Fasting is a physiological stimulus of vagus-mediated enhancement of nociception in the female rat.

The vagus nerve modulates nociception by a mechanism dependent upon gonadal hormones and the adrenal medulla. In the present study we tested the hypothesis that this modulation is dynamically controlled by physiological stimulation of structures innervated by the subdiaphragmatic vagus. Specifically, food deprivation (fasting) was employed to increase activity in the subdiaphragmatic vagus, and the experiments were performed mainly in female rats because our previous observations suggested that baseline activity in the pathway is lower in females than in males. Consistent with the hypothesis, after a 48-h fast, female rats exhibited increased nociceptive behavior in the formalin test. In contrast, fasting had no effect on formalin-evoked nociceptive behavior in male rats. The fasting-induced effect on nociception appears to be mediated by the vagus nerve since it is prevented by subdiaphragmatic vagotomy. Also similar to the previously characterized vagus-mediated modulation, the effect of fasting in the female is blocked by gonadectomy or adrenal medullectomy, and hormone replacement with 17beta-estradiol in gonadectomized female rats restored the effect of fasting. Decreased glucose metabolism apparently does not play a significant role in the effect of fasting on nociception, since the effect was unchanged when 5% glucose was provided in the drinking water throughout the fasting period. On the other hand, increasing the bulk content of the stomach (without providing nutrients) by infusion of petrolatum significantly attenuated the effect of fasting during the interphase period of the formalin response, suggesting that decreased gut distention, and possibly motility, are important in fasting-induced enhancement of nociception. These results indicate that fasting is a physiological activator of the vagus-mediated pain modulation pathway. This suggests the possibility that, especially in females, natural periodic changes in gut distention and motility may control an ongoing vagus-mediated adjustment in the organism's nociceptive sensitivity.

Role of adrenal medulla in development of sexual dimorphism in inflammation.

Many inflammatory diseases show a female predilection in adults, but not prepubertally. Because sex differences in the inflammatory response in the adult rat are mediated, in part, by sexual dimorphism in adrenal medullary function, we investigated the contribution of the adrenal medulla to the ontogeny of sexual dimorphism in inflammation. Whilst there was no sex difference in the magnitude of the plasma extravasation (PE) induced by the potent inflammatory mediator bradykinin (BK) in prepubertal rats, in adult rats BK-induced PE was markedly greater in males. Also, adult male rats, gonadectomized prior to puberty, had a lower magnitude of BK-induced PE than did adult male controls, whilst adult females gonadectomized prepubertally had higher BK-induced PE than did controls. In rats gonadectomized after puberty, the magnitude of BK-induced PE in adult males was not affected, whilst in females it resulted in significantly higher BK-induced PE, similar to the effect of prepubertal gonadectomy. When tested prepubertally, adrenal denervation increased the magnitude of BK-induced PE in females, but not in males. In contrast, in both males and females tested as adults, but castrated prepubertally, and in gonad-intact adult females, adrenal denervation significantly increased the magnitude of BK-induced PE. Adrenal denervation in prepubertal females given adult levels of 17beta-oestradiol produced a marked enhancement in the denervation-induced increase in magnitude of BK-induced PE compared to females not exposed prematurely to sex hormones. These studies suggest that an adrenal medulla-dependent inhibition of BK-induced PE is present in female but not male rats, and is enhanced by oestrogen but suppressed by testosterone.

Sex hormones regulate the contribution of PKCepsilon and PKA signalling in inflammatory pain in the rat.

We have evaluated the contribution of differences in second messenger signalling to sex differences in inflammatory pain and its control by sex hormones. In normal male but not female rats, epinephrine-induced mechanical hyperalgesia was antagonized by inhibitors of protein kinase Cepsilon (PKCepsilon), protein kinase A (PKA) and nitric oxide synthetase (NOS). Similarly, in PKCepsilon knockout mice, a contribution of PKCepsilon to epinephrine-dependent mechanical hyperalgesia occurred in males only. In contrast, hyperalgesia induced by prostaglandin E2, in both females and males, was dependent on PKA and NO. In both sexes, inhibitors of mitogen-activated protein kinase/extracellular-signal related kinase kinase (MEK) inhibited epinephrine hyperalgesia. In gonadectomized females, the second messenger contributions to epinephrine hyperalgesia demonstrated the pattern seen in males. Administration of oestrogen to gonadectomized females fully reconstituted the phenotype of the normal female. These data demonstrate gender differences in PKCepsilon, PKA and NO signalling in epinephrine-induced hyperalgesia which are oestrogen dependent and appear to be exerted at the level of the beta-adrenergic receptor or the G-protein to which it is coupled.

