Myosin in cultured vascular smooth muscle cells: immunofluorescence and immunochemical studies of alterations in antigenic expression.

Vascular smooth muscle cells (VSMC) in the rat mesenteric artery show specific immunofluorescent staining with antisera against purified human uterine myosin (ASMM) but not human platelet myosin (APM). However, in primary cultures produced by enzymatic dissociation of this vessel, VSMC stain specifically with both ASMM and APM within 5 h after plating and throughout growth to confluence (4-10 d). In confluent cultures, APM staining remains bright while ASMM staining is reduced in intensity in most cells. In contrast, cellular myosin content, determined by quantitative SDS PAGE, is comparable in confluent and growing cultures. Immunoprecipitation of high salt extracts of cultured VSMC with ASMM and APM yields myosins with the same mobilities on SDS PAGE. When serial, exhaustive precipitations are performed with one antiserum, followed by reprecipitation with the other, myosin in subconfluent and confluent VSMC cultures is exhaustively precipitated by either antiserum, thus indicating complete immunological cross-reactivity. These results might be explained by synthesis of a new myosin isoform reactive with both ASMM and APM. However, the development of APM staining in cultured VSMC did not require protein synthesis. Therefore, it is more likely that the changes in immunofluorescent staining observed in vitro reflect conformational alterations, perhaps related to cytoskeletal rearrangements. These changes in myosin antigenic expression may be relevant to the problem of VSMC phenotypic modulation both in vitro and in vivo.

Functional angiotensin II receptors in cultured vascular smooth muscle cells.

To study cellular mechanisms influencing vascular reactivity, vascular smooth muscle cells (VSMC) were obtained by enzymatic dissociation of the rat mesenteric artery, a highly reactive, resistance-type blood vessel, and established in primary culture. Cellular binding sites for the vasoconstrictor hormone angiotensin II (AII) were identified and characterized using the radioligand 125I-angiotensin II. Freshly isolated VSMC, and VSMC maintained in primary culture for up to 3 wk, exhibited rapid, saturable, and specific 125I-AII binding similar to that seen with homogenates of the intact rat mesenteric artery. In 7-d primary cultures, Scatchard analysis indicated a single class of high-affinity binding sites with an equilibrium dissociation constant (Kd) of 2.8 +/- 0.2 nM and a total binding capacity of 81.5 +/- 5.0 fmol/mg protein (equivalent to 4.5 x 10(4) sites per cell). Angiotensin analogues and antagonists inhibited 125I-AII binding to cultured VSMC in a potency series similar to that observed for the vascular AII receptor in vivo. Nanomolar concentrations of native AII elicited a rapid, reversible, contractile response, in a variable proportion of cells, that was inhibited by pretreatment with the competitive antagonist Sar1,Ile8-AII. Transmission electron microscopy showed an apparent loss of thick (12-18 nm Diam) myofilaments and increased synthetic activity, but these manifestations of phenotypic modulation were not correlated with loss of 125I-AII binding sites or hormonal responsiveness. Primary cultures of enzymatically dissociated rat mesenteric artery VSMC thus may provide a useful in vitro system to study cellular mechanisms involved in receptor activation-response coupling, receptor regulation, and the maintenance of differentiation in vascular smooth muscle.

Angiotensin II stimulation of prostaglandin production in cultured human vascular endothelium.

Immunoreactive material resembling prostaglandin E accumulates in the medium of cultured human umbilical vein endothelial cells. Prodcution is inhibited by indomethacin and stimulated by angiotensin II. Prostaglandin secretion by endothelium may be important in platelet-dependent thrombotic phenomena, and in local control of vascular permeability and tone in vivo.

Vasoconstriction: a new activity for platelet-derived growth factor.

Platelet-derived growth factor (PDGF) is a potent mitogen for vascular smooth muscle cells that has been implicated in the pathogenesis of atherosclerosis. The potential role of PDGF in the altered vasoreactivity of atherosclerotic vessels has been studied through an examination of its effects on contractility in the rat aorta. PDGF caused a concentration-dependent contraction of aortic strips and was significantly more potent on a molar basis than the classic vasoconstrictor peptide angiotensin II. Furthermore, PDGF increased the cytosolic free calcium concentration in cultured rat aortic smooth muscle cells. These observations suggest a new biological activity for PDGF that may contribute to the enhanced vasoreactivity of certain atherosclerotic vessels.

