The effect of prostaglandin I2 and 6a-Carba-PGI2 on the motility of isolated osteoclasts

We have recently found that calcitonin (CT), a hormone which inhibits osteoclastic bone resorption, completely abolishes the normally intense cytoplasmic movement of isolated osteoclasts. We have also found that prostaglandin (PG)I2 causes an identical change in behaviour. In this paper we extend our investigations into the mode of action of PGI2 and a stable analogue, 6a-Carba-PGI2. We found that, unlike CT which causes prolonged immotility in osteoclasts, the effect of PGI2 and 6a-Carba-PGI2 were transient. Our results suggest that the transient nature of the inhibition was neither caused by inactivation of these compounds, nor was it due to production in the cultures of an osteoclastic stimulator. CT, PGI2 and 6a-Carba-PGI2 all appear to operate by increasing the intracellular cyclic AMP level. We found no refractoriness to either CT, dibutyryl cyclic AMP or 8-bromo cyclic AMP, and neither PGI2 nor 6a-Carba-PGI2 affected the sensitivity of osteoclasts to CT or dibutyryl cyclic AMP. This implies that refractoriness of osteoclasts to PGI2 and 6a-Carba-PGI2 develops at some stage in the interaction between PG and cell proximal to cyclic AMP production. We also found that there was cross-tachyphylaxis between PGI2 and 6a-Carba-PGI2, and this suggests that these two compounds share a receptor site on osteoclasts.     

A comparison of the effects of PGI2, PGE2 and PGH2 on the cyclic nucleotide levels in rat anterior pituitary glands

Rat anterior pituitary explants were incubated with PGI₂, PGH₂ and PGE₂ in the presence of theophylline (1mM) and the production of cyclic AMP was measured. PGE₂ was found to be about 20 times more potent than PGI₂ while PGH₂ was slightly more effective than PGI₂. The results suggest that PGI₂ does not play a physiological role in cyclic AMP mediated events in the rat anterior pituitary.     

A comparison of the effects of PGI2, PGE2 and PGH2 on the cyclic nucleotide levels in rat anterior pituitary glands in vitro

Rat anterior pituitary explants were incubated with PGI₂, PGH₂ and PGE₂ in the presence of theophylline (1mM) and the production of cyclic AMP was measured. PGE₂ was found to be about 20 times more potent than PGI₂ while PGH₂ was slightly more effective than PGI₂. The results suggest that PGI₂ does not play a physiological role in cyclic AMP mediated events in the rat anterior pituitary.     

Prostaglandin I2 stimulation of granulosa cell cyclic AMP production

The ability of prostaglandin I₂ (PGI₂) to stimulate cyclic AMP production by granulosa cells, isolated from intact immature rats, has been demonstrated in vitro. The minimal effective dose was 15 ng/ml, which was comparable to the minimal effective dose for PGE₂. However, a concentration of 15 μg/ml PGI₂ was required to stimulate cyclic AMP production maximally, compared to a concentration of 1 μg/ml PGE₂, which produced the maximum response. It therefore appears that PGI₂ is not more effective than PGE₂ in stimulating cyclic AMP production in granulosa cells, and is possibly less effective. Submaximal concentrations of PGI₂ appeared to be able to modify the stimulation of cyclic AMP production by follicle- stimulating hormone (FSH), but whether or not PGI₂ plays any role in follicular function remains to be established.     

Effects of prostaglandin E1, I2 and isoproterenol on the tissue cyclic AMP content in longitudinal muscle of rabbit intestine

Effects of prostaglandin E₁(PGE₁) and prostaglandin I₂(PGI₂) on the mechanical activity and tissue cyclic AMP content of the longitudinal muscle of rabbit intestine were examined, comparing that of isoproterenol. PGE₁ or PGI₂ caused a contraction and did not affect the tissue cyclic AMP content. Isoproterenol caused a relaxation and increasedtissue cyclic AMP content.     

Endogenous synthesis of prostaglandins E2 and I2 in 3T3 fibroblasts transformed by polyoma virus: Effects on adenosine 3′:5′-monophosphate production

Prostaglandins (PG)E₁, E₂ and I₂ were produced by polyoma virus transformed (py) 3T3 fibroblasts. The levels of PGE₁, PGE₂ and 6-keto-PGF_1α (degradation product of PGI₂) were 22.7, 225 and 33.2 ng/ml medium, respectively, 72 h after medium change. The stimulatory potencies of exogenous PGE₁, PGE₂ and PGI₂ on adenosine 3′:5′-monophosphate (cyclic AMP) formation were similar. Therefore, the prostaglandin mediated increase in cyclic AMP levels observed during growth of these cells (Claesson, H.-E., Lindgren, J.Å. and Hammarström, S. (1977) Eur. J. Biochem. , 13) is largely (>80%) mediated by PGE₂ and to lesser extents by PGE₁ and PGI₂.     