Gender and gonadal hormone effects on vagal modulation of tonic nociception.

We studied the influence of gender and gonadal hormones on modulation of tonic nociception exerted by vagal activity. In male rats, subdiaphragmatic vagotomy resulted in significantly reduced nociceptive behavior during phase 2 of the formalin test. Whereas gonadectomy alone had no effect, it completely eliminated the suppressive effect of subdiaphragmatic vagotomy; however, sex hormone replacement with either testosterone or dihydrotestosterone did not restore the ability of subdiaphragmatic vagotomy to suppress nociceptive behavior. These results suggest that, in males, a gonad-dependent but androgenic gonadal hormone-independent mechanism contributes to pronociceptive effects of vagal afferent activity. Although neither gonadectomy nor subdiaphragmatic vagotomy alone affected the response to formalin in females, gonadectomy plus vagotomy resulted in significantly reduced nociceptive behavior during phase 2. Reconstitution with 17 beta-estradiol implants in gonadectomized females not only prevented suppression of nociceptive behavior seen with gonadectomy plus vagotomy, but also led to increased nociceptive behavior in the interphase between phases 1 and 2. However, placement of 17 beta-estradiol implants in gonad-intact females had no effect on formalin-induced nociceptive behavior. The finding that estrogen produced an increase in nociceptive behavior in gonadectomized female rats after vagotomy but not in normal female rats (with intact gonads and subdiaphragmatic vagus) suggests that the interaction between estrogen and nociceptive afferent activity is suppressed by vagal function. In conclusion, a nonandrogenic action of testicular function in male rats and estrogen in females seems to influence the effect of vagal activity on formalin-induced nociceptive behavior.

Sex steroid regulation of the inflammatory response: sympathoadrenal dependence in the female rat.

To investigate the role of sex steroids in sex differences in the response of rats to the potent inflammatory mediator bradykinin (BK), we evaluated the effect of sex steroid manipulation on the magnitude of BK-induced synovial plasma extravasation (PE). The magnitude of BK-induced PE is markedly less in females. Ovariectomy of female rats increased BK-induced PE, and administration of 17beta-estradiol to ovariectomized female rats reconstituted the female phenotype. Castration in male rats decreased BK-induced PE, and administration of testosterone or its nonmetabolizable analog dihydrotestosterone reconstituted the male phenotype. The results of these experiments strongly support the role of both male and female sex steroids in sex differences in the inflammatory response. Because the stress axes are sexually dimorphic and are important in the regulation of the inflammatory response, we evaluated the contribution of the hypothalamic-pituitary-adrenal and the sympathoadrenal axes to sex differences in BK-induced PE. Neither hypophysectomy nor inhibition of corticosteroid synthesis affected BK-induced PE in female or male rats. Adrenal denervation in females produced the same magnitude increase in BK-induced PE as adrenalectomy or ovariectomy, suggesting that the adrenal medullary factor(s) in females may account for the female sex steroid effect on BK-induced PE. Furthermore, we have demonstrated that in female but not male rats, estrogen receptor alpha immunoreactivity is present on medullary but not cortical cells in the adrenal gland. These data suggest that regulation of the inflammatory response by female sex steroids is strongly dependent on the sympathoadrenal axis, possibly by its action on estrogen receptors on adrenal medullary cells.

A novel nociceptor signaling pathway revealed in protein kinase C epsilon mutant mice.

There is great interest in discovering new targets for pain therapy since current methods of analgesia are often only partially successful. Although protein kinase C (PKC) enhances nociceptor function, it is not known which PKC isozymes contribute. Here, we show that epinephrine-induced mechanical and thermal hyperalgesia and acetic acid-associated hyperalgesia are markedly attenuated in PKCepsilon mutant mice, but baseline nociceptive thresholds are normal. Moreover, epinephrine-, carrageenan-, and nerve growth factor- (NGF-) induced hyperalgesia in normal rats, and epinephrine-induced enhancement of tetrodotoxin-resistant Na+ current (TTX-R I(Na)) in cultured rat dorsal root ganglion (DRG) neurons, are inhibited by a PKCepsilon-selective inhibitor peptide. Our findings indicate that PKCepsilon regulates nociceptor function and suggest that PKCepsilon inhibitors could prove useful in the treatment of pain.

