Role of prostaglandins and cyclic nucleotides in the mechanism of development of drug-induced thrombocytopenia

Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 115, N ^o 4, pp. 354–356, April, 1993     

Pyeloureteral motility and ureteral peristalsis: essential role of sensory nerves and endogenous prostaglandins

The cellular mechanisms that underlie the initiation and propagation of the peristaltic contractions, which transport urine from the kidney to the bladder for storage, remain little understood. Extracellular and intracellular microelectrode recordings have identified two populations of smooth muscle cells as well as a population of renal interstitial cells (RICs) that all display spontaneous electrical activity. By analogy with the heart it has been proposed that atypical smooth muscle cells, preferentially located in the very proximal regions of the renal pelvis, generate the essential pacemaker signal. These pacemaker potentials propagate to neighbouring typical smooth muscle cells or RICs to trigger action potential discharge. These action potentials then propagate distally to trigger other bundles of typical smooth muscle cells. The frequency of action potential discharge and contraction decreases as the relative number of RICs and atypical smooth muscle cells compared to typical smooth muscle cells decreases with distance from the renal fornix. It is clear that functional capsaicin-sensitive sensory afferents and the endogenous release of both tachykinins and prostaglandins are essential in the maintenance of normal peristalsis, as well as in monitoring and responding to any chemical or mechanical stimulation. However, the cellular mechanisms underlying the action of these endogenously-released agents remain to be elucidated.     

Effect of thymulin on intracellular cyclic nucleotides and prostaglandins E2 in peanut agglutinin-fractionated thymocytes

Prostaglandin E2 (PGE2) and other selected agents which elevate intracellular cyclic AMP (cAMP) levels have been demonstrated to induce the appearance of surface markers in immature T lymphocytes. Thymic hormones, which are the natural inducers of these markers, have long been hypothesized to act through the increase of cAMP levels. We have approached this area of investigation by studying the effects of thymulin (a serum thymus-derived factor, coupled with Zinc) on intracellular cAMP and cyclic GMP (cGMP) levels (expressed as cAMP/cGMP ratio) and on the release of PGE2 in different thymocyte subpopulations. Thymocytes were fractionated by the peanut agglutinin (PNA) technique into cortical immature PNA+ and medullary mature PNA- thymocytes. The data presented in this report show that thymulin is able to increase the cAMP/cGMP ratio in PNA+ and in unfractionated thymocytes, depending on its concentration, but not in PNA- thymic cells. Conversely, it is able to increase the release of PGE2 by PNA- thymocytes but not by PNA+ and unfractionated thymic lymphocytes. These results are consistent with the hypothesis that thymulin could act through different mechanisms depending on the differentiation stage of its target cells. In fact, it could be suggested that immature T cells could be activated by thymulin thereby increasing the cAMP/cGMP ratio, whereas more mature T cells would be further differentiated by thymulin through enhanced release of PGE2.     

Ischemia-induced vascular changes: role of xanthine oxidase and hydroxyl radicals

The results of previous studies indicate that oxygen-derived free radicals are responsible for the increased vascular permeability produced by 1 h of intestinal ischemia. The aims of this study were 1) to test the hypothesis that the enzyme xanthine oxidase is the source of oxygen radicals in the ischemic bowel and 2) to assess the role of the hydroxyl radical in the ischemia-induced vascular injury. The capillary osmotic reflection coefficient was estimated from lymphatic protein flux data in the cat ileum for the following conditions: ischemia, ischemia plus pretreatment with allopurinol (a xanthine oxidase inhibitor), and ischemia plus pretreatment with dimethyl sulfoxide (a hydroxyl radical scavenger). The increased vascular permeability produced by ischemia was largely prevented by pretreatment with either allopurinol or dimethyl sulfoxide. These findings support the hypothesis that xanthine oxidase is the source of oxygen radicals produced during ischemia. The results also indicate that hydroxyl radicals, derived from the superoxide anion, are primarily responsible for the vascular injury associated with intestinal ischemia.     

