Novel vitamin K-dependent pathways regulating cell survival.

Historically, the vitamin K1-dependent proteins have been associated primarily with blood coagulation and secondarily with bone formation. Recent identification of K1-dependent proteins as specific ligands for the receptor tyrosine kinases (RTKs) that can stimulate cell replication and transformation and participate in cell survival highlighted a previously unrecognized and potentially important role for vitamin K1 in cell signaling. Growing evidence suggests that most normal and tumor cells possess an active K1-dependent gamma-carboxylation mechanism necessary for the production of gamma-carboxyglutamic acid (Gla)-containing proteins. Gla residues in proteins facilitate calcium-dependent protein/phospholipid interaction. Recent studies demonstrating the potentially positive effects of a vitamin K-dependent receptor:ligand system on cell growth and survival in general and the effects of the overexpression of these RTKs on malignant cell survival provide a new perspective on the role of vitamin K1, its dependent protein ligands, and their receptors. These cumulative observations also provide an explanation for the rigidly controlled K1 levels in the mammalian fetus and the minimal hepatic stores in the adult.

Circadian periodicity of urinary volume, creatinine and 5-hydroxyindole acetic acid excretion in healthy Indians.

The circadian periodicity of urinary output, creatinine (Cr) and 5-hydroxyindole acetic acid (5-HIAA) excretion was studied under near-tropical conditions in 130 healthy volunteers (65 men and 65 women, 16-75 years of age) with a diurnal activity from about 06:30 to about 22:00 and nocturnal rest. These volunteers were divided into 4 groups, 16-30, 31-45, 46-60 and 61-75 years of age, comprising 20, 20, 15 and 10 participants of each gender, respectively. A marked circadian rhythm was recorded for urine volume, Cr and 5-HIAA excretion in healthy Indians of different ages. The acrophase tended to be delayed in the older age group. The relative amplitude decreased with advancing age, notably in women. Overall, men produced a larger urine volume as compared to women. Excretions of Cr and 5-HIAA in healthy Indian volunteers of different ages may be influenced by diet, societal relations, climate and/or geographic location. The contribution of such factors in metabolism and degradation warrants further study.

The riddle of vitamin K1 deficit in the newborn.

Vitamin K in the fetus and newborn is maintained at levels less than that necessary to achieve full gamma-carboxylation of the K-dependent proteins, including those required for hemostasis. As the infant matures and even into adulthood, there is no significant storage pool for this vitamin, and a K1-deficient state can be produced by placing an adult on a K-deficient diet for 7 to 10 days. Questions arise as to why the level of vitamin K is so rigidly controlled and why the placental gradient in humans and other mammals maintains the fetus in a K-"deficient" state. The evidence is reviewed that suggests that K-dependent proteins are ligands for receptor tyrosine kinases, which, in the rapidly proliferating cell milieu of the fetus, control growth regulation. Increased stimuli may result in growth dysregulation whereas conversely, the further depletion of vitamin K-dependent proteins, as in warfarin toxicity, depletes the required stimuli for normal embryogenesis. These findings argue for the need for tightly controlled levels of vitamin K consistent with normal embryogenesis.

A novel role for vitamin K1 in a tyrosine phosphorylation cascade during chick embryogenesis.

The development of the embryo is dependent upon a highly coordinated repertoire of cell division, differentiation, and migration. Protein-tyrosine phosphorylation plays a pivotal role in the regulation of these processes. Vitamin K-dependent gamma-carboxylated proteins have been identified as ligands for a unique family (Tyro 3 and 7) of receptor tyrosine kinases (RTKs) with transforming ability. The involvement of vitamin K metabolism and function in two well characterized birth defects, warfarin embryopathy and vitamin K epoxide reductase deficiency, suggests that developmental signals from K-dependent pathways may be required for normal embryogenesis. Using a chick embryogenesis model, we now demonstrate the existence of a vitamin K1-dependent protein-tyrosine phosphorylation cascade involving c-Eyk, a member of the Tyro 12 family, and key intracellular proteins, including focal adhesion kinase (pp125FAK), paxillin, and pp60src. This cascade is sensitive to alteration in levels or metabolism of vitamin K1. These findings provide a major clue as to why, in the mammalian (and human) fetus, the K-dependent proteins are maintained in an undercarboxylated state, even to the point of placing the newborn at hemorrhagic risk. The precise regulation of vitamin K1-dependent regulatory pathways would appear to be critical for orderly embryogenesis.