Effects of etidronate and lovastatin on the regression of atherosclerosis in cholesterol-fed rabbits.

Eighty New Zealand rabbits in eight groups (10 each) were fed a 0.5% cholesterol diet for 12 weeks. One group served as a control and was sacrificed at the end of 12 weeks. Seven other groups were shifted to a normal diet and received a drug(s) or placebo for the second 12 weeks. The high dose of etidronate (3 mg/kg/day) with lovastatin (6 mg/kg/day) significantly reduced the percent of aortic atherosclerotic lesions [56 +/- 21 vs. 77 +/- 17% (mean +/- SD), p < 0.05] in the regression study. Compared to the control groups for etidronate and lovastatin, the high or low dose (0.15 mg/kg/day) of etidronate significantly reduced aortic standardized plaque volume per unit (18.7 +/- 7.9 or 18.8 +/- 9.1 vs. 28.4 +/- 11.8 mm.%, p < 0.05). Lovastatin reduced pulmonary artery maximum plaque thickness (0.13 +/- 0.10 vs. 0.23 +/- 0.11 mm, p < 0.05). There were no differences in serum lipid and calcium levels in the control and treated groups. The high dose of etidronate inhibited bone mineralization as expected, whereas the low dose of etidronate did not. These data suggest that etidronate with lovastatin can regress aortic atherosclerosis in the cholesterol-fed rabbit placed on a normal diet.

Passive smoking increases experimental atherosclerosis in cholesterol-fed rabbits.

OBJECTIVES: We evaluated the influence of passive smoking on experimental atherosclerosis in cholesterol-fed rabbits. BACKGROUND: Exposure to environmental tobacco smoke (ETS) has been epidemiologically linked to death from ischemic heart disease in nonsmokers. METHODS: New Zealand male rabbits were randomly divided into three groups after 2 weeks of a 0.3% cholesterol diet. Sixteen rabbits were exposed to a high and 16 rabbits to a low dose of ETS; 32 rabbits located in another room served as an unexposed control group. After 10 weeks of ETS exposure, all rabbits were killed, and the percent of aortic and pulmonary artery endothelial surfaces covered by lipid lesions was measured by staining and planimetry. RESULTS: Average air nicotine, carbon monoxide and total particulate concentrations were 1,040 micrograms/m3, 60.2 ppm and 32.8 mg/m3 for the high dose ETS group, 30 micrograms/m3, 18.8 ppm and 4.0 mg/m3 for the low dose ETS group and < 1 microgram/m3, 3.1 ppm and 0.13 mg/m3 for the control group. The percent atherosclerotic involvement of the aorta and pulmonary artery increased significantly with ETS exposure (for the aorta, 30 +/- 19% [mean +/- SD] for the control group, 36 +/- 14% for the low dose ETS group and 52 +/- 21% for the high dose ETS group, p < 0.001; for the pulmonary artery, 22 +/- 15% for the control group, 29 +/- 25% for the low dose ETS group, and 45 +/- 12% for the high dose ETS group, p < 0.001). Bleeding time was significantly shorter in the two ETS groups than in the control group (86 +/- 17 vs. 68 +/- 15, 68 +/- 18 s, p < 0.001). There were no significant differences in serum triglycerides, cholesterol and high density lipoprotein cholesterol at the end of the study. CONCLUSIONS: Environmental tobacco smoke affects platelet function and increases aortic and pulmonary artery atherosclerosis. This increase of atherosclerosis was independent of changes in serum lipids and exhibited a dose-response relation. These results are consistent with data from epidemiologic studies demonstrating that ETS increases the risk of death due to heart disease.

Aspirin inhibits platelet activity but does not attenuate experimental atherosclerosis.