The importance of prostaglandins and cyclic nucleotides in the mechanism of action of the transfusion of blood and its components]

The authors studied changes in the content of groups E and F2 prostaglandins (PGE and PGF2) and cyclic nucleotides (cAMP and cGMP) in blood of dogs after 20 ml/kg blood loss as well as in transfusion of whole blood, plasma and 10 ml/kg packed erythrocytes after preliminary blood letting. It was established that after loss of blood the concentration of PGE, cAMP and cGMP increases while the concentration of PGF2 in the blood reduces. It is also shown that transfusion of whole blood facilitates correction of the content of prostaglandins and cyclic nucleotides in the blood. The posthemorrhagic changes of most of the studied indices are normalized in the 24 hours after transfusion of packed erythrocytes, transfusion of plasma fails to correct the revealed shifts.     

Ischemia-induced angiogenesis: role of inflammatory response mediated by P-selectin

P-selectin is a 140-kDa glycoprotein expressed on endothelial cells and platelets. P-selectin mediates the tethering and rolling of leukocytes along the endothelium, an early step of leukocyte extravasation. Although inflammation is a requisite process for ischemia-induced angiogenesis, little is known regarding the role of P-selectin in angiogenesis in the setting of tissue ischemia. We examined whether ischemia-induced angiogenesis is altered in P-selectin knockout (P-selectin(-/-)) mice. Angiogenesis was evaluated in a surgically induced hind-limb ischemia model using laser Doppler blood flowmetry (LDBF) and histological capillary density (CD). After left hind-limb ischemia, the ischemic/normal limb LDBF ratio was persistently lower in P-selectin(-/-) mice compared with wild-type (WT) mice. CD was also significantly lower in P-selectin(-/-) mice than in WT mice on Postoperative Day 14. Fewer numbers of total CD45+ inflammatory leukocytes infiltrated into the ischemic tissues in P-selectin(-/-) mice than in WT mice, and immunohistochemical analysis revealed the number of infiltrated leukocytes expressing vascular endothelial growth factor was also decreased in P-selectin(-/-) mice. P-selectin mRNA expression was augmented after hind-limb ischemia in WT mice. In conclusion, P-selectin may play an important role in ischemia-induced angiogenesis by promoting early inflammatory mononuclear cell infiltration. P-selectin would become one possible target molecule for modulating inflammatory angiogenesis.     

Studies on thymus products. VI. The effects of cyclic nucleotides and prostaglandins on rosette-forming cells. Interactions with thymic factor

Cyclic AMP, its dibutyryl derivative and prostaglandins, PGE₁ and PGE₂, do not modify the numbers of rosette-forming cells (RFC), but they do alter these cells' sensitivity to in vitro inhibition by azathioprine (AZ) and anti-theta serum (ATS). In thymectomized mice, cyclic nucleotides and prostaglandins mimic the action of the thymic factor contained in thymic extracts and in normal serum, correcting the low spleen RFC sensitivity to AZ and ATS induced by thymectomy. Moreover, the mixture of infraliminal active doses of cyclic AMP and of thymic extracts (or normal mouse serum) also gives this effect. In normal mice, the nucleotides (but not prostaglandins) lower the AZ and ATS sensitivity of RFC, which remains insensitive to thymic extracts. Theophylline and isoproterenol have effects similar to those of cyclic AMP, a result in accord with their postulated interactions with the adenyl-cyclase system. Noncyclic AMP and cyclic GMP show no effect in the RFC assay system at doses 10–100 times higher than active doses of cyclic AMP. Other vasoactive compounds, including epinephrine, phentolamine, propanolol, and angiotensin, do not show any effect on RFC.     