Toxicological evaluation of 1-chloroacetophenone and dibenz[b,f]-1,4-oxazepine after repeated inhalation exposure in mice.

Toxicological evaluation was made on the effects of two peripheral sensory irritants (tear gases): 1-chloroacetophenone (CN) and dibenz[b,f]-1,4-oxazepine (CR). Animals had a 15-min daily inhalation exposure to average vapour concentrations of 87.6 mg CN m-3 or 1008 mg CR m-3 (both equal to 0.05 LC50) for 5 or 10 days and were sacrificed 24 h after the last exposure, when biochemical and histopathological observations were made. Both chemicals caused a significant decrease in body weight gain. Histological changes in lung, liver and kidneys were more severe after 10 than after 5 days of exposure and were more severe in CN-exposed than in CR-exposed mice. Organ weight to body weight ratios remained normal except for the spleen to body weight ratio, which decreased in CN-exposed mice after both 5 and 10 days of exposure. Biochemical indicators showed a toxic response only in CN-exposed mice, but the only consistent change was an increase in blood glucose. Hepatic alkaline phosphatase was not influenced, malondialdehyde concentration and acid phosphatase activity were increased only after 5 days of exposure and liver GSH concentration decreased after 10 days of exposure. Results indicate that CN is not only more toxic than CR in absolute terms but is also more toxic at the 5% level of their LC50.

Multiple cytokines activate phosphatidylinositol 3-kinase in hemopoietic cells. Association of the enzyme with various tyrosine-phosphorylated proteins.

Activation of phosphatidylinositol (PI) 3-kinase is a common sequel to tyrosine kinase activation and appears to be essential for tyrosine kinases to induce proliferation. Since multiple hemopoietic growth factors activate tyrosine kinases, we investigated whether these growth factors activate PI 3-kinase. We show that interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), granulocyte-macrophage colony stimulating factor (GM-CSF), and steel factor (SLF) all activate PI 3-kinase. These cytokines increased the amount of PI 3-kinase activity that could be immunoprecipitated with anti-phosphotyrosine antibodies from the MC-9 mast cell line or from the hemopoietic progenitor cell line FDC-P1. Increases in this assay frequently correlate with PI 3-kinase activation in vivo. To determine directly whether these factors activate PI 3-kinase in vivo, we measured the levels of 3-phosphorylated inositol phospholipids in intact 32P-labeled MC-9 cells. IL-3, IL-4, IL-5, GM-CSF, and SLF all caused increased synthesis of the PI 3-kinase products phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate with a relative potency of SLF >> IL-3 > IL-5, GM-CSF > IL-4. In contrast, IL-4 caused the largest increase in the in vitro anti-phosphotyrosine immune complex PI 3-kinase assay. Thus, the in vitro assay does not accurately reflect in vivo activation of PI 3-kinase. Cytokine treatment did not stimulate tyrosine phosphorylation of either the 85-kDa regulatory subunit or the 110-kDa catalytic subunit of PI 3-kinase and is therefore not required for activation of PI 3-kinase by these factors. Cytokine treatment did induce PI 3-kinase to associate with other tyrosine-phosphorylated proteins in a cytokine-specific manner. PI 3-kinase associated with c-kit after SLF stimulation, a 170-kDa protein after IL-4 stimulation, and a 70-kDa protein after treatment with IL-3 or GM-CSF. Thus, multiple hemopoietic growth factors that act through different types of receptors activate PI 3-kinase in vivo and induce factor-specific interactions of PI 3-kinase with other tyrosine-phosphorylated proteins.

Major histocompatibility complex genes and susceptibility to systemic lupus erythematosus in northern India.

Fifty-eight patients with systemic lupus erythematosus (SLE) from Northern India were tissue typed for HLA class I and II antigens. The results revealed an appreciable increase of HLA-DR4 (37.5%) among patients compared with controls (17.9%), P < 0.03. Additionally, haplotype B8-DR3 was encountered frequently in the patient group. The findings suggest an important role of MHC genes in influencing susceptibility to SLE.

Histamine as an intracellular messenger in human platelets.

The results of investigations in platelets provide evidence for an intracellular messenger role for histamine. Studies of neutrophils and of cellular proliferation suggest that there may be a wider role for histamine as an intracellular messenger modulating activation processes in cells.