We evaluated the antiatherosclerotic potential of aspirin, a platelet inhibitor, in lipid-fed rabbits (0.3% cholesterol diet). Seventy-five male New Zealand white rabbits were divided into treated or control groups. The treated groups were given aspirin by daily gavage for 12 weeks (1 mg/kg, 10 mg/kg, 30 mg/kg, and 60 mg/kg) and 10 rabbits served as controls. Increased bleeding time was observed in the aspirin-treated groups (average, 58 +/- 10 seconds to 75 +/- 17 seconds; p < 0.001). Only high-dose aspirin (60 mg/kg/day) significantly inhibited platelet aggregation (1.04 +/- 0.15 vs 0.67 +/- 0.14; p < 0.05). Seventeen additional rabbits had aortic endothelial injury produced by a balloon catheter. Eight of them were given aspirin (40 mg/kg/day), and the other nine served as controls. The average percent of surface lesions and lesion thickness of the aorta and pulmonary artery were not significantly reduced by aspirin. These results show that at doses that cause antiplatelet effects, aspirin does not attenuate atherosclerosis.

Effect of lovastatin on suppression and regression of atherosclerosis in lipid-fed rabbits.

The present study was designed to evaluate the effects of lovastatin on suppression and regression of atherosclerosis in the lipid-fed rabbit. Fifty-seven New Zealand rabbits in six groups were fed a 0.3% cholesterol diet for 10 weeks. In the progression phase of the study, group C10 served as a control and received 1 ml of DMSO daily by gavage. Two other groups, L10 and H10, received low (L)-dose (10 mg/day) or high (H)-dose (20 mg/day) lovastatin dissolved in 1 ml of DMSO for 10 weeks. In the regression phase of the study, three groups of rabbits received the high lipid diet for 10 weeks and were then shifted to a normal diet for the second 10 weeks. During the second 10 weeks, the control group C20 received 1 ml of DMSO daily, and groups L20 and H20 received 10 and 20 mg/day of lovastatin by gavage, respectively. In the progression phase of the study, lovastatin significantly attenuated the percent of aortic lesions in groups L10 (8 +/- 7%) and H10 (9 +/- 14%) vs. the control group C10 (31 +/- 17%; p less than 0.01). There was a similar reduction in pulmonary lesions in groups L10 (10 +/- 6%) and H10 (4 +/- 5%) compared to the control group C10 (30 +/- 16%; p less than 0.01). There was also a reduction in plaque thickness in both the aorta and pulmonary artery, and hence an even greater reduction in estimated plaque volume. In the regression phase of the study, lovastatin also significantly reduced the percent of aortic lesions (groups L20 and H20 vs. C20: 27 +/- 18 and 22 +/- 7% vs. 40 +/- 17%; p less than 0.05) and pulmonary lesions (21 +/- 10 and 17 +/- 6% vs. 26 +/- 9%; p greater than 0.05 and p less than 0.05, respectively); the average maximum plaque thickness of aorta (0.24 and 0.26 mm vs. 0.49 mm; p less than 0.01); and the standardized plaque volume per unit area of aorta (4.5 and 3.4 vs. 12.1 mm-%; p less than 0.05 and p less than 0.01, respectively). However, there was no significant difference in the percent of aortic lesions between groups L20 and H20 and group C10 (27 +/- 18 and 22 +/- 7 vs. 31 +/- 17%; p greater than 0.05).(ABSTRACT TRUNCATED AT 400 WORDS)

Endogenous arachidonic acid metabolism by calcium ionophore A23187-stimulated lamb lungs: effect of hypoxia.

We have determined eicosanoid production from endogenous arachidonic acid by neonatal lamb lungs stimulated with calcium ionophore A23187 during normoxia and hypoxia. Lungs of lambs 19 to 25 d of age were isolated and perfused with cell-free Krebs' bicarbonate buffer at a flow rate of 15 to 20 ml/kg/min. After 30 min of equilibration in a recirculating system, A23187 was added to the perfusate in a 5-microM concentration and perfusion continued for 15 min more. Eicosanoids were measured in perfusate and lung homogenate supernatant. Cyclooxygenase metabolites prostaglandin (PG) E2, thromboxane A2, and PGI2, were measured by radioimmunoassay, and 5-lipoxygenase metabolites leukotrienes (LT) B4, C4, D4, and E4 by high performance liquid chromatography. During normoxia, all three cyclooxygenase metabolites were present in perfusate, but only PGI2 and thromboxane A2 were present in lung homogenate supernatant. Prostacyclin constituted 50% of all the cyclooxygenase products measured. LTC4 and LTD4 were detected in both perfusate and lung homogenate supernatant with little production of LTE4 and LTB4. During hypoxia, the profile of cyclooxygenase products was unchanged and prostacyclin production was not increased. However, the profile of leukotriene metabolites was altered. LTC4 synthesis was markedly reduced. The synthesis of LTE4 and LTB4 was increased 10-fold, with most of the leukotrienes being retained in lung tissue. We conclude that hypoxia significantly alters leukotriene metabolism of endogenous arachidonic acid by calcium ionophore-stimulated lungs. The increased production by stimulated lungs during hypoxia of LTE4, a substance that may increase lung capillary permeability, and that of LTB4, a powerful chemoattractant, may be important contributing factors to lung injury.

