Smoking rate, carboxyhemoglobin, and body mass in the Second National Health and Nutrition Examination Survey (NHANES II).

The relationship of body mass and triceps skinfold thickness to both reported number of cigarettes smoked per day and carboxyhemoglobin levels was examined in healthy cigarette smokers in the NHANES II. Among both men and women, higher carboxyhemoglobin levels were related to lower body mass and thinner skinfolds, whereas higher levels of reported daily cigarette smoking were related to increased body mass and thicker skinfolds among men only. These relationships were independent of age, education, caloric intake, physical activity, and exercise. The opposite effects of number of cigarettes smoked per day and a biological index of cigarette smoke exposure on body mass suggest that increased cigarette smoking may covary with factors that would favor increased body weight among men, whereas decreases in body weight with increases in carboxyhemoglobin may reflect the effects of nicotine exposure on energy expenditure in both men and women.

Cigarette smoking and body weight in the Cancer Prevention Study I.

To investigate the generality of the association of heavy cigarette smoking with increased body weight, the relation of number of cigarettes smoked per day to relative body weight was examined in baseline data for 891,589 participants in a prospective study initiated in 1959. Although the relative weight of cigarette smokers was consistently lower than that of never and exsmokers, men and women smoking two or more packs of cigarettes per day were more likely to be categorized as moderately or severely overweight and less likely to be categorized as underweight than those smoking 10-20 cigarettes per day, despite somewhat greater educational attainment by heavier smokers. These analyses offer support for the temporal generality of the relation between heavier cigarette smoking and greater body weight, and suggest that this phenomenon cannot be explained by historic trends in the socioeconomic stratification of smoking prevalence or smoking dose.

Pathway for the formation of D-3 phosphate containing inositol phospholipids in PDGF stimulated NIH 3T3 fibroblasts.

PtdIns (3, 4, 5) P3 is formed rapidly in NIH-3T3 cells stimulated with platelet derived growth factor (PDGF). We have determined the pathway of formation of this lipid in these cells. Cells were labeled briefly with 32PO4 and then stimulated with PDGF under conditions where the specific activity of each phosphate group determines the order of its addition. The D-5 phosphate of this lipid contained approximately 42% of the total radioactivity present in the molecule, while approximately 32% was in the D-4 position, 25% in the D-3 position, and approximately 2% in the D-1 position. This indicates that PtdIns (3, 4) P2 and not PtdIns (4, 5)P2 is the immediate precursor of PtdIns (3, 4, 5) P3, and defines the pathway of formation of these lipids to be PtdIns (3) P----PtdIns (3, 4) P2----PtdIns (3, 4, 5) P3. This pathway is the same as that in thrombin-stimulated platelets and infers that the pathways are not different in non-growing and proliferating cells.

Pathway for the formation of D-3 phosphate containing inositol phospholipids in intact human platelets.

We have identified the structure of phosphatidylinositol 3-phosphate (PtdIns(3)P), phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) in human platelets. These lipids accounted for less than 2% of the total 32P incorporated into inositol phospholipids in the platelets. All three lipids were labeled in unstimulated platelets, but incorporation of 32P changed rapidly by 15 s after thrombin stimulation, suggesting that they are important in platelet activation. Specific inositol polyphosphate phosphatases were used to both identify the lipid structures and to determine the route of synthesis of these lipids. During 32P labeling and after thrombin stimulation of human platelets, as much as 60% of the total radioactivity present in PtdIns(3,4)P2 was found in the D-4 phosphate and only 35% in the D-3 phosphate indicating that PtdIns(3)P is the precursor of PtdIns(3,4)P2. In addition, the D-5 and D-4 phosphates of PtdIns(3,4,5)P3 each contained 35-40% of the total radioactivity in the molecule compared with only 18-28% in the D-3 position, suggesting that PtdIns(3,4)P2 and not PtdIns(4,5)P2 is the major precursor of this lipid. These results define the predominant pathway for synthesis of these lipids in platelets as PtdIns----PtdIns(3)P----PtdIns(3,4)P2----PtdIns(3,4,5)P3.

