Cell elongation induces laminin alpha2 chain expression in mouse embryonic mesenchymal cells: role in visceral myogenesis.

Bronchial smooth muscle (SM) mesenchymal cell precursors change their shape from round to spread/elongated while undergoing differentiation. Here we show that this change in cell shape induces the expression of laminin (LM) alpha2 chain not present in round mesenchymal cells. LM alpha2 expression is reversible and switched on and off by altering the cell's shape in culture. In comparison, the expression of LM beta1 and gamma1 remains unchanged. Functional studies showed that mesenchymal cell spreading and further differentiation into SM are inhibited by an antibody against LM alpha2. Dy/dy mice express very low levels of LM alpha2 and exhibit congenital muscular dystrophy. Lung SM cells isolated from adult dy/dy mice spread defectively and synthesized less SM alpha-actin, desmin, and SM-myosin than controls. These deficiencies were completely corrected by exogenous LM-2. On histological examination, dy/dy mouse airways and gastrointestinal tract had shorter SM cells, and lungs from dy/dy mice contained less SM-specific protein. The intestine, however, showed compensatory hyperplasia, perhaps related to its higher contractile activity. This study therefore demonstrated a novel role for the LM alpha2 chain in SM myogenesis and showed that its decrease in dy/dy mice results in abnormal SM.

Embryonic mesenchymal cells share the potential for smooth muscle differentiation: myogenesis is controlled by the cell's shape.

Undifferentiated embryonic mesenchymal cells are round/cuboidal in shape. During development, visceral myogenesis is shortly preceded by mesenchymal cell elongation. To determine the role of the cell's shape on smooth muscle development, undifferentiated embryonic mesenchymal cells from intestine (abundant visceral muscle), lung (some visceral muscle) or kidney (no visceral muscle) were plated under conditions that maintained cell rounding or promoted elongation. Regardless of their fate in vivo, all the cells differentiated into smooth muscle upon elongation as indicated by the expression of smooth muscle-specific proteins and the development of membrane potentials of -60 mV and voltage-dependent Ca2+ currents, characteristic of excitable cells. Smooth muscle differentiation occurred within 24 hours and was independent of cell proliferation. Regardless of their fate in vivo, all the round cells remained negative for smooth muscle markers, had membrane potentials of -30 mV and showed no voltage-activated current. These cells, however, differentiated into smooth muscle upon elongation. The role of the cell's shape in controlling smooth muscle differentiation was not overcome by treatment with retinoic acid, TGF-beta1, PDGF BB or epithelial-conditioned medium (all modulators of smooth muscle differentiation). These studies suggest that the mesenchymal cell shape plays a main role in visceral myogenesis.

Role of laminin polymerization at the epithelial mesenchymal interface in bronchial myogenesis.

Undifferentiated mesenchymal cells were isolated from mouse embryonic lungs and plated at subconfluent and confluent densities. During the first 5 hours in culture, all the cells were negative for smooth muscle markers. After 24 hours in culture, the mesenchymal cells that spread synthesized smooth muscle alpha-actin, muscle myosin, desmin and SM22 in levels comparable to those of mature smooth muscle. The cells that did not spread remained negative for smooth muscle markers. SM differentiation was independent of cell-cell contact or proliferation. In additional studies, undifferentiated lung mesenchymal cells were cocultured with lung embryonic epithelial cells at high density. The epithelial cells aggregated into cysts surrounded by mesenchymal cells and a basement membrane was formed between the two cell types. In these cocultures, the mesenchymal cells in contact with the basement membrane spread and differentiated into smooth muscle. The rest of the mesenchymal cells remained round and negative for smooth muscle markers. Inhibition of laminin polymerization by an antibody to the globular regions of laminin beta1/gamma1 chains blocked basement membrane assembly, mesenchymal cell spreading and smooth muscle differentiation. These studies indicated that lung embryonic mesenchymal cells have the potential to differentiate into smooth muscle and the process is triggered by their spreading along the airway basement membrane.

Laminin fragment E4 inhibition studies: basement membrane assembly and embryonic lung epithelial cell polarization requires laminin polymerization.