Monoclonal antibodies bound to subunits of the integrin GPIIb-IIIa are internalized and interfere with filopodia formation and platelet aggregation.

The monoclonal antibodies Tab and AP3 are directed, respectively, against GPIIb and GPIIIa, the subunits of the platelet fibrinogen receptor. When added together to platelets, these antibodies prevent adenosine diphosphate (ADP)-induced platelet aggregation, despite normal fibrinogen binding (Newman et al, Blood 69:668, 1987). To explore the cellular requirements of aggregation after fibrinogen binding, we used several techniques to study platelets treated with Tab and AP3, then stimulated with ADP. We used scanning and transmission electron microscopy to evaluate platelet morphology, immunolabel-surface replication to determine whether individual GPIIb-IIIa complexes clustered, immunocytochemistry on frozen thin sections to study the subcellular distribution of the integrin GPIIb-IIIa and fibrinogen, and biochemical methods to assess the activation of the platelet cytoskeleton. We found that the treated cells had short, blunted projections instead of normal filopodia. Other morphologic abnormalities, apparent in thin section, were aberrantly placed alpha-granules and microtubules, and a prominent, worm-like, fibrinogen-filled surface-connected canalicular system. Biochemical analysis suggested that such platelets undergo massive actomyosin-controlled membrane flow, which serves to sequester GPIIb-IIIa and makes the platelets refractory to aggregation. We conclude that aggregation requires the formation of long, slender filopodia, probably directed by cytoskeletal rearrangements after activation, and that the transmembrane GPIIb-IIIa complex may play a role in signaling these events.

Regression of atherosclerosis in cholesterol-fed rabbits: effects of fish oil and verapamil.

Previous studies have shown that either fish oil or verapamil can attenuate the development of atherosclerosis in the lipid-fed rabbit. The present study was designed to evaluate the individual and combined effects of these two interventions on regression. Seventy New Zealand rabbits in seven groups (10 each) were fed a 0.3% cholesterol diet for 10 weeks. Control group C10 was then killed. Control group C20 was fed a 0.3% cholesterol diet and the other five groups were fed a normal diet for an additional 10 weeks. Group F in three treated groups received 2 ml/day of fish oil (Proto-Chol, eicosapentaenoic acid, 180 mg/ml and docosahexaenoic acid, 120 mg/ml) by gavage. Group V received verapamil, 2 g/1,000 ml drinking water, and group FV received both fish oil and verapamil for an additional 10 weeks. Group CF (control for fish oil) received 2 ml/day of water by gavage and group CV (control for verapamil) received water without gavage for an additional 10 weeks. The percent of aortic and pulmonary atherosclerosis was measured by planimetry of sudanophilic lesions. The percent of aortic lesions in the four control groups (C20, C10, CF and CV) was 57 +/- 22, 40 +/- 15, 40 +/- 14 and 33 +/- 25%, respectively. The fish oil or verapamil groups (F, V, FV) showed a significant reduction in aortic lesions: 15 +/- 17%, p less than 0.05; 16 +/- 12%, p less than 0.05; and 26 +/- 24%, p = NS, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunogold-surface replica study of ADP-induced ligand binding and fibrinogen receptor clustering in human platelets.