Significance of serum immunoglobulin levels in conglomeration with trans-sternal phlebography in the phlebography management of breast cancer.

The study was conducted on 63 patients with breast lump and twenty normal healthy females. In benign breast disease, a significant (P < 0.001) rise in serum IgA, significant (P < 0.001) decrease in IgG and no change in IgM levels was seen before operation. A significant decrease (P < 0.001) in serum IgA and significant increase in IgG and IgM was observed post operatively. In carcinoma breast, a significant (P < 0.001) elevation in IgA, IgG and IgM levels was found pre-operatively with a concomitant decrease in serum IgA and IgG and increase in serum IgM levels, post operatively. Trans-sternal phlebography (TSP) carried out with 95.23% success has revealed significant (P < 0.001) change in the staging of carcinoma breast. The increased levels of serum immunoglobulins associated with the patients of carcinoma breast with metastasis has led to conclude that these levels, if punctuated with TSP findings can lead to better assessment of the staging of carcinoma breast and thereby its management.

Biochemical and ultrastructural studies suggest that the effects of thapsigargin on human platelets are mediated by changes in intracellular calcium but not by intracellular histamine.

The involvement of intracellular histamine in thapsigargin (Tg)-induced platelet aggregation was studied. Platelet aggregation induced by 0.25 and 0.5 microM Tg was not accompanied by a rise in intracellular histamine but a significant (p < 0.01) increase in the level of intracellular histamine was observed at 1 microM Tg. Preincubation of platelets with inhibitors of histamine metabolizing enzymes had little effect on intracellular histamine levels in platelets stimulated by 0.5 microM Tg. In addition, the inhibitors of histidine decarboxylase (HDC), alpha-methyl histidine (alpha-MH) and alpha-fluoromethyl histidine (alpha-FMH) failed to inhibit Tg-induced aggregation. The intracellular histamine receptor antagonist, N,N-diethyl-2-[4-(phenylmethyl)phenoxy] ethanamine. HCl (DPPE), inhibited Tg-induced aggregation but with IC50 values dependent on the concentration of agonist used. The inhibitory effects of DPPE on Tg-induced aggregation were not reversed by the addition of histamine to saponin-permeabilized platelets suggesting non-histamine mediated effects of DPPE on Tg-induced aggregation. Tg stimulated an increase in the cytosolic free calcium concentration which was unaffected by DPPE indicating that the effects of DPPE are also not due to the inhibition of mobilization of cytosolic calcium. The ultrastructural studies suggest that the major Tg-induced changes (pseudopod formation and granule centralization) are consistent with a primary role for Tg to mobilize calcium; DPPE had very little effect on these ultrastructural changes. The results indicate that the effects of Tg on human platelets are mediated by an increase in cytosolic calcium but not by intracellular histamine.

Prevalence, classification and natural history of gastric varices: a long-term follow-up study in 568 portal hypertension patients.

To determine the prevalence and natural history of gastric varices, we prospectively studied 568 patients (393 bleeders and 175 nonbleeders) with portal hypertension (cirrhosis in 301 patients, noncirrhotic portal fibrosis in 115 patients, extrahepatic portal vein obstruction in 117 patients and hepatic venous outflow obstruction in 35 patients). Primary (present at initial examination) gastric varices were seen in 114 (20%) patients; more were present in bleeders than in non-bleeders (27% vs. 4%, respectively; p < 0.001). Secondary (occurring after obliteration of esophageal varices) gastric varices developed in 33 (9%) patients during follow-up of 24.6 +/- 5.3 mo. Gastric varices (compared with esophageal varices) bled in significantly fewer patients (25% vs. 64%, respectively). Gastric varices had a lower bleeding risk factor than did esophageal varices (2.0 +/- 0.5 vs. 4.3 +/- 0.4, respectively) but bled more severely (4.8 +/- 0.6 vs. 2.9 +/- 0.3 transfusion units per patient, respectively). Once a varix bled, mortality was more likely (45%) in gastric varix patients. Gastric varices were classified as gastroesophageal or isolated gastric varices. Type 1 gastroesophageal varices (lesser curve varices) were the most common (75%). After obliteration of esophageal varices, type 1 gastroesophageal varices disappeared in 59% of patients and persisted in the remainder; bleeding from persistent gastroesophageal varices was more common than it was from gastroesophageal varices that were obliterated (28% vs. 2%, respectively; p < 0.001). Type 2 gastroesophageal varices, which extend to greater curvature, bled often (55%) and were associated with high mortality. Type 1 isolated gastric varices patients had only fundal varices, with a high (78%) incidence of bleeding.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis of intracellular histamine in platelets is associated with activation of protein kinase C, but not with mobilization of Ca2+.