Laminins (LMs), the main constituents of basement membranes (BMs), are heterotrimeric glycoproteins composed of alpha, beta, and gamma chains held together by disulfide bonds. In the presence of Ca2+, some laminins, such as laminin-1 self-assemble into a polymer through the interaction of their three NH2 termini. Here we exposed lung organotypic cultures to a proteolytic fragment of laminin-1 that blocks laminin polymerization. This fragment, referred as E4, comprises the outer globular region of laminin beta1 chain. Inhibition of laminin polymerization in lung organotypic cultures resulted in impaired basement membrane assembly and failure of epithelial cells to polarize. In addition, we found that in control organotypic cultures, the bronchial smooth muscle cells were arranged in concentric layers around the newly formed epithelium. However, in E4-treated cultures, the smooth muscle cells were in disarray. Exposure of organotypic cultures to laminin-1 proteolytic fragment P1', that comprises part of alpha1, beta1, and gamma1 chains, but does not overlap with fragment E4, had no effect in basement membrane assembly. Exposure to fragment E4 also caused an increased release of laminin-1 into the culture medium, suggesting a failure to retain laminin at the epithelial-mesenchymal interface. These studies provide the first direct evidence linking epithelial cell polarization to laminin polymerization at the epithelial-mesenchymal interface and assign a key role to the outer globular region of laminin beta1 chain.

Preferential interactions of the Escherichia coli LexA repressor with anions and protons are coupled to binding the recA operator.

The binding of Escherichia coli LexA repressor to the recA operator was examined as a function of the concentration of NaCl, KCl, NaF, and MgCl2 at pH 7.5, 21 degrees C. The effects of pH at 100 mM NaCl were also examined. Changes both in the qualitative appearance of the binding isotherms and in the magnitude of the apparent binding affinity with changes in solution conditions suggest that binding of anions and protons by LexA repressor is linked to oligomerization and/or operator binding. Binding of LexA repressor to the recA operator in the presence of NaCl ranging from 25 to 400 mM at picomolar DNA concentration showed a broad, apparently noncooperative, binding isotherm. Binding of LexA repressor in NaF at the same [DNA] yielded binding isotherms with a narrow transition, reflecting an apparently cooperative binding process. Also, the apparent binding affinity was weaker in NaF than in NaCl. Furthermore, the binding affinity and also the apparent binding mode, cooperative vs noncooperative, were pH dependent. The binding affinity of LexA repressor for operator was greatest near neutral pH. The apparent binding mode was noncooperative at pH 7-9 but was cooperative at pH 6 or 9.3. These observations suggest that the specific cation and anion composition and concentrations must be considered in understanding the details of regulation of the SOS system.

Azoxymethane enhances ligand-induced activation of EGF receptor tyrosine kinase in the colonic mucosa of rats.

Recent observations suggest that transforming growth factor alpha (TGF-alpha), which binds to the epidermal growth factor (EGF) receptor (EGFR), may induce neoplastic growth of the colonic mucosa through an autocrine mechanism. To assess the functional role of TGF-alpha in colonic carcinogenesis the present investigation examines the changes in TGF-alpha-and EGF-induced activation of intrinsic tyrosine kinase (Tyr-k) activity of EGFR in the colonic mucosa of rats after administration of the colonic carcinogen azoxymethane (AOM; 20 mg/kg body wt). Five days after a single injection of AOM to 4- to 5-month old rats proliferative activity (as assessed by 5-bromo-2'-deoxyuridine immunoreactivity) in the colonic mucosa was increased by approximately 700% over the corresponding saline-injected controls. This was accompanied by: (i) a marked rise in autophosphorylation of a number of mucosal proteins, including one with a M(r) of 170 kDa, a molecular mass that corresponds to EGFR; (ii) a 110-130% increase in basal EGFR Tyr-k activity. Despite this rise in basal EGFR Tyr-k activity, exposure of isolated colonocytes or detergent-solubilized colonic mucosa from AOM-treated animals to either 1 x 10(-8) M TGF-alpha or EGF caused a further 90-160% increase in EGFR Tyr-k activity over the corresponding basal levels. In contrast, bombesin produced no apparent change in EGFR Tyr-k activity. We conclude that increased ligand-induced activation of EGFR Tyr-k may be an important event for development of the hyperproliferative state associated with induction of colorectal neoplasia.

Increased activation of EGF-receptor tyrosine kinase by EGF and TGF-alpha in the colonic mucosa of aged rats.