Linoleic acid and its metabolites, hydroperoxyoctadecadienoic acids, stimulate c-Fos, c-Jun, and c-Myc mRNA expression, mitogen-activated protein kinase activation, and growth in rat aortic smooth muscle cells.

Previous studies from other laboratories suggest that linoleic acid and its metabolites, hydroperoxyoctadecadienoic acids, play an important role in modulating the growth of some cells. A correlation has been demonstrated between hydroperoxyoctadecadienoic acids and conditions characterized by abnormal cell growth such as atherosclerosis and psoriasis. To determine if linoleic acid and its metabolites modulate cell growth in atherosclerosis, we measured DNA synthesis, protooncogene mRNA expression, and mitogen-activated protein kinase (MAPK) activation in vascular smooth muscle cells (VSMC). Linoleic acid induces DNA synthesis, c-fos, c-jun, and c-myc mRNA expression and MAPK activation in VSMC. Furthermore, nordihydroguaiaretic acid, a potent inhibitor of the lipoxygenase system, significantly reduced the growth-response effects of linoleic acid in VSMC, suggesting that conversion of linoleic acid to hydroperoxyoctadecadienoic acids (HPODEs) is required for these effects. HPODEs also caused significant induction of DNA synthesis, protooncogene mRNA expression, and MAPK activation in growth-arrested VSMC, suggesting that linoleic acid and its metabolic products, HPODEs, are potential mitogens in VSMC, and that conditions such as oxidative stress and lipid peroxidation which provoke the production of these substances may alter VSMC growth.

Characterization of the human platelet alpha-adrenergic receptor. Correlation of [3H]dihydroergocryptine binding with aggregation and adenylate cyclase inhibition.

Human platelets aggregate and undergo a release reaction when incubated with catecholamines. Indirect evidence indicates that these events are mediated through alpha-adrenergic receptors. We used [(3)H]dihydroergocryptine, an alpha-adrenergic antagonist, to identify binding sites on platelets that have the characteristics of alpha-adrenergic receptors. Catecholamines compete for the binding sites in a stereo-specific manner with the potency series of (-) epinephrine > (-) norepinephrine > (+/-) phenylephrine > (-) isoproterenol. The dissociation constant (K(d)) of (-) epinephrine is 0.34 muM. Binding is saturable using a platelet particulate fraction but not with intact platelets. There are 0.130 pmol binding sites per milligram protein in fresh platelet membranes. This number represents approximately 100 binding sites per platelet. The K(d) for [(3)H]-dihydroergocryptine was 0.003-0.01 muM. The alpha-adrenergic antagonist phentolamine (K(d) = 0.0069 muM) was much more potent than the beta-adrenergic antagonist (+/-) propranolol (K(d) = 27 muM) in competing for the binding sites. The binding data were correlated with catecholamine-induced platelet aggregation and inhibition of basal and prostaglandin E(1)-stimulated adenylate cyclase. (-) Epinephrine was more potent than (-) norepinephrine in producing aggregation whereas (-) isoproterenol was ineffective. The threshold dose for inducing aggregation by (-) epinephrine was 0.46 muM. Phentolamine and dihydroergocyrptine blocked this response, whereas (+/-) propranolol had no effect. (-) Epinephrine and (-) norepinephrine inhibited basal and prostaglandin E(1)-stimulated adenylate cyclase in a dose-related manner. The concentration of (-) epinephrine inhibiting adenylate cyclase 50% was 0.7 muM. This inhibition was also blocked by phentolamine and dihydroergocryptine but not by (+/-) propranolol. The specificity of binding and the close correlation with alpha-adrenergic receptor-mediated biochemical and physiological responses suggest that the [(3)H]dihydroergocryptine binding site represents, or is closely related to, the human platelet alpha-adrenergic receptor. The ability to assay this receptor directly and to correlate these data with independently measured sequelae of receptor activation should facilitate increased understanding of the physiology and pathophysiology of the human platelet alpha-adrenergic receptor.