Platelet cohesion requires the binding of fibrinogen to its receptor, a heterodimer consisting of the plasma-membrane glycoproteins GPIIb and GPIIIa. Although the GPIIb-IIIa complex is present on the surface of unstimulated platelets, it binds fibrinogen only after platelet activation. We have used an immunogold-surface replica technique to study the distribution of GPIIb-IIIa and bound fibrinogen over broad expanses of surface membranes in unstimulated and ADP-activated human platelets. We found that the gold prove was monodispersed over the surface of unstimulated platelets, although the cell surface lacked immunoreactive fibrinogen. To ascertain whether the receptors clustered prior to ligand binding or as a consequence thereof, we studied the surface distribution of GPIIb-IIIa after stimulation with ADP, which causes activation of the fibrinogen receptor function of GPIIb-IIIa without inducing the secretion of fibrinogen. In the absence of added fibrinogen, the unoccupied, yet binding-competent receptors on ADP-stimulated platelets were monodispersed. The addition of fibrinogen caused the GPIIb-IIIa molecules to cluster on the cell surface. Clustering was also induced by the addition of the GPIIb-IIIa binding domains of fibrinogen--namely, the tetrapeptide Arg-Gly-Asp-Ser on the alpha-chain or the gamma-chain decapeptide gamma 402-411. These results show that receptor occupancy causes clustering of GPIIb-IIIa in activated platelets.

Mycobacterium kansasii diffuse pulmonary infection in a patient with acquired immune deficiency syndrome. Successful therapy with an antituberculous regimen.

Mycobacterium kansasii is a rare cause of disseminated mycobacterial infection in patients with the acquired immune deficiency syndrome (AIDS). It occurs as the index AIDS diagnosis in only 0.2% of AIDS cases. Previously reported cases of AIDS-associated M. kansasii infection have manifested as diffuse interstitial pneumonitis and diffuse small bowel inflammation and have been refractory to antimycobacterial therapy. The authors now report success in treating a hypoxemic patient with AIDS-associated M. kansasii diffuse granulomatous interstitial pneumonitis that was diagnosed by open lung biopsy. The patient has no evidence of mycobacterial disease after 12 months of therapy with isoniazid, rifampin, and ethambutol.

Inhibition of atherosclerosis by fish oil in cholesterol-fed rabbits.

To evaluate the effects of dietary fish oil on cholesterol-induced atherosclerosis, 36 New Zealand rabbits in four groups were fed a 0.3% cholesterol diet for 10 weeks. One group served as control, whereas groups I, II and III received 1, 2 and 3 ml/day, respectively, of fish oil (Protochol, eicosapentaenoic acid, 180 mg, and docosahexaenoic acid [DHA], 120 mg/ml). The percent of aortic and pulmonary atherosclerosis was measured by planimetry of sudanophilic lesions. The percent of aortic lesions in the control group was 59 +/- 22%. The two higher dose fish oil groups showed a significant reduction in aortic lesions: group I (40 +/- 26%, p = NS), group II (18 +/- 11%, p less than 0.01) and group III (36 +/- 22%, p less than 0.05). Area of pulmonary artery lesions was significantly higher in the control group (48 +/- 22%) as compared with group I (15 +/- 13%, p less than 0.01), group II (4 +/- 3%, p less than 0.01) and group III (8 +/- 9%, p less than 0.01). The high cholesterol diet in the control group decreased bleeding time from 82 +/- 17 to 59 +/- 22 s (p less than 0.05). Groups II and III showed an increased bleeding time (62 +/- 15 to 84 +/- 17 s and 66 +/- 22 to 95 +/- 27 s; p less than 0.05, respectively). Fish oil did not significantly alter total serum cholesterol and high density lipoprotein (HDL) cholesterol. In group II triglyceride decreased from 128 +/- 22 to 64 +/- 25 mg/dl (p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

The platelet fibrinogen receptor: an immunogold-surface replica study of agonist-induced ligand binding and receptor clustering.