Freshly isolated colonocytes as well as detergent-solubilized colonic mucosa and lectin purified receptor-enriched mucosal preparations were utilized to compare ligand-induced activation of EGF-receptor (EGF-R) tyrosine kinase (Tyr-k) activity between young (4 months) and aged (24 months) rats. In all three mucosal preparations, EGF and TGF-alpha produced a significantly greater stimulation in EGF-R Tyr-k activity in aged than in young rats, when compared with the corresponding basal levels. This was observed in spite of a significantly higher basal EGF-R Tyr-k activity in the colonic mucosa of aged rats than in young animals. Neither in young nor in aged rats did bombesin cause any significant change in EGF-R Tyr-k activity in the colonic mucosa. In aged rats, TGF-alpha also caused a stimulation in tyrosine phosphorylation of EGF-R and autophosphorylation of the 165 kDa band (a molecular mass that corresponds to EGF-R) and several other mucosal proteins (M, 120, 110, 70, 60, 55 and 50 kDa). We suggest that mitogenic activation of EGF-R Tyr-k may be an important event for the development of hyperproliferative state in the colonic mucosa of aged rats.

Induction of EGF-receptor tyrosine kinase during early reparative phase of gastric mucosa and effects of aging.

BACKGROUND: Although the gastric mucosa of adult healthy animals possesses a remarkable capacity to promptly repair its mucosal architecture after an acute injury, aging attenuates this process. We hypothesize that certain tyrosine kinases (Tyr-k), specifically the enzyme associated with EGF-receptor (EGF-R), may play a role in this process. The present investigation was undertaken to evaluate the role of this enzyme in the early reparative phase of the gastric mucosa in young and aged rats. EXPERIMENTAL DESIGN: In our initial effort to test the hypothesis, we examined the changes in both total and EGF-R-associated Tyr-k activities in the gastric mucosa of young adult rats (4-months old) during the first 60 minutes after hypertonic saline (2 M NaCl; 1.5 ml/130 g body weight)-induced injury. Because the maximal stimulation (90-100% over the controls) in both total and EGF-R-associated Tyr-k occurred at 30 minutes after injury, we used this time point to perform the next experiment, in which groups of young and aged rats were given (intragastically) 2 M NaCl or water. One of the young and aged groups of rats was also injected (i.p.) with the Tyr-k inhibitor tyrphostin-51 (300 micrograms/kg body weight) 60 minutes before injury. The gastric mucosa was assayed for EGF-R Tyr-k activity and tyrosine phosphorylation and expression of EGF-R, phospholipase C (PLC) activity and relative concentration and tyrosine phosphorylation of PLC-gamma 1, as well as transforming growth factor-alpha (TGF-alpha) levels. RESULTS: Basal EGF-R Tyr-k activity and the extent of tyrosine phosphorylation of EGF-R, as well as PLC activity, were all found to be higher in the gastric mucosa of aged than in young rats. Although 30 minutes after injury, EGF-R Tyr-k activity, tyrosine phosphorylation of EGF-R, and relative abundance of the receptor were all increased in the gastric mucosa of both young and aged rats, the magnitude of stimulation of each of the parameters was found to be considerably lower in aged than in young rats, compared with the corresponding basal levels. A similar phenomenon was also observed for PLC activity and tyrosine phosphorylation of PLC-gamma 1. The relative concentration of mucosal PLC-gamma 1 level was, however, not affected by injury in either young or aged rats. Tyrphostin greatly attenuated the injury-induced increases in the above mentioned parameters in both young and aged rats. In young but not in aged rats, injury caused a significant increase in mucosal TGF-alpha levels. CONCLUSIONS: We conclude that (a) activation of EGF-R Tyr-k is an important event in the early reparative process of the gastric mucosa, and (b) local production of TGF-alpha may play an important role in regulating the activation of EGF-R Tyr-k.

Identification and evaluation of the role of endogenous tyrosine kinases in azoxymethane induction of proliferative processes in the colonic mucosa of rats.

