Histamine as an intracellular messenger.
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Animals↗
Biomedical subjects
J M Gerrard
Publications and source records attributed to J M Gerrard.
Studies in patients with bleeding disorders show that platelet-vessel interaction is important for thromboxane formation in bleeding time wounds.
The production of thomboxane B2, the primary metabolite of thromboxane A2, and 6-keto prostaglandin F1 alpha, the primary metabolite of prostacyclin, were measured in response to a standardized vascular injury, the bleeding time, in patients with von Willebrand's disease and in patients with platelet function defects. Compared to controls, thromboxane B2 levels in bleeding time blood were significantly lower in subjects with von Willebrand's disease. In patients with platelet function defects associated with a deficient response to thromboxane A2, thromboxane B2 production in bleeding time blood was similar to controls. In subjects with other platelet function defects, thromboxane production was significantly lower than normal. 6-keto PGF1 alpha production in bleeding time blood was not significantly different in patients compared to controls. The results suggest that bleeding time thromboxane production is influenced by the extent of platelet-vessel interaction.
6-Ketoprostaglandin F1 alpha↗
Inhibition of intravascular platelet aggregation by endothelium-derived relaxing factor: reversal by red blood cells.
Studies were performed to determine whether endothelium-derived relaxing factor (EDRF) can inhibit platelet aggregation within the vascular lumen, and if so, whether the inhibition persists in the presence of red blood cells (RBCs). Canine femoral arteries mounted in an organ bath were perfused with physiologic saline solution to which acetylsalicylic acid was added to block prostacyclin formation. During contraction with phenylephrine, addition of acetylcholine to the perfusing solution to evoke EDRF release relaxed the vessel wall. Washed human platelets labeled with 14C-5-hydroxytryptamine were added to the perfusing solution, and activated by thrombin infused via a branch vessel. The perfusate was collected downstream and centrifuged; the fraction of 14C-5-hydroxytryptamine appearing in the supernatant reflected the degree of platelet activation. Stimulation of EDRF release with acetylcholine inhibited 14C-5-hydroxytryptamine release. Hemoglobin (Hb) (10(-5) mol/L) blocked vascular relaxation and platelet-inhibition. RBCs at a hematocrit of 10% (treated with echothiophate to block erythrocyte cholinesterase) did not prevent relaxation but reversed the platelet inhibition. Lower hematocrits did not completely block the inhibition. Thus, erythrocyte Hb may modulate the inhibition of intraluminal platelet aggregation by EDRF.
Animals↗
A role for intracellular histamine in collagen-induced platelet aggregation.
We previously demonstrated that newly formed intracellular histamine mediates platelet aggregation in response to phorbol-12-myristate-13-acetate (PMA). We now report further investigations of the role of histamine during physiological activation of platelets by collagen. Platelets stirred with collagen produced histamine; the rise in histamine precedes the onset of aggregation. The dose response for collagen stimulation of histamine synthesis and platelet aggregation is similar. Inhibitors of histidine decarboxylase (HDC) block both aggregation and histamine synthesis in parallel. Histamine production is not dependent on aggregation; both the intracellular histamine receptor antagonist, N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine-HCl (DPPE), and the cyclooxygenase inhibitors, aspirin and indomethacin, inhibit collagen-induced aggregation but not histamine synthesis. DPPE also inhibits collagen-induced serotonin secretion and thromboxane production. The effects of DPPE and HDC inhibitors are significantly reversed by the addition of histamine (0.1 to 10 mumol/L) to saponin-permeabilized platelets, though histamine alone has no pro-aggregatory effects. The results suggest that newly synthesized intracellular histamine has a role in collagen-induced platelet activation and that it may act to promote the generation of thromboxane and the secretion responses of platelet granules.
Aspirin↗
Studies of the mechanism of natural killer (NK) degranulation and cytotoxicity.