6-Keto-prostaglandin E1 is not equipotent to prostacyclin (PGI2) as an antiaggregatory agent

A direct comparison of the relative potencies of the two anti-aggregatory prostaglandins PGI₂ and 6-keto-PGE₁ showed PGI₂ was at least 20 times more potent than 6-keto-PGE₁ when tested against ADP-induced human platelet aggregation. This marked difference in potency was even more evident when the ability of PGI₂ and 6-keto-PGE₁ to stimulate platelet cyclic AMP levels was determined. When cyclic AMP levels were measured direct comparisons were difficult because the respective dose response curves were not parallel, but 10 ng of PGI₂ was equivalent to 300 ng of 6-keto-PGE₁.PGI₂ was also more potent (10–20 times) than 6-keto-PGE₁ as a disaggregatory agent, and the disaggregatory activity of both prostaglandins was enhanced by the phosphodiesterase inhibitor 1-methyl-3-isobutylmethylxanthine.PGI₂ was also more active than 6-keto-PGE₁ as an inhibitor of thrombus formation in dog coronary arteries in vivo. In vivo, 6-keto-PGE₁ was at least 10 times less potent than PGI₂, the exact difference could not be determined because 6-keto-PGE₁ caused significant falls in blood pressure before anti-platelet activity could be detected.PGI₂ is an intrinsically more potent anti-aggregatory molecule than 6-keto-PGE₁, but these data do not rule out the possibility that some of the activities attributed to PGI₂ could be the result of the conversion of PGI₂ and/or 6-keto-PGF_1α to 6-keto-PGE₁.     

Prostaglandins E1 and E2 stimulate release of intestinal brush border enzymes

Rat small bowel was perfused and in the absence of biliary and pancreatic secretion. Intraluminal release of sucrase, alkaline phosphatase, aminopeptidase and enterokinase was significantly increased after administration of PG E₁ and E₂ 1 and 5 μg/kg. This suggests a direct stimulation of the intestinal mucosa, which might be mediated through cyclic AMP ; dibutyryl cAMP significantly stimulates intraluminal release of proteins, sucrase and enterokinase.     

Regulations of macrophage-mediated tumor cell killing by prostaglandins: Comparison of the effects of PGE2 and PGI2

Mouse resident peritoneal macrophages stimulated by purified bacterial lipopolysaccharide (LPS) produced both prostaglandin E₂ (PGE₂) and prostaglandin I₂ (PGI₂), the latter detected as its stable metabolite, 6-keto PGF_1α. Maximum production, induced in each case by 1 ng/ml purified LPS, was in the range of 10⁻⁷M for PGI₂ and 3 × 10⁻⁸M for PGE₂. A quantitatively similar increase in intracellular levels of macrophage cyclic AMP (measured on a whole cell basis), with a similar duration of effect, was stimulated by PGE₂ and PGI₂; however, only PGE₂ had a negative regulatory effect on macrophage activation for tumor cell killing. These data confirm that more than a whole cell increase in the concentration of cyclic AMP is needed to shut off nonspecific tumor cell killing mediated by LPS-activated resident peritoneal macrophages.     

Prostacyclin-induced hypothermia: Involvement of central histamine H2-receptors

Some of the biological activities of prostacyclin (PGI₂) are known to be mediated through cyclic AMP (cAMP). The purpose of this study was to assess the involvement of histamine and serotonin receptors as well as cAMP in the PGI₂-induced hypothermia in conscious guinea pig. Intracerebroventricular administration of 50–500 μg/kg PGI₂ produced a dose-related hypothermia, whereas its stable metabolite 6-keto prostaglandin F₁α had an insignificant effect. Low central doses (10–50 μg/kg) of dibutyryl cAMP (DBC) were hyperthermic, but high doses (100–500 μg/kg) caused hypothermia. Theophylline and low doses of DBC given centrally attenuated the PGI₂-induced hypothermia. Mepyramine and methysergide did not antagonize the effects of PGI₂ or DBC. However, central administration of metiamide (10–100 μg/kg) attenuated the hypothermic responses to both PGI₂ and DBC. These results suggest that histamine H₂-receptors are involved in the hypothermia induced by PGI₂.     