The role of prostaglandins in endotoxic activities

Summary Endotoxins elicit an extraordinary variety of biological effects in higher organisms. Mononuclear phagocytes are believed to be the cellular source of secondary mediators responsible for the host's reaction. Several findings indicate that among these endogenous mediators prostaglandins are of importance. Macrophages of different origin synthesize prostaglandins when stimulated with LPS. The prostaglandin-inducing activity is located in the lipid A part of the LPS molecule. Macrophages from an LPS-resistant mouse strain (C3H/HeJ) and cells from mice rendered tolerant to LPS do not produce prostaglandins in vitro when incubated with LPS, a phenomenon paralleling the lack of in vivo activity. Certain prostaglandins (TxA₂ and PGI₂) have been shown to be of importance in endotoxicosis. We found that macrophages do not produce TxA₂ and PGI₂ on incubation with LPS in vitro, although they possess the potential to synthesize these metabolites.Thus it remains to be elucidated which role macrophages, their prostaglandin production and/or other factors play in endotoxicosis.     

The role of prostaglandins in bone formation

Prostaglandins of the E series have been shown to be effective inducers of bone formation in vivo. In this study, the effects of PGE2 were evaluated in vivo using subcutaneous administration (3 mg/kg/d for 25 days) to ovariectomized rats or local application in the marrow cavity of tibiae of rats using biodegradable implants (0.13, 1.4 and 32 microg released over 8 days). Systemic treatment of rats with PGE2 stimulated cancellous bone formation in the metaphysis of the proximal tibiae as well as endocortical bone formation and de novo trabecular bone formation in the marrow cavity. Local delivery of PGE2 increased cancellous bone volume in the secondary spongiosa and cortical thickness (at 32 microg). Comparisons of prostanoid effects in vitro, in a bone-derived cell line, showed that PGF2alpha was a better stimulator of DNA synthesis than PGE2. PGF2alpha increased the steady state levels of IGF-I receptor mRNA while PGE2 increased IGF-I expression. Although the mechanism of bone formation by PGE2 is not known at this time, it is clear that PGE2 has powerful local anabolic effects on bone formation in vivo possibly by mediating responses to signals such as changes in mechanical force.     

Microassay of cyclic nucleotides in vessel wall: V. Simultaneous assay of cyclic AMP and cyclic GMP

Cyclic GMP (cGMP) is an important key substance in cell function; however, the related mechanisms have not been entirely elucidated, as the content of this nucleotide within the cell is so small that an accurate estimation is difficult. We designed a microassay for cyclic GMP in 500 μg dry weight of tissue samples, using succinylation, and were able to assay, separately, the content of cyclic GMP in the intima or media of the arterial wall. In addition, we attempted to develop a method for a simultaneous determination of both cyclic AMP and cyclic GMP in a small amount of the same assay mixture. Five hundred micrograms dry weight of intima or media of rabbit aorta was prepared under a stereomicroscope. After deproteinization and lyophilization, the samples were dissolved with 60 μl of H₂O and 30 μl of succinylation mixture. After succinylation, 40 μl of each of the tissue extractions was sampled to determine the levels of cyclic AMP and cyclic GMP. The radioimmunoassay was performed, using Yamasa's kit. The recovery rates of cyclic AMP and cyclic GMP in our method were 86.2 ± 1.2 and 87.6 ± 1.3%, respectively. The levels of cyclic GMP in the intima and media of aorta of cow, pig, and rabbits were estimated to be, intima: 0.18 ± 0.02, 0.06 ± 0.03, and 0.20 ± 0.01 pmole/mg protein, media: 0.08 ± 0.01, 0.04 ± 0.01, and 0.09 ± 0.01. The cyclic GMP levels in the intima of aorta of cow and rabbits were high compared to levels in the media and this difference was statistically significant (P < 0.05).     

The influence of cyclic nucleotides on DNA synthesis in skin

Total saline-soluble protein, total DNA, and 3H-thymidine labelling of DNA were measured in cultured guinea-pig skin during a 3-day incubation period. An early rise in total soluble protein and a fall in total DNA indicated loss of the cells damaged in cutting the skin samples. Samples incubated with dibutyryl cyclic adenosine monophosphate (db cAMP) showed an early stimulation of DNA synthesis. Cyclic guanosine monophosphate (cGMP) stimulation of DNA synthesis showed a peak after 48 h incubation, probably reflecting a slower rate of entry of the compound into the epidermal cells.     