Recent insights in phosphatidylinositol signaling.

Studies of phosphatidylinositol signaling pathways are entering a new phase in which molecular genetic techniques are providing powerful tools to dissect the functions of various metabolites and pathways. Studies with phospholipase C are most advanced and clearly indicate that phosphatidylinositol turnover is critical for vision in Drosophila and cell proliferation in various cultured cells. Expression of cDNA constructs and microinjection of PLC or antibodies against it clearly establish a role for PtdIns signaling distinct from its role in calcium mobilization and protein kinase C activation. The importance of inositol cyclic phosphates is also beginning to be realized from the study of cyclic hydrolase using similar techniques. Elucidation of the function of the 3-phosphate inositol phospholipid pathway awaits similar studies. The recent cDNA cloning of inositol monophosphatase (Diehl et al., 1990), Ins(1,4,5)P3 3-kinase (Choi et al., 1990), and inositol polyphosphate 1-phosphatase (York and Majerus, 1991) should provide tools to define further the cell biology of the phosphatidylinositol signaling pathway.

Regulation of superoxide responses of human neutrophils by adenine compounds. Independence of requirement for cytoplasmic granules.

Recent evidence suggests that the adenine compounds ATP, adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S), and adenosine have important regulatory effects on O2- responses of human neutrophils stimulated with the chemotactic peptide N-formyl-Met-Leu-Phe (fMLP). Because of evidence that receptors on neutrophils may be modified by granule fusion events, we assessed the extent to which these adenine compounds affect fMLP and CR3 (C3bi) receptors on neutrophils and whether cytoplasmic granules are required for the ability of the adenine compounds to modify O2- responses in neutrophils stimulated with fMLP. Incubation of neutrophils with ATP gamma S or adenosine led to a decrease in numbers of fMLP receptors (17 and 9.2%, respectively) but no change in receptor affinity (Kd). Paradoxically, ATP gamma S caused an increase in CR3 receptors (Mo1, CD-11b antigen), suggesting that fMLP and CR3 receptors may be under separate control. The ability of the adenine compounds to modify O2- responses in fMLP-stimulated cells was equivalent in both neutrophils and cytoplasts, suggesting that the regulatory effect of ATP, ATP gamma S, and adenosine do not require the presence of cytoplasmic granules. ATP gamma S caused enhancement of O2- responses of neutrophils to phorbol 12-myristate 13-acetate, raising the possibility that ATP gamma S may be affecting late events in the signal transduction pathway.

Treatment of A431 cells with epidermal growth factor (EGF) induces desensitization of EGF-stimulated phosphatidylinositol turnover.

Epidermal growth factor (EGF) stimulates the turnover of phosphoinositides in A431 cells. In cells that were pretreated with EGF for 30 min at 37 degrees C and then washed to remove surface-bound hormone, a 70-100% decrease in the EGF-stimulated production of inositol monophosphate, inositol bisphosphate, and inositol triphosphate was noted when the cells were exposed to the agonist a second time. Since only a 15% decrease in receptor number was observed in these pretreated cells, the loss of responsiveness to EGF for the production of inositol phosphates could not be attributed to a down-regulation of the EGF receptors. These data suggest that pretreatment of A431 cells with high concentrations of EGF leads to a desensitization of the EGF receptor. This desensitization of the receptor by EGF is apparent within 10-15 min of the addition of EGF and is maximal by 30 min. The desensitization appears to be homologous in nature since pretreatment of cells with EGF did not diminish their responsiveness to bradykinin; and conversely, pretreatment with bradykinin did not diminish the subsequent responsiveness of the cells to EGF. Desensitization to EGF was observed in cells in which protein kinase C had been down-regulated by prolonged treatment with 12-O-tetradecanoylphorbol-13-acetate, implying that EGF receptor desensitization is independent of protein kinase C. The desensitizing effects of EGF on growth factor-induced phosphatidylinositol turnover could be prevented by pretreatment of the cells with the calmodulin antagonist trifluoperazine, suggesting that calmodulin may be involved in the regulation of EGF receptor sensitivity.