Contribution of lipoxygenase and cyclooxygenase metabolites in the modulation of cyclic nucleotides in the guinea pig mymotrium. Differential effects of carbachol and ionophore A23187

Accumulation of cyclic GMP in estradiol-treated immature guinea pig myometrium was enhanced by carbachol, ionophore A₂₃₁₈₆, unsaturated fatty acids and their hydroperoxides. Cyclic AMP content was elevated only by arachiodonic acid, A₂₃₁₈₇ and PGI₂. Eicosatetraynoic acid (TYA), but not indomethacin prevented all cyclic GMP responses. The effects of A₂₃₁₈₇ and arachidonate on cyclic AMP were accompanied by a parallel increase (2–3 fold) on the generation of PGI₂ by the myometrium. Both events were similarly reduced by indomethacin, TYA, 15-hydroperoxyarachidonic acid and tranylcypromine, suggesting that PGI₂ was involved. Omission of Ca²⁺ or addition of mepacrine of p-bromophenacylbromide abolished the stimulatory effects of A₂₃₁₈₇ and carbachol on cyclic GMP as well as the A₂₃₁₈₇-induced elevations in both PGI₂ and cyclic AMP generation. Thus, with both exogenous arachidonate as well as with endogenous fatty acid, released through an apparent phospholipase A₂-induced activation process, the lipoxygenase pathway was associated with an activation of the cyclic GMP system and the cyclooxygenase pathway, via PGI₂ generation, with an activation of the cyclic AMP system. Carbachol failed to alter both cyclic AMP content and the release of PGI₂ suggesting a cholinergic receptor-mediated fatty acid release process, selectively coupled to the lipoxygenase route.     

Concerning the biosynthesis of prostaglandin I2

Two diastereoisomers, 5R,6R-5-hydroxy-6(9α)-oxido-11α,15S-dihydroxyprost-13-enoic acid (7) and 5S,6S-5-hydroxy-6(9α)-oxido-11α,15S-dihydroxyprost-13-enoic acid (10) were synthesized for evaluation as possible biosynthetic intermediates in the enzymatic transformation of PGH₂ or PGG₂ into PGI₂. The synthetic sequence entails the stereospecific reduction of the 9-keto function in PGE₂ methyl ester after protecting the C-11 and C-15 hydroxyls as tbutyldimethylsilyl ethers. The resulting PGF_2α derivative was epoxidized exclusively at the C-5 (6) double bond to yield a mixture of epoxides, which underwent facile rearrangement with SiO₂ to yield the 5S,6S and 5R,6R-5-hydroxy-6(9α)-oxido cyclic ethers. It was found that dog aortic microsomes were unable to transform radioactive 9β-5S,6S[³H] or 9β-5R,6R[³H]-5-hydroxy-6(9α)-oxido cyclic ethers into PGI₂. Also, when either diastereoisomer was included in the incubation mixture, neither isomer diluted the conversion of [1-¹⁴C]arachidonic acid into [1-¹⁴C]PGI₂.     

Agonist-specific desensitization of PGI2-stimulated cyclic AMP accumulation by PGE1 in human foreskin fibroblasts

Agonist-specific desensitization of prostaglandin I₂-stimulated (PGI₂)¹ adenosine 3′:5′-monophosphate (cyclic AMP) accumulation can be demonstrated in intact human foreskin fibroblasts (HFF) following a single exposure to PGE₁ or a stable PGI₂ analog (nitrilo-PGI₂). A single PGI₂-stimulation of HFF cells does not result in desensitization. Continuous re-addition of PGI₂ over a 4 hr period does induce desensitization to subsequent PGI₂-stimulation. HFF cells that are desensitized to PGI₂ are also desensitized to PGE₁ or nitrilo-PGI₂ stimulation indicating that these agonists share a common adenylate cyclase complex. Desensitization to PGI₂ can be measured after a 60 min, but not after a 30 min, exposure to PGE₁ or nitrilo-PGI₂. Once HFF cells are desensitized, a 12–24 hr period is required for the recovery of PGI₂ sensitivity.The adenylate cyclase in membranes prepared from intact cells that were preincubated with PGE₁ is also desensitized to subsequent PGI₂-stimulation. Preincubation of cells with PGI₂ does not induce desensitization of PGI₂-stimulated adenylate cyclase. These data suggest that HFF cells must be constantly exposed to a biologically active prostaglandin for desensitization to occur. The intrinsic chemical lability of PGI₂ may be a biochemical protection mechanism against desensitization in cells that normally respond to PGI₂.     