In previous reports, we have provided evidence indicating that newly formed histamine is an intracellular messenger in human platelets. The involvement of protein kinase C (PKC) and intracellular calcium (Ca2+i) in the synthesis of histamine was investigated. Human platelets were stimulated by phorbol 12-myristate 13-acetate (PMA), collagen and the Ca2+ ionophore A23187, with or without the PKC inhibitor staurosporine. Aggregation, histamine synthesis and phosphorylation of pleckstrin (47 kDa; P47) and myosin light chain (20 kDa; P20) proteins were monitored. Staurosporine inhibited PMA- and collagen-induced aggregation, histamine synthesis and phosphorylation of 47 kDa and 20 kDa proteins in a dose-dependent manner. For PMA, median inhibitory concentrations (IC50 values) for staurosporine inhibition of aggregation, histamine synthesis and phosphorylation were similar, suggesting that histamine synthesis induced by this agonist may be a consequence of PKC activation. Conversely, collagen-stimulated histamine synthesis was inhibited by staurosporine at concentrations significantly higher than those required to inhibit aggregation (P less than 0.005) or pleckstrin phosphorylation (P less than 0.01), indicating the possible involvement of non-PKC mechanism(s) in the synthesis of histamine induced by this agonist. A23187 failed to induce the synthesis of intracellular histamine in platelets, whereas staurosporine blocked A23187-induced aggregation and phosphorylation of the 20 kDa protein at significantly higher concentrations than those needed to inhibit PKC. When platelets were stimulated with a combination of A23187 and PMA, the increase in platelet histamine was less than that with PMA alone. The results provide evidence that the synthesis of intracellular histamine in platelets occurs as a consequence of PKC activation and may be down-regulated under conditions where there is a substantial rise in [Ca2+]i.

Further Studies on the Effects of the Intracellular Histamine Antagonist DPPE on Platelet Function.

SUMMARY. The role of intracellular histamine in the activation of human platelets was explored using the novel histamine antagonist N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine (DPPE). DPPE inhibited aggregation, [(32)P]-phosphatidic acid production (an index of phospholipase C activity) and the increase in cytosolic calcium ([Ca(2+)]i) in response to collagen. In contrast, while at higher concentrations DPPE inhibited aggregation in response to ADP and the thromboxane mimetic EP171, it failed to inhibit EP171-induced [(32)P]-phosphatidic acid production or increases in [Ca(2+)]i elicted by either ADP or EP171. Collagen but neither ADP nor EP171 caused the significant formation of intracellular histamine. The biochemical changes induced by collagen have been shown previously to be, at least partly, secondary to thromboxane generation. Collagen-induced arachidonic acid release was therefore assessed. DPPE inhibited the release of arachidonic acid in response to collagen but had no effect on its subsequent conversion to thromboxane. The findings imply that intracellular histamine acts at an early stage in collagen-induced platelet activation, most likely prior to arachidonic acid release, and further that DPPE inhibits ADP and EP171-induced activation by a non-histamine related effect.

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.

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.

A role for intracellular histamine in ultrastructural changes induced in platelets by phorbol esters.

In human platelets, phorbol esters, such as phorbol-12-myristate-13-acetate (PMA), induce morphological changes, including pseudopod formation and the swelling and fusion of intracellular granule membranes with those of the surface-connected canalicular system, effects which have been attributed to activation of protein kinase C. However, a novel intracellular histamine antagonist, N,N-diethyl-2-[4-(phenylmethyl)phenoxy]-ethanamine. HCl (DPPE), previously has been shown to block PMA-induced aggregation independently of protein kinase C interaction, an effect reversible in permeabilized platelets by the addition of histamine. We now demonstrate that DPPE inhibits, in a concentration-dependent manner, the effects of PMA on human platelet ultrastructure. In permeabilized platelets, histamine reverses this inhibition, although it alone induces minimal effects on morphology. The results support a role for this amine to promote the labilization of platelet granules and pseudopod formation induced by PMA, presumably by acting in concert with additional PMA-activated pathways.

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.