Platelet aggregation requires the binding of fibrinogen to its receptor, a heterodimer consisting of the plasma-membrane glycoproteins (GP) IIb and IIIa. Although the GPIIb-IIIa complex is present on the surface of unstimulated platelets, it binds fibrinogen only after platelet activation. We have used an immunogold-surface replica technique to study the distribution of GPIIb-IIIa and bound fibrinogen over broad areas of surface membranes in unstimulated, as well as thrombin-activated and ADP-activated human platelets. We found that the immunogold-labeled GPIIb-IIIa was monodispersed over the surface of unstimulated platelets, although the cell surface lacked immunoreactive fibrinogen. On thrombin-stimulated platelets, approximately 65% of the GPIIb-IIIa molecules were in clusters within the plane of the membrane. Fibrinogen, which had been released from the alpha-granules of these cells, bound to GPIIb-IIIa on the cell surface and was similarly clustered. To determine whether the receptors clustered before ligand binding, or as a consequence thereof, we studied the surface distribution of GPIIb-IIIa after stimulation with ADP, which causes activation of the fibrinogen receptor function of GPIIb-IIIa without inducing the release of fibrinogen. In the absence of added fibrinogen, the unoccupied, yet binding-competent receptors on ADP-stimulated platelets were monodispersed. The addition of fibrinogen caused the GPIIb-IIIa molecules to cluster on the cell surface. Clustering was also induced by the addition of the GPIIb-IIIa-binding domains of fibrinogen, namely the tetrapeptide Arg-Gly-Asp-Ser on the alpha-chain or the gamma-chain decapeptide gamma 402-411. These results show that receptor occupancy causes clustering of GPIIb-IIIa in activated platelets.

Topographic distribution of a granule membrane protein (GMP-140) that is expressed on the platelet surface after activation: an immunogold-surface replica study.

Monoclonal and polyclonal antibodies have been developed that recognize a 140 kD glycoprotein on the plasma membrane of activated, but not unstimulated, platelets. This glycoprotein is found in resting platelets as an alpha-granule membrane protein and has therefore been named GMP-140. After thrombin stimulation, alpha-granules fuse with the surface-connected canalicular system and GMP-140 is redistributed to the plasma membrane. In the present study, we immunolabeled unstimulated and activated human platelets and analyzed the distribution of GMP-140 over broad expanses of the plasma membrane using surface replication techniques. Fixed platelets were allowed to settle onto poly-L-lysine-coated coverslips and immunolabeled with polyclonal anti-GMP-140, followed by protein A gold. After critical-point drying, rotary-shadowed surface replicas were made. GMP-140 was not present on the surfaces of unstimulated platelets, but thrombin stimulation resulted in the massive expression of GMP-140 on the cell surface, with the immunogold label monodispersed. In contrast, we recently found that GPIIb-IIIa, the fibrinogen receptor, is monodispersed on unstimulated platelets and clustered on activated platelets. Although GMP-140's hemostatic function is unknown, its monodispersed surface pattern implies significant differences form GPIIb-IIIa with respect to ligand binding and/or cytoskeletal interaction.

Asbestos burden and the pathology of lung cancer.

To determine whether we could distinguish asbestos-related lung cancers from unrelated ones, we typed and quantified by electron-optical methods the asbestos fibers in the lungs of 75 men with lung cancer. All but eight men had some history of asbestos exposure. On the basis of combined amosite and crocidolite (AC) concentrations, we divided the subjects into three groups (AC fibers per gram of dry lung): low (less than 10(5)); intermediate (10(5) to 10(6)); and high (greater than 10(6)). Age, smoking history, latent period, and type and location of tumors were similar in all three groups. Of 62 evaluated subjects, zero of 14 in the low group, seven of 29 in the intermediate group, and five of 19 in the high group had asbestosis. Epidemiologic studies suggest that persons exposed to concentrations of asbestos that can cause asbestosis are at increased risk for lung cancer. Thus, the subjects in our intermediate and high concentration groups may have been at increased risk for cancer, even when they did not have asbestosis. Because large burdens of asbestos do not always cause pulmonary fibrosis, asbestosis may be a poor marker of fiber-related lung cancer.

Blood vessel invasion and axillary lymph node involvement as prognostic indicators for human breast cancer.

Blood vessel invasion and axillary lymph node involvement were examined in 175 breast cancer patients. The incidence of blood vessel invasion was 35%. The presence of blood vessel invasion was highly associated with early disease recurrence. The association of poor prognosis with blood vessel invasion was independent of clinical stage, menopausal status, node status, tumor size, or postsurgical treatment. Those patients with blood vessel invasion and two or more positive nodes were at extremely high risk for early recurrence (70% recurrence by two years compared with 15% recurrence in the remainder of the patients). Thus, blood vessel invasion is a useful indicator of early recurrence in patients with primary breast cancer and, in combination with node status, is a prognostic indicator with high discriminatory power.