Growth Of Primary Cultures Research Articles

Nitric oxide production in SJL mice bearing the RcsX lymphoma: a model for in vivo toxicological evaluation of NO.

SJL mice spontaneously develop pre-B-cell lymphoma that we hypothesized might stimulate macrophages to produce nitric oxide (NO.). Transplantation of an aggressive lymphoma (RcsX) was used to induce tumor formation. Urinary nitrate excretion was measured as an index of NO. production and was found to increase 50-fold by 13 days after tumor injection. NO. production was prevented by the addition of a nitric oxide synthase (NOS) inhibitor. The expression of inducible NOS (iNOS) in various tissues was estimated by Western blot analysis and localized by immunohistochemistry. The synthase was detected in the spleen, lymph nodes, and liver of treated but not control mice. To assess whether the iNOS-staining cells were macrophages, spleen sections from ResX-bearing animals were costained with anti-iNOS antibody and the anti-macrophage antibody moma-2. Expression of iNOS was found to be limited to a subset of the macrophage population. The concentration of gamma-interferon, a cytokine known to induce NO. production by macrophages, in the serum of tumor-bearing mice, was measured and found to be elevated 25-fold above untreated mice. The ability of ResX-activated macrophages to inhibit splenocyte growth in primary culture was estimated and macrophage-derived NO. was found to inhibit cell division 10-fold. Our findings demonstrate that ResX cells stimulate NO. production by macrophages in the spleen and lymph nodes of SJL mice, and we believe this experimental model will prove useful for study of the toxicological effects of NO. under physiological conditions.

Human keratinocyte culture from the peritonsillar mucosa.

Tonsillectomy tissue can be used as a routine source for cultures of oropharyngeal keratinocytes. In so doing, a peritonsillar strip of unaltered mucosa was dissected in the upper submucosa. Subsequent trypsinization yielded 7.0 +/- 3.4 x 10(6) keratinocytes per bilateral tonsillectomy. Keratinocyte attachment and growth in primary culture were promoted by sublethally irradiated 3T3 murine fibroblasts. Three subcultures could be performed without a feeder layer and were characterized by a population doubling time of 4.5 days during log growth phase. Electrophoretic and immunoblot analysis of the third subculture revealed a strong expression of keratin pairs 5/14 and 6/16 as well as keratins 7 and 19, whereas keratins 8/18 were expressed less intensely. The lowest intensity, was found for keratin 13, which is known to be indicative of the differentiated mucosa. The culture technique thus provides an easily available in vitro model for morphological and functional studies on the epithelial compartment of human oropharyngeal mucosa.

Intratracheal instillation of keratinocyte growth factor decreases hyperoxia-induced mortality in rats.

Alveolar type II cell proliferation occurs after many forms of lung injury and is thought to play a critical role in alveolar epithelial repair. Keratinocyte growth factor/fibroblast growth factor 7 (KGF) has been shown to promote alveolar type II cell growth in primary culture and alveolar epithelial hyperplasia in vivo. In this study, we used immunohistochemical analysis to determine the intrapulmonary distribution and cellular localization of recombinant human KGF (rhKGF) instilled into the trachea of rats. 6 h after administration, immunoreactive KGF was observed within the lung parenchyma and along alveolar epithelial cell membranes. By 18-24 h, KGF was detected intracellularly in alveolar epithelial cells and intraalveolar macrophages. Immunoreactive KGF was not demonstrable 48 h after delivery or in lung sections from PBS-treated animals. Intratracheal instillation of 5 mg/kg rhKGF stimulated a marked, time-dependent increase in the alveolar type II cell specific labeling index to a maximum level of 33 +/- 3% 48 h after rhKGF administration compared with 1.3 +/- 0.3% after PBS instillation. In addition, this increase in type II cell proliferation in vivo was documented by flow cytometric analysis of isolated type II cells which revealed a nearly fivefold increase in the proportion of cells traversing through the S and G2/M phases of the cell cycle. To test the hypothesis that KGFs effects on type II cells in vivo might affect the response to lung injury, rats were treated with rhKGF and exposed to hyperoxia. Animals that received 1 or 5 mg/kg rhKGF exhibited dramatically reduced mortality (P < 0.001, for both doses). Survival for animals treated with 0.1 mg/kg rhKGF was not significantly different from either untreated rats or animals treated with heat-denatured rhKGF. The lungs of rhKGF-treated animals that survived hyperoxia exposure had minimal hemorrhage and no exudate within the intraalveolar space. These experiments established that intratracheal administration of rhKGF stimulated alveolar type II cell proliferation in vivo and reduced hyperoxia-induced lung injury in rats. Directed delivery of KGF to the lungs may provide a therapeutic strategy to preserve or restore the alveolar epithelium during exposure to hyperoxia or other injurious agents.