Effects of thyrotropin and N6,O2′-dibutyryl cyclic 3′,5′-adenosine monophosphate on prostaglandin levels in thyroid

We have shown that TSH increases PG levels in isolated bovine thyroid cells. We now report that TSH also increases PG levels in rat and mouse thyroid, and that these effects may be mediated via cyclic AMP. PG and cyclic AMP levels in intact rat and mouse thyroid lobes were measured by radioimmunoassay. During 60-min incubations at 37°C, 25 mU/ml TSH effected a 75–83% increase in PGE₁ and PGF_2α ”equivalents“ in rat thyroid; parallel measurements of endogenous cyclic AMP in these intact thyroid lobes revealed that maximal TSH-induced increase in cyclic AMP also required 60-min incubations. In mouse thyroid, 5 mU/ml TSH increased PGE₁ and PGF_2α levels 38–82% above basal; this TSH effect was evident within 15 min of incubation, thus mimicking the time-course of TSH-induced increase in mouse thyroid cyclic AMP. Exogenous DBcAMP, 0.5 to 3 mM, effected a dose-related increase in mouse thyroid PG levels. The stimulatory effects of both TSH and DBcAMP on mouse thyroid PG levels were abolished by aspirin and indomethacin. These studies suggest that TSH-induced increase in endogenous PG levels in thyroid may be mediated by cyclic AMP.     

Prostanoids and hemostasis in chickens: Anti-aggregating activity of the prostaglandins E1 and E2, but not of prostacyclin and prostaglandin D2

Aggregation of chicken thrombocytes was studied in whole blood using an electronic aggregometer. Serotonin (5-hydroxytryptamine, 5HT), arachidonic acid (AA) and collagen, but not adenosinediphosphate (ADP) induced aggregation. Prostaglandin (PG) endoperoxides were essential for arachidonic acid-induced aggregation, but were not involved in 5HT-induced aggregation, as indicated by inhibitory studies with indomethacin. Similar experiments indicated that biosynthesis of endogenous PG endoperoxides contributed to the aggregation induced by low concentrations of collagen, but was of little importance when high collagen doses were employed. PGE₁ and PGE₂ could abolish all types of aggregation studied, whereas prostacyclin (PGI₂) and PGD₂ were without any anti-aggregatory activity at 1 μg/ml. Between 1 and 100 ng/ml PGE₁ and PGE₂ inhibited arachidonic acid- and 5HT-induced aggregation dose-dependently.The lack of any hemostatic function of PGI₂ in chickens was also indicated by the absence of biosynthesis of endogenous PGI₂ in chicken aorta. PGI₂ was assessed as anti-aggregating activity, released by aortic fragments stirred in rabbit platelet rich plasma. Still, the presence of chicken aortic tissue i chicken whole blood inhibited 5HT-, but not arachidonic acid-induced aggregation. This inhibition was not affected by pretreatment of the aortic fragments with indomethacin or pargyline.     

Histamine releases PGI2 from human pulmonary artery

Histamine caused a triphasic response of human pulmonary artery strips in vitro, consisting of a small initial contraction followed by pronounced relaxation preceding a second contractile response. These characteristics were not seen with other contractile stimuli including 5-hdyroxytryptamine, leukotriene D₄, and KC1. The relaxant component of this response was ablated by removal of endothelium from the vascular strips or by pretreatment of the tissues with 1μM indomethacin. Measurement of the PGI₂ degradation product 6-keto-PGF_1α in supernatants from histamine-challenged tissues confirmed the synthesis of PGI₂. Supernatants from unstimulated or leukotriene-challenged tissues contained no detectable amounts of 6-keto-PGF_1α. The histamine H₁ antagonist diphenhydramine inhibited both the contractile and relaxant responses to histamine whereas the H₂ antagonist cimetidine affected neither component. The released PGI₂ significantly altered the dose-respons curve to histamine without inhibiting the maximal contractile responses. We conclude that histamine induces PGI₂ formation from pulmonary arterial endothelium via an H₁ receptor.     