The role of extracellular Ca2+ and cyclic nucleotides in the mechanism of enzyme secretion from the cat pancereas

The role of Ca2+ in protein secretion from the isolated perfused cat's pancreas, the effect of the dibutyryl analogues of cAMP and cGMP, and the interrelation of Ca2+ and the nucleotides were studied. The following results were obtained: 1. Pancreatic enzyme secretion can be elicited by CaCl2 injections into the pancreatic arteries and is linearily related to the peak Ca2+ concentration in the effluent perfusate. Different background Ca2+ concentrations in the perfusate (3 mM or 0.125 mM) do not disturb this relation, indicating that no adaptation occurs. The effect of Ca2+ injections is of the same magnitude as that evoked by the hormones pancreozymin or acetylcholine. 2. Injections of Ca2+ potentiate the effects of dibutyryl adenosine 3',5'-cyclic monophosphate (dbcAMP), dibutyryl guanosine 3',5'-cyclic monophosphate (dbcGMP) or theophylline. 3. Infusion of low doses of pancreozymin increases the Ca2+ effect. The findings indicate that extracellular Ca2+ is involved in the mechanism of enzyme secretion and that Ca2+ and cyclic nucleotides have a synergistic action on the target.     

The role of prostaglandins in experimental ocular inflammations

Intraocular inflammations (uveitis) were produced in rabbits by intravitreal injection of killed and driedMycobacterium butyricum orE. coli endotoxin, or paracentesis of the anterior chamber. The increase in permeability of the blood-aqueous barrier and the leucocyte migration into the aqueous humor were observed after these stimuli, although the leucocyte did not migrate after paracentesis. Topically applied indomethacin reduced these inflammatory parameters in the latter two models. However, the increased permeability afterM. butyricum injection was not affected by indomethacin, though the leucocyte migration was reduced. On the other hand, prostaglandin-like substances in the aqueous humor were significantly elevated in the latter models, whereas only traces of these substances were detected in the former model. These results indicate that, unlike the increase in the permeability of the blood-aqueous barrier after endotoxin injection and paracentesis, the response afterM. butyricum injection is not mediated by prostaglandins. The role of prostaglandins in the leucocyte migration in these ocular inflammations was also discussed.     

The role of the lysosome in natural killing: inhibition by lysosomotropic vital dyes.

Recent evidence has indicated the importance of lysosomes in natural killing (NK). The lysosomotropic dye neutral red, at concentrations greater than 2 X 10(-5) M, inhibits NK activity of large granular lymphocytes (LGL). Inhibition is at the level of effector cell activity rather than target cell susceptibility. Neutral red inhibits NK activity without affecting the viability of the effector cell population or the response of lymphocytes to the mitogen, phytohaemagglutinin (PHA). While the mechanism of this inhibition is unknown, neutral red-treated peripheral blood lymphocytes (PBL) do not suppress untreated effector cell populations. Furthermore, neutral red does not influence the binding of effector cells to target cells. Since natural killing by neutral red-treated PBL recovers slowly upon incubation of the cells at 37 degrees, inhibition is not due to selective toxocity. Trypan blue, another frequently used lysosomotropic vital stain, has no effect when effector cells are pretreated with 10(-3) M dye for 30 min or less. However, effector cells first treated with neutral red lose red granular staining following treatment with trypan blue for as little as 10 min at room temperature. Natural killing by neutral red pretreated PBL also recovers more quickly following brief incubation with trypan blue. Treatment of effector cells with trypan blue and neutral red simultaneously prevents accumulation of neutral red in cytoplasmic granules and subsequent inhibition of NK.     

Content of prostaglandins and cyclic AMP in rat tissue

The content of prostaglandins (PG) and cyclic adenosine-3,5-monophosphate (cyclic AMP) in rat tissues was studied by a radioisotope method. The maximal content of both PG and cyclic AMP was found in the same organs, those functioning most actively: the brain, endocrine glands, and small intestine, and the minimal content in adipose tissue. The investigations show close functional interdependence between PG synthesis and adenylate cyclase activity in the tissues of the body.Kiev Scientific-Research Institute of Endocrinology and Metabolism. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 4, pp. 416–418, April, 1977.