Modified low density lipoprotein and its constituents augment cytokine-activated vascular cell adhesion molecule-1 gene expression in human vascular endothelial cells.

Early features in the pathogenesis of atherosclerosis include accumulation of oxidized LDL (oxLDL) and endothelial expression of the vascular adhesion molecule VCAM-1. Because antioxidants inhibit endothelial VCAM-1 expression, we tested the hypothesis that oxLDL functions as a prooxidant signal in atherogenesis to augment VCAM-1 activation by inflammatory signals. Cultured human aortic endothelial cells (HAECs) or human umbilical vein endothelial cells (HUVECs) were incubated with unmodified LDL, oxLDL, or glycated LDL for 48 h. No change in VCAM-1, intercellular cell adhesion molecule-1 (ICAM-1), or E-selectin expression from control was observed by ELISA. However, dose-response and time course studies demonstrated that oxLDL enhanced VCAM-1 expression induced by the cytokin tumor necrosis factor alpha (TNF alpha) 63% in HAECs and 45% in HUVECs over unmodified LDL or control. Using flow cytometry analysis, oxLDL augmented TNF alpha-induced VCAM-1 expression in a uniform HAEC population. oxLDL had no effect on E-selection induction. oxLDL augmented TNF alpha-induced ICAM-1 expression 44% in HAECs but not in HUVECs. Glycated LDL augmented TNF alpha-induced VCAM-1 expression 35% in HAECs but not HUVECs. Similar results were obtained with 13-HPODE or lysophosphatidylcholine, significant components of oxLDL. 13-HPODE augmented TNF alpha-induced mRNA accumulation and transcriptional activation of VCAM-1 in HAECs. These results suggest that as long-term regulatory signals, specific oxidized fatty acid and phospholipid components of oxLDL augment the ability of vascular endothelial cells to express cytokine-mediated VCAM-1. These studies link oxidant signals conferred by oxLDL to oxidation-sensitive regulatory mechanisms controlling the expression of endothelial cell adhesion molecules involved in early atherosclerosis.

Effects of dietary sodium and of acute saline infusion on the interrelationship between dopamine excretion and adrenergic activity in man.

The effects of dietary sodium and of saline infusion on urinary dopamine and norepinephrine and on the relationship of these catecholamines to adrenergic activity were determined. In seven normal subjects on a 9-meq sodium intake, urinary dopamine and norepinephrine were 136+/-18 (SE) and 37.4+/-5.3 mug/day, respectively. When sodium intake was increased to 209 or 259 meq/day, urinary dopamine increased to 195+/-20 mug/day (P<0.01) whereas urinary norepinephrine decreased to 21.1+/-3.0 mug/day (P<0.01). Infusion of saline in seven subjects increased sodium excretion and urinary dopamine (from 2.18+/-0.22 to 2.79+/-0.19 mug/20 min, P<0.01), but decreased plasma dopamine-beta-hydroxylase by 33% and urinary norepinephrine insignificantly. The clearance of inulin and p-aminohippurate did not change significantly and filtration fraction was the same. The data indicate that an increase in dietary sodium or infusion of saline results in an apparent decrease in adrenergic activity and an increase in urinary dopamine. Dopamine excretion would thus appear to relate inversely to adrenergic activity and to parallel sodium excretion. These findings suggest a possible role for dopamine and norepinephrine in the regulation of renal sodium excretion.

Spontaneously hypertensive rat vascular smooth muscle cells in culture exhibit increased growth and Na+/H+ exchange.

