat osteogenic sarcoma, was stimulated by PGE₁, PGE₂ and to a less extent by PGF_2α and PGA₂. In the case of PGE₂, the cyclic AMP content of cells was miximal within 5 min. The 13, 14-dihydro derivatives of PGE₁, PGE₂ and PGF_2α had approximately 40% of the activity of the parent prostaglandin whilst, in every case, the metabolites (15-keto and 13,14-dihydro-15-keto) had very little activity. Two prostaglandin endoperoxide analogues (U44069 and U46619) had only 10% of the activity of an equimolar dose of PGE₂. The data presented in this paper demonstrates similarities between the responses of these cells and cells derived from bony tissue in terms of the ability of prostaglandins to stimulate bone resorption in tissue culture.     

The effect of locally administered PGE2 on the contractility of the nonpregnant human uterus

The effect of locally administered prostaglandin E₂ on the sensitivity and reactivity of the nonpregnant human uterus during the menstrual cycle was studied in seven women. An increase in uterine contractility in response to as little as 0.25 μg PGE₂ could be observed during both the mid-proliferative and mid-secretory phases of the menstrual cycle, but around ovulation a marked decrease in sensitivity to PGE₂ was noted. An inhibition of uterine motility was observed during menstruation in response to 30–40 μg PGE₂. Endogenous E prostaglandin normally occurs in the secretory endometrium in levels comparable to the amount of exogenous PGE₂ which elicited increased or decreased uterine activity in this study. These findings suggest that PGE₂ may play an important role in the cyclical regulation of uterine motility during the menstrual cycle.     

Functional characterization of prostaglandin E2 inducible osteogenic colony forming units in cultures of cells isolated from the neonatal rat calvarium

Prostaglandin E₂ (PGE₂) increases the number of mineralized nodules that form in cultures of rat calvarial (RC) cells. The purpose of our study was to characterize PGE₂-inducible osteogenic colony forming units (CFU-Os) by determining their number, the cell populations from which they were released, their specific responsive period to PGE₂, and their proliferating and differentiating characteristics under the stimulation of PGE₂. Limiting dilution analysis was used to determine the number of PGE₂-inducible CFU-Os. Sequential digestion of intact rat parietal bones with collagenase isolated 5 subpopulations of RC cells that were used to estimate the cell populations where PGE₂-inducible CFU-Os resided. The responsive period of PGE₂-inducible CFU-Os to PGE₂ was evaluated by treating cultures of mixed RC cells for all possible combinations of days 1–10, 11–20, and 21–30. PGE₂ effects on proliferation and differentiation of CFU-Os were evaluated by comparing the DNA synthesis and AP activity in subpopulations I and IV on days 3, 6, and 9. Results showed: (1) PGE₂-inducible CFU-Os represent 0.27% of cells in the mixed RC population, (2) the majority of determined and PGE₂-inducible CFU-Os were found in the subpopulations released during the 60–100 min digestion periods, (3) the response of PGE₂-inducible CFU-Os is limited to the first 10 days of culture, and (4) PGE₂-stimulated nodule formation is associated with an early increase in DNA synthesis and a sustained increase in alkaline phosphatase activity. We conclude that, functionally, PGE₂-inducible CFU-Os are slowly proliferating AP negative cells primarily found in the subpopulations III-V. PGE₂ stimulates them to proliferate and become AP+, and function as determined CFU-Os to form mineralized nodules in vitro. © 1996 Wiley-Liss, Inc.