Although tyrosine kinases (Tyr-k) are known to play a role in regulating proliferation of normal, preneoplastic and neoplastic cells, little is known about the identity of different species of Tyr-k involved in this process. Utilizing a non-denaturing polyacrylamide gel electrophoresis system, in which the separated proteins from tissue extracts are assayed directly for Tyr-k, we attempted to identify the species of Tyr-k that may be involved in azoxymethane (AOM) induction of colonic mucosal ornithine decarboxylase (ODC) activity, an enzyme whose activity is known to rise in rapidly proliferating cells. We have observed that 5 days after a single injection of the colonic carcinogen AOM (20 mg/kg body wt) to 3-4-month old rats, a significant 230% rise in colonic mucosal proliferative activity (as evidenced by 5-bromo-2'-deoxyuridine (BrdU) immunoreactivity) was also accompanied by a 550% increase in ODC activity. This was also associated with a marked rise (140-240%) in the relative activity of Tyr-k of three mucosal proteins with MI of 165, 145 and 125 kDa. Since the molecular mass of one of the Tyr-k (165 kDa) corresponded to that of EGF-receptor (EGF-R), this led us to examine the role of EGF-R Tyr-k in AOM induction of colonic mucosal ODC. We observed that a 320% increase in mucosal ODC activity, 5 days after AOM injection, was accompanied by over 200% rise in Tyr-k activity of EGF-R. Daily injection of tyrphostin (300 micrograms/kg body wt.), a Tyr-k inhibitor with a higher specificity for EGF-R Tyr-k, significantly attenuated AOM-induced stimulation of both ODC and Tyr-k activity of EGF-R. Administration of AOM also stimulated the rate of synthesis and secretion of TGF-alpha in isolated colonocytes. In addition, the levels of TGF-alpha and its mRNA in the colonic mucosa were also found to be 100% and 250% higher, respectively, in AOM-treated rats when compared with the controls. We suggest that (a) activation of intrinsic Tyr-k of EGF-R is an important event in AOM induction of colonic mucosal proliferative processes, and (b) this activation is thought to be mediated by TGF-alpha through an autocrine mechanism.

Increased expression of pp60c-src in gastric mucosa of aged rats.

The relationship between proliferative activity and the expression of pp60c-src in gastric mucosa (oxyntic gland area) of young (4-month) and aged (24-month) Fischer 344 rats was examined. Gastric mucosal proliferative activity, as assessed by 5-bromo-2'-deoxyuridine (BrdU) immunoreactive cells, was found to be 115% (p < .001) higher in aged than in young rats. This was associated with a 47% rise (p < .025) in overall tyrosine kinase (Tyr-k) activity and a 5-7-fold increase in autophosphorylation of four prominent protein bands with M(r) of 40, 55, 60, and 70 kDa in gastric mucosal membranes. A similar phenomenon was also observed for Tyr-k activity of pp60c-src in that the aged rats revealed a 69% (p < .025) higher enzyme activity and a 5-fold rise in the extent of autophosphorylation of this protein when compared with the corresponding values from young animals. Increased Tyr-k activity of pp60c-src in the gastric mucosa of aged rats could in part be due to higher levels of this protein because the relative concentration of pp60c-src, as assessed by Western blot analysis, showed a 2-5-fold increase over the young animals. In addition, the relative concentration of c-src mRNA in the gastric mucosa of aged rats was also found to be 5-6-fold higher than in young animals. We suggest that pp60c-src may play a role in regulating gastric mucosal proliferative processes in the gastric mucosa of aged rats.

Aging diminishes gastric mucosal regeneration: relationship to tyrosine kinases.