The cellular mechanisms responsible for abnormalities in spontaneously hypertensive rat (SHR) vascular smooth muscle cell (VSMC) growth and vasoreactivity are not defined. Because Na+/H+ exchange, which we have previously demonstrated in cultured VSMC, plays an essential role in mediating growth factor responses, we hypothesized that abnormalities in SHR growth regulation might be reflected in the activity of this transporter. To test this hypothesis, we studied DNA synthesis and Na+/H+ exchange (measured as the rate of amiloride-sensitive intracellular alkalinization or Na+ influx) in early subcultures (less than 6) of aortic VSMC from 12-wk-old SHR and Wistar Kyoto (WKY) animals. Serum-deprived SHR VSMC grew more rapidly in response to 10% serum with an increase in [3H]thymidine incorporation of 439% compared with 191% in WKY controls. Basal intracellular pH (pHi) values determined by fluorescent pH measurements were 7.37 +/- 0.04 and 7.27 +/- 0.03 (P less than 0.05) in early passage SHR and WKY, respectively. Acid recovery (initial pHi = 6.8) by SHR VSMC was faster than by WKY VSMC as measured by alkalinization (1.8 +/- 0.6 vs. 0.8 +/- 0.2 mmol H+/liter.min, P less than 0.05) or by amiloride-sensitive 22Na+ influx (14.5 +/- 1.2 vs. 4.0 +/- 0.5 nmol Na+/mg protein.min, P less than 0.05). In comparison to WKY cells early passage SHR VSMC exhibited 2.5-fold greater alkalinization and amiloride-sensitive 22Na+ influx in response to 100 nM angiotensin II. During serial passage, WKY cells acquired enhanced Na+/H+ exchange and growth rates so that by passage 6, these differences were no longer present. These findings in early cultures of SHR VSMC, removed from the in vivo neurohumoral milieu, suggest that increased Na+/H+ exchange in SHR may reflect alterations in Na+ homeostasis that might contribute to altered SHR VSMC function such as enhanced growth and vasoreactivity.

Regulation of postocclusive hyperemia by endogenously synthesized prostaglandins in the dog heart.

Experiments were performed to evaluate the role of prostaglandin synthesis in the regulation of coronary blood flow in dog hearts. The left main coronary artery was cannulated and flow measured both in otherwise intact animals and in canine heart-lung preparations. Prostaglandin E was measured by radioimmunoassay. Reactive hyperemia (flow after occlusion release) was induced by coronary occlusion for 10, 15, and 20 s and was 39 plus or minus 13 (mean plus or minus SEM), 66 plus or minus 21, and 82 plus or minus 24 ml, respectively. Indomethacin, an inhibitor of prostaglandin synthetase, reduced reactive hyperemia at 10, 15, and 20 s to 15 plus or minus 5, 33 plus or minus 11, and 47 plus or minus 17 ml, respectively (P smaller than 0.05). Meclofenamate, a different prostaglandin synthetase inhibitor, gave similar results. In a second group of five dogs, prostaglandin production of the heart was examined in response to 20-s occlusions. There was a significant increase in prostaglandin production from a basal level of 18.6 plus or minus 4.9 mg/min to 35.3 plus or minus 5.8 ng/min after occlusion of the coronary artery for 20 s (P smaller than 0.05). After indomethacin, this increase in prostaglandin production was not observed and reactive hyperemia was significantly reduced. Thus, prostaglandin synthesis appears to be important to modulating canine coronary blood flow in response to brief periods of coronary occlusion.

Vascular cell adhesion molecule-1 (VCAM-1) gene transcription and expression are regulated through an antioxidant-sensitive mechanism in human vascular endothelial cells.

Oxidative stress and expression of the vascular cell adhesion molecule-1 (VCAM-1) on vascular endothelial cells are early features in the pathogenesis of atherosclerosis and other inflammatory diseases. Regulation of VCAM-1 gene expression may be coupled to oxidative stress through specific reduction-oxidation (redox) sensitive transcriptional or posttranscriptional regulatory factors. In cultured human umbilical vein endothelial (HUVE) cells, the cytokine interleukin 1 beta (IL-1 beta) activated VCAM-1 gene expression through a mechanism that was repressed approximately 90% by the antioxidants pyrrolidine dithiocarbamate (PDTC) and N-acetylcysteine (NAC). Furthermore, PDTC selectively inhibited the induction of VCAM-1, but not intercellular adhesion molecule-1 (ICAM-1), mRNA and protein accumulation by the cytokine tumor necrosis factor-alpha (TNF alpha) as well as the noncytokines bacterial endotoxin lipopolysaccharide (LPS) and double-stranded RNA, poly(I:C) (PIC). PDTC also markedly attenuated TNF alpha induction of VCAM-1-mediated cellular adhesion. In a distinct pattern, PDTC partially inhibited E-selectin gene expression in response to TNF alpha but not to LPS, IL-1 beta, or PIC. TNF alpha and LPS-mediated transcriptional activation of the human VCAM-1 promoter through NF-kappa B-like DNA enhancer elements and associated NF-kappa B-like DNA binding proteins was inhibited by PDTC. These studies suggest a molecular linkage between an antioxidant sensitive transcriptional regulatory mechanism and VCAM-1 gene expression that expands on the notion of oxidative stress as an important regulatory signal in the pathogenesis of atherosclerosis.