Exocytosis of cytotoxic granule contents towards bound target cells is thought to be of central importance in natural killer (NK) cell-mediated killing. Although cellular cytotoxicity involving degranulation is thought to be calcium-dependent, the biochemical mechanisms that mediate this granule mobilization are unknown. Inositol-1,4,5-tris-phosphate (IP3), which acts to elevate internal calcium levels, and 1,2-diacylglycerol (DAG), which activates protein kinase C, are potent second messengers that have been shown to synergistically mediate secretion in other cell types. Production of these products of inositol phospholipid metabolism has previously been demonstrated in a rat NK cell line RNK upon exposure to susceptible tumor targets. We therefore investigated the role of IP3 and DAG in NK-mediated cytotoxicity, specifically at the level of degranulation. Pretreatment of RNK cells with neomycin, a drug that interferes with the hydrolysis of inositol phospholipids and thus inhibits the formation of second messengers, inhibited RNK cytotoxicity against a susceptible tumor target and also inhibited RNK production of DAG in response to a similar target. Natural killing exhibited by normal rat nylon wool-nonadherent splenocytes was also inhibited by neomycin. Phorbol-12-myristate, 13-acetate (PMA), a phorbol ester that acts like DAG to activate protein kinase C, markedly enhanced lysis of a susceptible target cell by RNK. We evaluated whether modulation of lysis by these drugs was associated with effects on RNK degranulation by assaying the release of a granule-specific serine esterase (BLTE) in response to PMA and the calcium ionophore A23187. These agents synergized to promote the release of BLTE, and the extent of release was dependent on the concentrations of both agents. D2O and cytochalasin B, which enhance secretion in other cells, both enhanced BLTE release from RNK cells, indicating that we were detecting BLTE released via granule secretion and not due to nonspecific causes such as cell lysis. Our findings lead us to propose that NK cells form IP3 and DAG in response to susceptible target cells and that a major function of these second messengers is to mediate the exocytosis of cytotoxic granules towards the bound target cells.
Animals↗
Platelet storage pool deficiency: diagnosis in patients with prolonged bleeding times and normal platelet aggregation.
We evaluated 46 patients with prolonged bleeding times, and no demonstrable abnormalities of either von Willebrand factor or platelet aggregation, for possible deficiency of platelet storage pool. Studies of ATP release from thrombin-stimulated platelets and enumeration of dense granules in platelet whole mounts were performed in these patients. Seventeen patients (35%) had both decreased ATP release and decreased numbers of dense granules, suggesting the presence of a storage pool defect. Thus, storage pool deficiency may be present in the absence of the classical aggregation abnormalities. Evidence of storage pool deficiency should be considered in all patients with an isolated unexplained prolongation of the bleeding time. The methods used in this study are readily applicable to most clinical laboratories.
Adenosine Triphosphate↗
Long-term cultures of human peripheral blood lymphocytes with recombinant human interleukin-2 generate a population of virtually pure CD3+ CD16- CD56- large granular lymphocyte LAK cells.
It has been reported that lymphocytes from peripheral blood (PBL) cultured with interleukin-2 (IL-2) produce predominantly CD16+ lymphokine-activated killer (LAK) cells. We developed a two-step method to generate LAK cells from human PBL in long-term cultures (10-12 days) with recombinant human IL-2 (rhIL-2) and characterized the evolving LAK cell population by testing its phenotype and cytotoxic activity as a function of time. The starting PBL displayed some natural killer (NK) cytotoxicity but no LAK activity. At day 6, the cells were a mixed population of about 80% CD3+ and 6% CD16+ cells. Little proliferation was evident but strong LAK activity was detected. After 10-12 days, major cell expansion had occurred and they were essentially a pure (greater than 90%) CD3+ CD16- CD56- cell population large granular lymphocyte (LGL) by morphology that displayed strong non-MHC-restricted killing activity (greater than 200 lytic units). Over the same period of time, the CD16+ cells had almost completely regressed in these cultures. This preferential induction of CD+ LAK cells was not an effect of IL-2 concentration as 10 U/ml was as effective as 500 U/ml. Further characterization revealed a major population of CD4+ (60%) and CD8+ (30%) with a smaller fraction (less than 9%) of gamma delta + cells. These results indicate that a virtually pure CD3+ LAK cells population was produced with long-term cultures of lymphocytes from peripheral blood in rhIL-2, in which active proliferation of the CD3+ but not CD16+ cells occurred.
Antigens, Differentiation↗
Characterization of the gastroprotective effects of N,N-diethyl-2-[4-(phenylmethyl)phenoxy]-ethanamine hydrochloride, a non-H1/non-H2 histamine antagonist.