Signal transduction events in stimulated rat neutrophils: effects of adenine nucleotides.

When rat neutrophils were stimulated with chemotactic peptide N-formyl-Met-Leu-Phe; fMLP), phorbol myristate acetate (PMA), or immune complexes in the presence of homologous platelets, O2- responses were enhanced. Secretion products of thrombin-stimulated platelets as well as ATP, ATP gamma S, or ADP enhanced O2- responses of fMLP-stimulated neutrophils, although these nucleotides did not, by themselves, initiate an O2- response. Calcium transients were measured in neutrophils which were stimulated with fMLP under a variety of conditions (+/- ATP gamma S, +/- cytochalasin B) which enhance O2- responses. Neutrophils incubated with ATP gamma S alone developed brief calcium transients similar to those produced by fMLP. As compared to changes in intracellular calcium in fMLP-stimulated neutrophils, the copresence of ATP gamma S with fMLP did not cause a statistically significant difference in the calcium transients, even though O2- production was enhanced. In contrast, in the presence of cytochalasin B, the addition of ATP gamma S to fMLP-stimulated neutrophils produced greatly sustained and protracted elevations in intracellular calcium, correlating with further enhancement of O2- responses. In fMLP-stimulated neutrophils the latter phases of the calcium transients appeared to be dependent in part on the availability of extracellular calcium. These data suggest that ATP gamma S-induced enhancement of O2- responses in fMLP-stimulated rat neutrophils may be related to two mechanisms, one being independent of fMLP-induced intracellular calcium transients and the other, which is related to the presence of cytochalasin B, being linked to sustained elevations in intracellular calcium associated with mobilization of extracellular calcium.

Differing calcium requirements for regulatory effects of ATP, ATP gamma S and adenosine on O2.- responses of human neutrophils.

In formyl peptide stimulated human neutrophils the enhancement of O2.- responses by ATP and ATP S requires extracellular calcium. In contrast, the inhibitory effects of adenosine are independent of a calcium requirement. Rates of O2.- generation are not affected by these adenine compounds. Rather, ATP and ATP S cause a sustained period of generation whereas adenosine causes an abrupt early termination of the O2- response. The differing calcium requirements for regulatory effects of adenine compounds on O2.- responses of stimulated neutrophils suggests that ATP (or ATP gamma S) and adenosine may exert their effects at different points in the pathway of signal transduction events.

Regulatory effects of adenosine and adenine nucleotides on oxygen radical responses of neutrophils.

Our recent studies have indicated that release of ATP/ADP from platelets causes enhanced O2-. responses in stimulated neutrophils. The current investigations were designed to provide further details of this phenomenon, to determine the structure-function correlates of the adenine compounds, and to assess if the results might be explained by the formation of a single metabolic product of ATP. ATP, ADP, AMP and adenosine enhanced O2-. responses of rat neutrophils stimulated with immune complexes or formyl chemotactic peptide (FMLP) but had no effect on responses of phorbol ester-stimulated neutrophils. Similar results were obtained in human neutrophils stimulated with immune complexes; when FMLP was the agonist, the results were divergent: ATP and ADP enhanced the responses, whereas AMP and adenosine were inhibitory. In structure-function studies, hydrolytically resistant forms of ATP (and other adenine nucleotides) containing blocked or cross-linked phosphate groups were active, suggesting that hydrolysis of these compounds to a common metabolic product is not required for their effects on O2-. responses. In contrast, other chemical modifications of the ribose ring or adenine base of ATP resulted in greatly diminished activity. To further pursue the question of whether metabolism of the adenine compounds via the adenosine pathway was related to the observed effects on O2-. responses, addition to rat neutrophils of inhibitors of adenosine deaminase, S-adenosyl homocysteine hydrolase, or xanthine oxidase failed to reproduce or augment the enhancement effects of the adenine compounds on O2-. responses, suggesting that metabolism of the adenine compounds to a common product may not be a requirement for the observed effects. Although the manner by which the adenine compounds affect O2-. responses is not known, the data suggest that adenosine and adenine nucleotides have important regulatory effects on oxygen radical responses of stimulated neutrophils.