Responses of coronary arteries of cardiac transplant patients to acetylcholine.

Accelerated coronary atherosclerosis is a major cause of graft failure after heart transplantation. Graft atherosclerosis is typically diffuse and difficult to detect even with coronary arteriography. Recently, acetylcholine was shown to dilate blood vessels by releasing a vasorelaxant substance from the endothelium (endothelium-derived relaxing factor). We have demonstrated paradoxical vasoconstriction induced by acetylcholine both early and late in the course of coronary atherosclerosis in patients, suggesting an association of endothelial dysfunction and atherosclerosis. In this report, we tested the hypothesis that coronary arteries of heart transplant patients can show endothelial dysfunction before or in the early stages of angiographically evident coronary atherosclerosis. Acetylcholine was infused into the left anterior descending artery of 13 heart transplant patients at 12 (n = 9) and 24 (n = 4) mo after transplantation. Vascular responses were evaluated by quantitative angiography. Among patients with angiographically smooth coronary arteries, relatively few (6/25) arterial segments had preserved vasodilator responses, while the majority failed to dilate (10/25) or paradoxically constricted (9/25). Angiographically irregular coronary arteries were present in three patients, in whom 8/10 segments showed marked paradoxical constriction and the remaining 2/10 failed to dilate. Only 1 of 13 patients retained appropriate dilation to acetylcholine in all segments. Nitroglycerin, which acts directly on vascular smooth muscle, dilated nearly all segments. No clinical features of the patients, including myocardial rejection appeared to correlate with the impaired functional response of vessels. Thus impaired response to acetylcholine is a common early finding in heart transplant patients and emphasizes the potential importance of endothelial dysfunction in the development of atherosclerosis.

Molecular cloning and characterization of the constitutive bovine aortic endothelial cell nitric oxide synthase.

The constitutive endothelial cell nitric oxide synthase (NOS) importantly regulates vascular homeostasis. To gain understanding of this enzyme, a pEF BOS cDNA library of 5 x 10(5) clones was prepared from bovine aortic endothelial cells (BAEC) and screened with a 2.8-kb cDNA BamHI fragment of rat brain NOS. Clone pBOS13 was found to express NO synthase activity when transfected into COS-7 cells. Sequence analysis revealed sequences compatible with binding domains for calcium/calmodulin, flavin mononucleotide, flavin adenine nucleotide and NADPH. The deduced amino acid sequence revealed a protein with a relative mol mass of 133,286, which is 58% homologous to the rat cerebellar NOS and 51% homologous to the mouse macrophage NOS. The amino-terminal portion of the protein exhibits several characteristics peculiar to the endothelial cell NOS. These include a proline-rich region and several potential sites for proline-directed phosphorylation as well as a potential substrate site for acyl transferase. Northern hybridization to mRNA from cultured BAEC revealed an abundant 4.8-kb message, which was not increased by coincubation with tumor necrosis factor alpha, but was markedly increased by exposure to shear stress for 24 h. The unique features of the endothelial cell NO synthase, particularly in the amino terminal portion of the molecule, may provide for novel regulatory influences of enzyme activity and localization.

Atherosclerosis influences the vasomotor response of epicardial coronary arteries to exercise.