BACKGROUND: Increased incidence of gastric ulcer observed in the aged could be partly attributed to increased susceptibility of the mucosa to various damaging agents together with impediment of the repair process. The present investigation was undertaken to compare the rate of mucosal regeneration and the role of tyrosine kinases in regulation of this process between young (4-month-old) and aged (24-month-old) rats during the first 24 hours after injury. EXPERIMENTAL DESIGN: Groups of young and aged rats were given intragastrically with either 2 M NaCl (1.5 ml/130 gm body weight), or an equivalent volume of water and killed 1, 6, and 24 hours later. Each animal was injected intraperitoneally with 5-bromo-2'-deoxyuridine (BrdU; 50 mg/kg) 1 hour before killing to assess proliferative activity by immunocytochemistry. The stomach (oxyntic gland area) was also evaluated by light microscopy for the extent of injury and subsequent regeneration, and mucosa assayed for ornithine decarboxylase and tyrosine kinase (Tyr-k) activity and tyrosine phosphorylation of membrane proteins. RESULTS: Although 2 M NaCl caused extensive damage to the gastric mucosa in both young and aged rats, as evidenced by the total loss of the surface epithelium at 1 hour postinjury, the degree of regeneration was faster in young animals. In young rats, gastric epithelium showed signs of regeneration at 6 hours postinjury and was essentially complete by 24 hours. In contrast, in aged rats, only intermittent surface cells were seen 24 hours after injury. In both age groups, injury resulted in stimulation of mucosal proliferative activity. However, whereas ornithine decarboxylase activity in both age groups was maximally stimulated (350% in young versus 80% in aged) at 6 hours after injury, the number of BrdU-positive cells in young rats increased steadily with time after injury. In contrast, aged rats showed a biphasic pattern in that the number of BrdU-positive cells/gland remained decreased for up to 6 hours, whereafter a steep rise occurred. At 24 hours after injury, the number of BrdU-positive cells/gastric gland in aged rats were found to be higher than in young rats (6 +/- 1.5 cells/gland in young rats versus 9 +/- 2.1 cells/gland in aged rats). The pattern of Tyr-k activity in young and aged rats after injury was found to be quite different from that observed for proliferative activity. In young rats, mucosal Tyr-k activity increased by about 60% at 1 hour after injury, then decreased slightly over the next 5 hours and increased again revealing a 120% rise at 24 hours postinjury. This was associated with a concomitant change in tyrosine phosphorylation of six membrane proteins with molecular weight (in kilodalton) of 30, 35, 50, 55, 60 and 70. In contrast, in aged rats, Tyr-k activity was increased only marginally (about 20%) during the first 6 hours, but at 24 hours postinjury it was found to be 70% above the control. In aged rats, injury produced no significant stimulation in tyrosine phosphorylation of gastric mucosal membrane proteins. CONCLUSIONS: We conclude that aging is associated with the diminished regenerative capacity of the gastric mucosa. This could partly be attributed to diminished activation of mucosal Tyr-k and decreased tyrosine phosphorylation of certain membrane proteins.

Acyl-CoA ligases from rat brain microsomes: an immunochemical study.

Acyl-CoA ligase activities, solubilized from rat brain microsomes, were fractionated into three different peaks by hydroxyapatite chromatography. Based on physical and chemical properties, we suggested that peak A (pamitoyl-CoA ligase) and peak C (lignoceroyl-CoA ligase) were two different enzymes (A. Bhushan, R. P. Singh, and I. Singh (1986) Arch. Biochem. Biophys. 246, 374-380). We raised antibodies against purified liver microsomal palmitoyl-CoA ligase (EC 6.2.1.3) and examined the effect of this antibody on acyl-CoA ligase activities for palmitic, arachidonic and lignoceric acids in microsomal enzyme extract and different acyl-CoA ligase peaks from the hydroxyapatite column. In an enzyme activity assay system in microsomal extract, the antisera inhibited the palmitoyl-CoA ligase activity but had very little effect on the acyl-CoA ligase activities for arachidonic and lignoceric acids. This antisera inhibited the acyl-CoA ligase activities for these three fatty acids in peak A and had no effect on these activities in peak B or peak C. Western blot analysis demonstrated that antibody to liver microsomal palmitoyl-CoA ligase cross-reacted with only peak A (palmitoyl-CoA ligase), but not with peak B or peak C. This immunochemical study demonstrates that palmitoyl-CoA ligase does not share immunological determinants with acyl-CoA ligases in peaks B or C, thus demonstrating that palmitoyl-CoA ligase (peak A) is different from the arachidonoyl-CoA and lignoceroyl-CoA ligase activities in peaks B or C.

Energy metabolism of the brain in malnutrition. An experimental study in young rhesus monkeys.

An experimental model of protein-calorie malnutrition (PCM) has been successfully produced in young rhesus monkeys. The model showed close resemblance to the energy kinetics of the brain as observed in human PCM inasmuch as oxygen consumption was decreased while glucose uptake was increased. The cerebral blood flow was also diminished. The observations suggest that a significant part of glucose is perhaps diverted to long-chain fatty acids, etc.