Macrophage-derived cytokines amplify immune complex-triggered O2-. responses by rat alveolar macrophages.

Interleukin-1 (IL-1) and tumor necrosis factor (TNF) are monocyte macrophage-derived hormonelike regulatory proteins that participate in many physiologic and pathophysiologic processes. Several proinflammatory activities have been attributed to these cytokines, but their importance in anatomically compartmentalized inflammatory processes is unclear. The current in vitro studies have been designed to examine modulatory influences of these cytokines on O2-. responses of rat phagocytes implicated as effector cells in immune complex mediated lung injury. Purified human IL-1, recombinant human TNF (rTNF), and culture supernatant from zymosan-activated alveolar macrophages significantly amplified O2-. responses of immune complex-stimulated alveolar macrophages but did not enhance the responses of neutrophils. Equivalent concentrations of IL-1, rTNF, and alveolar macrophage culture supernatant had no direct stimulatory effect on alveolar macrophages as measured by O2-. production. Culture media from unstimulated alveolar macrophages exerted negligible effects on O2-. generation by immune complex-activated alveolar macrophages. These data indicate that O2- responses of immune complex alveolar macrophages can be enhanced by the presence of IL-1, TNF, or media from activated macrophages. It is possible that macrophage products may greatly amplify tissue injury through the enhancement of oxygen radical production.

Platelet enhancement of O2-. responses in stimulated human neutrophils. Identification of platelet factor as adenine nucleotide.

Human neutrophils stimulated with immune complexes in the presence of platelets show enhanced superoxide anion (O2-.) responses that are proportional to the amount of agonist present and the number of platelets added. Platelet related enhancement of O2-. responses also occurs with the neutrophil agonists phorbol ester, formyl chemotactic peptide and zymosan particles. Pretreatment of platelets with cycloheximide does not alter their ability to enhance O2-. responses of neutrophils. In parallel with platelet-related enhancement of O2-. responses of immune complex-stimulated neutrophils, secretory release of myeloperoxidase is also increased. The platelet effects on O2-. responses can be reproduced with platelet lysates or with supernatant fluids which have been obtained from thrombin or immune complex-stimulated platelets and are rich in ATP and ADP content. Solutions containing ATP and ADP in amounts similar to those found in supernatant fluids of activated platelets reproduce the enhancement of O2-. responses in N-formyl methionyl leucyl phenylalanine (FMLP) or immune complex-activated neutrophils. The platelet factor responsible for the effects of neutrophils is heat-stable, elutes in gel sieving chromatography near the position of phenol red and does not, in the absence of a neutrophil agonist, trigger an O2-. response. With formyl peptide-stimulated neutrophils, ATP and ADP enhance O2-. responses while the responses are depressed by addition of AMP or adenosine. In immune complex-stimulated neutrophils, adenosine and all adenine nucleotides enhance the O2-. responses. Taking advantage of this information, treatment of ATP or of supernatant fluids from thrombin-stimulated platelets with alkaline phosphatase (resulting in formation of adenosine) converts the O2-. enhancing activity for formyl peptide-activated neutrophils into an inhibitory activity. In contrast, using immune complex-activated neutrophils, similar manipulations of ATP or supernatant fluids from stimulated platelets result only in enhanced O2-. responses. These data support the conclusion that the platelet-derived factor responsible for enhanced O2-. responses in neutrophils is ATP/ADP. In FMLP stimulated neutrophils, the presence of ATP or ADP leads to enhanced increases in intracellular levels of Ca++ as determined by the fura-2 probe, while the presence of AMP or adenosine results in inhibition of the increases in FMLP induced elevations in cytosolic Ca++. These data demonstrate a direct relationship between effects of adenine compounds on FMLP induced changes in cytosolic Ca++ and the associated O2-. responses.