We studied the vasomotion of epicardial coronary arteries during exercise and tested the hypotheses that abnormal vasoconstriction is related to the presence of atherosclerosis and may be related to endothelial dilator dysfunction. During cardiac catheterization quantitative coronary angiography was performed in 21 patients during supine bicycle exercise. 21 of 28 smooth, angiographically normal vessel segments dilated (14.0 +/- 1.8%) during exercise; four smooth segments did not change whereas only three constricted. In contrast, 15 of 16 vessel segments with irregularities constricted in response to exercise (17.0 +/- 0.1%) with only one segment dilating. All 10 stenotic segments constricted to exercise (23 +/- 4%). Six patients also received intracoronary acetylcholine before exercise to test endothelium-dependent dilator function. In five of six patients all nine vessel segments showed the same directional response to acetylcholine and exercise. Three irregular and two stenotic segments constricted with acetylcholine (51 +/- 21%) and exercise (9.0 +/- 0.6%). In contrast, four smooth segments dilated to acetylcholine (19 +/- 6%) and exercise (9 +/- 1%). Both exercise and acetylcholine generally dilated smooth but constricted irregular and stenosed coronary segments. It appears likely that atherosclerosis plays an important role in the abnormal vasomotion of diseased coronary arteries during exercise and the pattern of abnormality suggests impairment of vasodilator function.

Epidermal growth factor, a vascular smooth muscle mitogen, induces rat aortic contraction.

Atherosclerotic arteries have enhanced reactivity to vasoconstrictors, which suggests that features of the atherosclerotic process itself may result in this abnormal responsiveness. Since vascular smooth muscle proliferation is a prominent feature of atherosclerosis, we postulated that vasoactive agonists and smooth muscle mitogens may share certain common cellular mechanisms of action which potentially contribute to this hyperreactivity. To test this hypothesis, we studied the effects of epidermal growth factor (EGF), a well-characterized mitogen, on rat aortic vascular smooth muscle, both in intact aortic strips and in culture. EGF caused contraction (EC50 = 19 nM) of rat aortic strips which maximally was equivalent to 40% of that induced by angiotensin II, a potent vasoconstrictor. EGF increased 45Ca efflux (EC50 = 3 nM) from cultured rat aortic smooth muscle cells, which was an effect shared by angiotensin II and thought to reflect increased cytosolic-free calcium concentration. EGF (7.5 nM) also stimulated growth of these cultured cells to the same extent as 10% calf serum. These results demonstrate that EGF is both a vasoconstrictor and mitogen for rat aortic smooth muscle cells. The similarities in the effects of EGF and angiotensin II suggest that certain common intracellular mechanisms of action may exist for vasoactive agonists and growth factors which may contribute to the altered vasoreactivity of atherosclerotic vessels.

Avian oncovirus Mill Hill No. 2: pathogenicity in chickens.

Mill Hill No. 2 (MH2), an avian tumor virus, was studied for its transforming and oncogenic effects. In tissue culture it induced transformation of chicken fibroblasts and yolk sac macrophages. When injected into the chicken, the main feature of this virus was its ability to cause liver and kidney carcinoma, in addition to sarcoma. MH2-associated viruses did not transform cell cultures but were able to cause only lymphoma in the birds. Light and electron microscopy were used in a detailed histologic study of the tumors induced by MH2 virus. An unclassified round cell sarcoma was produced in soft tissues at sites of injection; there was no evidence of origin from endothelium. In the kidney, carcinomas mixed with a malignant stroma were found. Hepatocarcinomas were the dominant tumors found in the liver. The lymphomas produced by the associated virus were poorly differentiated and highly malignant. The study illustrated the highly oncogenic potential of this virus and offered a model for the analysis of the carcinogenic events in a more specific way.

Domain-domain communication in aminoacyl-tRNA synthetases.

Aminoacyl-tRNA synthetases are modular proteins, with domains that have distinct roles in the aminoacylation reaction. The catalytic core is responsible for aminoacyl adenylate formation and transfer of the amino acid to the 3' end of the bound transfer RNA (tRNA). Appended and inserted domains contact portions of the tRNA outside the acceptor site and contribute to the efficiency and specificity of aminoacylation. Some aminoacyl-tRNA synthetases also have distinct editing activities that are localized to unique domains. Efficient aminoacylation and editing require communication between RNA-binding and catalytic domains, and can be considered as a signal transduction system. Here, evidence for domain-domain communication in aminoacyl-tRNA synthetases is summarized, together with insights from structural analysis.