N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine HCl (DPPE) is an antihistamine with a unique profile of activity in the stomach. It is antisecretory and blocks the formation of experimental cold/stress- and ethanol-induced gastric lesions, as well as cysteamine-induced duodenal ulcers in a fashion more potent than observed with histamine H2 antagonists such as cimetidine. We now demonstrate that the antiulcer effects of DPPE are associated with a dramatic (10-fold) rise in the stable prostacyclin hydration product 6-keto-prostaglandin F1 alpha in gastric secretion collected from conscious rats. Cyclooxygenase inhibitors such as acetylsalicylic acid, indomethacin and sodium meclofenamate abolish high-dose DPPE-induced gastroprotection, whereas sodium salicylate, a lipoxygenase inhibitor, does not. These data suggest that DPPE-induced gastroprotection is mediated, at least in part, through an increase in endogenous prostacyclin (prostaglandin I2) synthesis in the gastric mucosa. These data are not consistent with an effect of DPPE primarily at the H2 receptor, but are consistent with the recent suggestion that DPPE antagonizes histamine at HIC, an intracellular histamine site.
6-Ketoprostaglandin F1 alpha↗
A role for protein kinase C in the membrane fusion necessary for platelet granule secretion.
The addition of 1-oleoyl-2-acetylglycerol (OAG), or phorbol-12-myristate-13-acetate (PMA) to platelets induced the phosphorylation of a 47,000 dalton protein (47 Kd), fusion of granule membranes with membranes of the surface connected canalicular system, the formation of large vesicles and the secretion of serotonin. 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine (H7), and sphingosine, inhibitors of protein kinase C, significantly inhibited the ultrastructural changes and the phosphorylation of 47 Kd. N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004), structurally similar to H7, but a weaker inhibitor of protein kinase C, did not attenuate these responses to OAG or to PMA. H7, but not HA1004, also markedly inhibited secretion induced by the synergistic combination of OAG and the calcium ionophore A23187. Amiloride and 5-(N,N dimethyl)-amiloride, inhibitors of the Na+/H+ transporter, did not inhibit the ultrastructural response and the protein phosphorylation induced by OAG, or the secretion induced by the combination of A23187 and OAG. The results link the activation of protein kinase C by diglycerides to the labilization and fusion of granule membranes important for secretion.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
Histamine formed in stimulated human platelets is cytoplasmic.
The localization of histamine formed by human platelets in response to agonists was evaluated. 87 +/- 5% of the histamine in a suspension of platelets exposed to phorbol-12-myristate-13-acetate (PMA) was associated with the platelet pellet. Incubation of saponin-permeabilized platelets with the intracellular histamine antagonist, N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine.HCl (DPPE), released 75 +/- 3.9% of the histamine into the supernatant. Under conditions where 90% of platelet serotonin was secreted into the supernatant, the majority (80%) of platelet histamine remained associated with the pellet. The results suggest that histamine synthesized in response to agonists is largely cytoplasmic.
Blood Platelets↗
Histamine is an intracellular messenger mediating platelet aggregation.
Inhibition of human platelet aggregation by N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine-HCl (DPPE), a novel antagonist of histamine binding, suggested that histamine might serve a critical role in cell function. Phorbol-12-myristate-13-acetate (PMA) or collagen was found to increase platelet histamine content in parallel with promotion of aggregation. Inhibitors of histidine decarboxylase (HDC) suppressed both aggregation and the elevation of histamine content, whereas DPPE inhibited aggregation only. In saponin-permeabilized platelets, added histamine reversed the inhibition by DPPE or HDC inhibitors on aggregation induced by PMA or collagen. The results indicate a role for histamine as an intracellular messenger, which in platelets promotes aggregation.
Blood Platelets↗
Identification of the molecular species of lysophosphatidic acid produced when platelets are stimulated by thrombin.
Platelets, when stirred with 3 U thrombin/10(9) platelets, produced significant quantities of palmitoyllysophosphatidic acid (2.17 ng/10(9) platelets), stearoyllysophosphatidic acid (2.11 ng/10(9) platelets), and arachidonoyllysophosphatidic acid (1.06 ng/10(9) platelets). When platelets were pretreated with 100 microM of the phospholipase A2 inhibitor U10029A, there was a significant decrease in thrombin-stimulated production of stearoyllysophosphatidic acid (to 0.16 ng/10(9) platelets), while arachidonoyllysophosphatidic acid production was unchanged. U10029A concomitantly increased thrombin-stimulated production of stearoyl-containing phosphatidic acid species (primarily stearoylarachidonoylphosphatidic acid) from 5.99 to 9.71 ng/10(9) platelets. The results are consistent with the concept that stearoyllysophosphatidic acid production in platelets occurs via phospholipase A2 degradation of phosphatidic acid.
Blood Platelets↗
The effect of exercise on bleeding time and local production of prostacyclin and thromboxane.