Effect of angiotensin II and norepinephrine on release of prostaglandins E2 and I2 by the perfused rat mesenteric artery

Prostaglandin E₂ (PGE₂) and 6 keto-PGF_1α, the stable metabolite of prostacyclin (PGI₂), have been measured in the effluent of perfused rat mesenteric arteries by the use of a sensitive and specific radioimmunoadday (RIA) method. The PGE₂ and 6-keto-PGF_1α were continuousyl released by the unstimulated mesenteric artery over a period of 145 min. After 100 min of perfusion the release of PGE₂ and 6-keto-PGF_1α was 4.5 ± 8.4 pg/min and 254 ± 75 pg.min respectively, which is in accord with the general belief that PGI₂ is the major PG synthesized by arterial tissue. Angiotensin II (AII) 5 ng/ml) induced an increased of PGE₂ and 6-keto-PGF_1α release without changing the perfusion pressure. The effect of norepinephrine (NE) injections on release of PGs depended on the duration of the stabilization period. The changes of perfusion pressure induced by NE were not related to changes in release of PGs. Thus, it seems that the increase of PG release induced by AII and NE was due to a direct effect of the drugs on the vascular wall. This may represent an important modulating mechanism in the regulation of vascular tone.     

A comparison of some pharmacological actions of prostaglandin E1, 6-OXO-PGE1 and PGI2

Some pharmacological actions of prostaglandin E₁ (PGE₁), 6-oxo-PGE₁ and PGI₂ have been studied. 6-oxo-PGE₁ and PGI₁ relaxed guinea-pig tracheal muscle in vitro and increased nasal patency in normal volunteers and in subjects with vasomotor rhinitis whereas PGI₂ produced opposite effects. All three compounds produced bronchodilatation in the anaesthetised guinea-pig and relaxed human respiratory tract muscle in vitro.PGI₂ was several times more potent than either 6-oxo-PGE₁ or PGE₁ against ADP-induced aggregation of human and baboon platelets in vitro. Intravenous 6-oxo-PGE₁ in the baboon caused an ex vivo inhibition of platelet aggregation, but the EC₅ was 7.8 times that of PGI₂. As a vasodepressor in the baboon 6-oxo-PGE₁ and PGE₂ were equipotent. Thus with the exception of the vasodepressor effect, the actions of 6-oxo-PGE₁ qualitatively and quantitatively resembled those of the structurally related PGE₁ rather than those of PGI₂.     

N6,O2′-dibutyryl adenosine 3′,5′-monophosphate-stimulated release of proteoglycans from cultured immature rabbit ear cartilage

The stimulatory effects of N⁶,O^2′-dibutyryl adenosine 3′,5′-monophosphate on proteoglycans released from immature rabbit ear cartilage were studied in vitro. Cartilage incubated in medium containing dibutyryl cyclic AMP resulted in a significant increase of proteoglycans released in concentrations above 0.5 mM. Theophylline (1 mM) which did not significantly stimulate proteoglycans released alone, was found to potentiate the action of this nucleotide. ATP, 5′-AMP and butyric acid in the presence of theophylline, did not stimulate proteoglycans released. The addition of protein or RNA synthesis inhibitors depressed proteoglycans released by dibutyryl cyclic AMP and theophylline.Gel chromatographic and chemical investigations of the proteoglycans released into the culture media in the presence of dibutyryl cyclic AMP indicated a reduction in the proportion of protein associated with these complexes. This result, together with enzyme inhibitor studies, leads us to speculate that the observed action of dibutyryl cyclic AMP on rabbit ear cartilages may be mediated by the neural proteases.     

Opposing actions of methylxanthines and dibutyryl cyclic AMP on 1,25 dihydroxyvitamin D3 production and calcium fluxes in isolated chick renal tubules

In contrast to dibuturyl cyclic AMP, the methylxanthine phosphodiesterase inhibitors theophylline and caffeine were found to inhibit the conversion of 25 hydroxyvitamin D₃ to 1,25 dihydroxyvitamin D₃ in isolated renal tubules from vitamin D deficient chicks. This inhibition occurred at concentrations of methylxanthines which were shown to increase renal tubule cyclic AMP levels. No effect of theophylline or caffeine on 25 hydroxyvitamin D₃ metabolism in isolated chick renal mitochondria was detected. Because of a demonstrated inhibitory action of calcium (10 and 20 μmol/l) on renal mitochondrial conversion of 25 hydroxyvitamin D₃ to 1,25 dihydroxyvitamin D₃, the effect of theophylline and dibutyryl cyclic AMP on cellular calcium-45 efflux and total renal tubule calcium content was estimated. Theophylline 10 mmol/l was found to inhibit renal tubular calcium efflux and to increase total cellular calcium content, while dibutyryl cyclic AMP 1 mmol/l had the reverse effect on both parameters. Divergent actions of the methylxanthines and dibutyryl cyclic AMP on the formation of 1,25 dihydroxyvitamin D₃ and renal tubule calcium efflux and content support the hypothesis that intracellular calcium is an important regulator of renal vitamin D metabolism. The results indicate that observed actions of methylxanthines cannot always be ascribed to cyclic AMP accumulation.