Immunity to cell surface antigens and immune complex glomerulonephritis in hamsters bearing human epithelial tumors.

A glomerulopathy in hamsters bearing human epithelial tumors is described. The tumors resulted from the s.c. implantation of human heteroploid cells (MA160) derived originally from a benign prostatic adenoma. The renal immunomorphological and ultrastructural changes include expansion of mesangial matrix and cellularity, electron-dense mesangial deposits, and mesangial deposits of immunoglobulin G and C'3. Renal eluates obtained from the tumor-bearing animals contained substantial amounts of immunoglobulins. Our immunopathological studies demonstrated that virtually all of the eluate immunoglobulin G was tumor-specific antibody directed exclusively against surface determinants of the MA160 cells. There was no demonstrable activity against cytoplasmic or nuclear constituents of the MA160 cells or against certain hamster antigenic determinants. The antibody in the eluate could be removed by repetitive absorptions with viable MA160 cells but not with similar absorptions with either normal human diploid fibroblasts, human heteroploid HeLa cells, or BHK21 hamster cells. Our data support the concept of a specific nephritogenic immunopathological process occurring as a result of immunity to tumor-specific surface determinants.

Effects of bombesin on growth of human small cell lung carcinoma in vivo.

Bombesin-like peptides are found in many different human tumors and are thought to function as an autocrine growth factor for small cell lung cancer in humans. In this study, a human small cell lung carcinoma (NCI-H69) was s.c. implanted bilaterally into the flanks of 12 nude mice. The mice were randomized and divided into two groups and given either bombesin (20 micrograms/kg) or saline i.p. 3 times a day. Tumor areas were measured twice weekly for 6 wk. At sacrifice, the tumors and normal pancreas were excised, weighed, and assayed for DNA, RNA, and protein content. Significant stimulation of tumor growth was observed at weeks 4, 5, and 6. Tumor weight at sacrifice was significantly elevated (77%) above the control, as was DNA content (78%). Bombesin significantly increased the weight (42%), DNA (48%), and protein (61%) contents of the normal mouse pancreas. We conclude that bombesin may act as an autocrine growth factor, or indirectly through the release of other growth factors, on human small cell lung carcinoma.

Hydrogen peroxide stimulates transcription of c-jun in vascular smooth muscle cells: role of arachidonic acid.

We reported previously that hydrogen peroxide induces DNA synthesis in rat aortic smooth muscle (RASM) cells. In the present paper we studied the mechanism by which hydrogen peroxide induces c-jun mRNA, an early response gene whose activation is required for mitogen-stimulated cell growth. Hydrogen peroxide induced c-jun mRNA in growth-arrested RASM cells in a time dependent manner. This stimulation was significantly inhibited by mepacrine, a phospholipase A2 (PLA2) inhibitor. Arachidonic acid, a PLA2 product, also increased c-jun mRNA with a time course similar to that of hydrogen peroxide. The increases in c-jun mRNA induced by hydrogen peroxide and arachidonic acid were significantly reduced (55%) by down-regulation of protein kinase C with a phorbol ester. Furthermore, the effect of hydrogen peroxide on c-jun mRNA was also reduced by NDGA, an inhibitor of the lipoxygenase-cytochrome P450 mono-oxygenase system, suggesting that metabolism of arachidonic acid through this pathway is required for the induction of c-jun mRNA by oxidants. Both hydrogen peroxide and arachidonic acid significantly increased c-jun transcription as demonstrated by nuclear run-on assays. Together these observations suggest that: (1) the induction of c-jun mRNA by hydrogen peroxide is mediated by PLA2-dependent arachidonic acid release and metabolism through the lipoxygenase-cytochrome P450 mono-oxygenase system; (2) PKC appears to be involved in this signaling pathway and (3) the induction of c-jun mRNA by hydrogen peroxide in RASM cells is due to increased transcription.