Exercise at a heart rate corresponding to 30% VO2max for 15 min was associated with an increase in the volume of bleeding time blood from a mean of 133 microliters before exercise to a mean of 218 microliters during and immediately after the exercise. There was similarly an increase in thromboxane B2 production from 6.40 nmol.l-1 before to 11.50 nmol.l-1. Most subjects also showed an increase in the length of the bleeding time and in the production of bleeding time 6-keto-PGF1 alpha. The extent of increase in the bleeding time and in production of 6-keto-PGF1 alpha was quite variable, with subjects showing the largest increases in bleeding time also demonstrating the greatest increases in 6-keto-PGF1 alpha (r = 0.76, P = 0.004). The ingestion of aspirin before exercise markedly inhibited basal bleeding time thromboxane B2 production and blocked the exercise-associated increments in thromboxane B2 and 6-keto-PGF1 alpha production. While the aspirin itself increased the length of the bleeding time, there was not any further increase associated with exercise. In contrast to the effects of acute short-term exercise, long-distance running was associated with a significant decrease in bleeding time, but no change in bleeding time blood volume, bleeding time thromboxane B2, or bleeding time 6-keto-PGF1 alpha. The results show that acute low-level exercise can be associated with significant changes in the volume of blood oozing from a bleeding time incision and in the amount of thromboxane production stimulated at the incisional site. Following exhaustive exercise of long duration, the above changes are no longer seen.
6-Ketoprostaglandin F1 alpha↗
Fatty acid composition of umbilical arteries and veins: possible implications for the fetal EFA-status.
Fatty acid compositions were determined of phospholipids, isolated from umbilical arteries and veins, obtained from Dutch neonates after vaginal delivery, terminating normal pregnancy. The fatty acid profiles of the cord vessels were characterized by the absence of eicosapentaenoic (timnodonic) acid, a low (2-3%) content of linoleic acid and reasonable amounts of arachidonic acid (10-15%) and docosahexaenoic (cervonic) acid (3-5%). Significant amounts of Mead acid (1-4%) and its direct elongation product (0.5-2%) were also observed. In each cord, the efferent blood vessels contained significantly more Mead acid and other fatty acids of the oleic acid (n-9) family and less fatty acids of the linoleic (n-6) and linolenic (n-3) families than the afferent blood vessel. This indicates that the essential fatty acid (EFA) status of 'downstream' neonatal tissue may be marginal. No signs of EFA-deficiency were observed in endothelial and smooth muscle cells in culture, or in blood vessels from adults. In all cords 22:5(n-6) was significantly higher in the artery compared to the vein, whereas for all other (n-6) fatty acids this difference was negative. Since the synthesis of 22:5(n-6) is known to be stimulated when the required amount of cervonic acid, 22:6(n-3), is too low, our observations also suggest that the cervonic acid status of the neonates investigated was not optimal. Further studies are in progress to relate these findings to maternal EFA status and complications of pregnancy.
Adult↗
Comparison of the effect of canola oil and sunflower oil on plasma lipids and lipoproteins and on in vivo thromboxane A2 and prostacyclin production in healthy young men.
The effect of dietary fatty acid composition on plasma lipids and eicosanoid production was studied in normolipidemic men. Approximately 75% of the fat in the diet was provided by a mixture of fats (MF) during the 6-d pre and postperiods and either canola oil (CO) or sunflower oil (SO) during the two 18-d experimental periods. Saturated, monounsaturated, and polyunsaturated fatty acids provided 14%, 15%, and 7%; 5%, 20%, and 10%; and 7%, 7%, and 22%, respectively, of total dietary energy in the MF, CO, and SO diets, respectively. The CO and SO diets produced similar decreases in plasma total (20% and 15%, respectively) and low-density lipoprotein (25% and 21%, respectively) cholesterol. Plasma high-density lipoprotein cholesterol and triglyceride concentrations were not altered by either fat source. Bleeding time was longer and in vivo 1-keto-PGF1 alpha production was greater (p less than 0.05) when on the CO diet than when on the MF diet. The hypocholesterolemic and antithrombotic effects of the CO diet were equivalent to those of the SO diet.
6-Ketoprostaglandin F1 alpha↗
Evidence for two mechanisms of thrombin-induced platelet activation: one proteolytic, one receptor mediated.
The possibility that thrombin-induced platelet reactivity could occur via both a receptor-related and a proteolytic process was examined. Thrombin elicited the formation of considerably more [32P]phosphatidic acid (an index of phospholipase C catalysed phosphoinositide metabolism) than did platelet activating factor, 5-hydroxytryptamine, ADP, and the thromboxane A2 analogue EP171, when these agents were added either alone or in combination. Co-addition of thrombin and EP171 did not evoke significantly more [32P]phosphatide acid than did thrombin alone. The protease inhibitor leupeptin, decreased but did not abolish [32P]phosphatidic acid formation elicited by either thrombin alone or thrombin in combination with EP171. The serine protease, trypsin, stimulated an increase in [32P]phosphatidic acid and this effect was additive with that of EP171. This augmentation by trypsin of EP171-induced [32P]phosphatidic acid formation was inhibited by leupeptin. These results are consistent with the concept that thrombin-induced activation of phospholipase C occurs by two distinct mechanisms: one via proteolysis, which is sensitive to leupeptin, and the other via receptor activation, a process shared by EP171. The individual components of this dual mechanism can be mimicked by the co-addition of a receptor-directed agonist (EP171) and a proteolytic agent (trypsin).
Adenosine Diphosphate↗
Measurements of 6-keto-prostaglandin F1 alpha and thromboxane B2 in bleeding time blood: relation to bleeding and vascular disorders?
The body's ability to produce prostacyclin and thromboxane by blood vessels and platelets may be important in hemostatic and thrombotic disorders and in blood pressure regulation. There are limitations to the information that can be derived from measurement of the active substances or metabolites in plasma and urine. Assays for thromboxane and prostacyclin in bleeding time blood reflect production in response to a single standardized vascular injury, and show considerable promise in furthering our understanding of the production of these chemicals in vivo. These assays may improve the assessment of risk of developing thrombotic disorders and improve the ability to monitor treatment. Studies to date have focused largely on the influences of various doses of aspirin on the production of prostacyclin and thromboxane in bleeding time blood, but also suggest that smokers are high thromboxane producers. In addition, individuals who exhibit type A behavior, a behavior pattern characterized by a relatively high level of ambitiousness, hostility, and competitive drive and a chronic sense of urgency appear to be low prostacyclin producers. Diets enriched in sunflower oil were found to diminish thromboxane production, while diets high in canola oil enhanced prostacyclin formation.
6-Ketoprostaglandin F1 alpha↗
In vivo measurement of thromboxane B2 and 6-keto-prostaglandin F1 alpha in humans in response to a standardized vascular injury and the influence of aspirin.
The effects of smoking, aspirin ingestion, and sex differences on bleeding times and bleeding time thromboxane B2 and 6-keto-prostaglandin (PG)F1 alpha production were examined. Nonsmoking men produced more thromboxane B2 (3.99 +/- 0.76 ng/ml) than nonsmoking women (2.13 +/- 0.24 ng/ml). Female smokers produced more thromboxane B2 (5.01 +/- 0.97 ng/ml) than nonsmoking women. Twenty-four hours after a single dose of 600 mg aspirin, in vitro production of thromboxane B2 in response to collagen fell by 95%, whereas in vivo production of thromboxane B2 and 6-keto-PGF1 alpha in bleeding time blood fell by 87% and 66%, respectively. Subjects with the lowest absolute levels of thromboxane B2 24 hours after aspirin were also those with the longest postaspirin bleeding times. Recovery of 6-keto-PGF1 alpha production was faster than recovery of thromboxane B2 production, but 6-keto-PGF1 alpha production for most subjects was still below basal 72 hours after aspirin. The influence of two different doses of long-term aspirin (80 mg every other day and 325 mg daily) on the in vivo production of thromboxane B2 and 6-keto-PGF1 alpha was studied in normals and diabetics. After 14 days of 80 mg aspirin every other day, thromboxane B2 and 6-keto-PGF1 alpha production were both substantially inhibited (93% and 78%, respectively). After 14 days of 325 mg aspirin daily, thromboxane B2 production was similarly substantially inhibited (93%), whereas 6-keto-PGF1 alpha was significantly less affected (only 45% inhibition). Study of a second group of five normal subjects confirmed that 6-keto-PGF1 alpha production was significantly inhibited 24 hours after the first dose of 325 mg aspirin but was not significantly less than basal after 14 days of 325 mg aspirin. The results suggest that 325 mg aspirin daily is more antithrombotic compared with 80 mg every other day due to the superior preservation of prostacyclin production.
6-Ketoprostaglandin F1 alpha↗