Modulators of growth in primary culture of rat proximal tubular cells II

Summary: The present studies assessed the effects of manipulating extracellular sodium (Na) concentration and Na transport on cellular hypertrophy and hyperplasia in primary culture of rat proximal tubular cells. A concentration‐dependent effect on thymidine incorporation and protein content was observed with cell culture media Na concentration of 130, 140 and 150 mmol/L. This effect was independent of osmolality (matched with mannitol) and no stimulatory effect occurred if choline was substituted for Na. Cells derived from sham‐operated (Sx) animals exposed to a higher media concentration of Na (150 vs 140 mmol/L) had both stimulated thymidine incorporation to 186.8 ± 35.41% (P&lt;0.05) and enhanced cell protein content to 134.7 ± 135% (P&lt;0.05). This effect was more pronounced in cell cultures derived from unilaterally nephrectomized (Nx) animals, being 212.8 ± 31.5% (P&lt;0.01) for thymidine incorporation (P&lt;0.05 vs cells from sham‐operated animals grown in high Na media) and 114.4 ± 3.2% (P&lt;0.001) for protein content (P=0.11 vs sham‐operated cells grown in similar conditions). the addition of 10−4 mmol/L ethylisopropyl amiloride hydrochloride (EIPA) to Nx cells in a normal or high Na concentration media resulted in a decrease in cellular protein content to 82.6 ± 6.8% (P&lt;0.05) and 85.5 ± 0.2% (P&lt;0.0001) compared to respective controls. 10−4 mol/L EIPA in media supplemented with insulin‐like growth factor (IGF‐1) blocked the proliferative response normally seen in response to this growth factor from 156.6 ± 13.7 to 27.5 ± 3.1% (P&lt;0.0001) compared to control. However, the presence of EIPA did not abrogate the hypertrophic response elicited by IGF‐1 (cell protein content 128.1 ± 13.1% of control with IGF‐1 vs 124.9 ± 12.5 with IGF‐1 and EIPA; P= n.s.). Addition of 10−4 mol/L EIPA to 10% serum derived from either Sx or Nx animals blocked the growth response to the sera, limiting the cellular protein content to 76.6 ± 5.5% (P&lt;0.0001) and 89.7 ± 4.4% (P&lt;0.0001) and thymidine incorporation to quiescent levels of 0.2 ± 0.1% (P&lt;0.0001) and 0.4 ± 0.1% (P&lt;0.0001) compared to respective controls. In summary, rat renal proximal tubular cell growth is influenced by Na concentrations in the cell culture environment and inhibited in the presence of EIPA. This supports a role for altered epithelial transport in the cellular growth response to a number of stimuli.

ALTERED GROWTH-REGULATION OF PROSTATIC EPITHELIAL-CELLS BY HUMAN PAPILLOMAVIRUS-INDUCED TRANSFORMATION

Six lines of human papillomavirus (HPV)-transformed prostatic epithelial cells, clonally-derived from transfection of three different parental cell strains, were analyzed for their ability to respond to stimulatory and inhibitory factors known to regulate prostatic cell growth. The cell lines were tested in a serum-free medium that was developed specifically for analysis of low-density growth of prostatic cells in order to mitigate any effects of autocrine factors. This medium has been used previously to characterize the growth regulatory pathways of primary cultures of prostatic epithelial cells derived from normal, benign prostatic hyperplasia (BPH), or adenocarcinoma tissues, as well as an SV40-transformed cell line (pRNS-1-1) that was derived from the same parental cell strain as four of the HPV-transformed lines used in the present study. The responses of the HPV-transformed cell lines to three essential mitogens in the medium - epidermal growth factor (EGF), bovine pituitary extract (BPE) and insulin-like growth factor (IGF) - were similar to those of primary cultures and pRNS-1-1, except that two of the HPV-transformed cell lines were IGF-independent for growth. The presence or absence of cholera toxin (CT), which acts synergistically with peptide growth factors, only mildly affected the proliferation of HPV-transformed cells, which is also true for primary cultures and pRNS-1-1. However, while hydrocortisone (HC) also has only minimal effect on the growth of primary cultures and pRNS-1-1, four of the HPV-transformed lines were very dependent on HC for growth. With regard to growth-inhibitory factors, all of the cell lines (HPV- or SV40- transformed) were insensitive to tumor necrosis factor-alpha (TNFalpha), which is a common feature of transformed cells. However, the cell lines remained sensitive to the growth-inhibitory properties of retinoic acid (RA) and transforming growth factor-beta (TGF(beta)). The HPV-transformed cell lines were also inhibited by 1,25(OH)(2) vitamin D-3 [1,25(OH)(2)D-3], although pRNS-1-1 cells were not. Perhaps the most unexpected finding in our study was the apparent loss of responsiveness of all of the transformed lines to fibroblast growth factor (FGF). Alterations in FGF pathways have been described for prostatic cancer cell lines and changes in expression of FGF or receptors may be involved in prostatic carcinogenesis. Our study demonstrates that the availability of primary cultures, SV40- and HPV- transformed cell lines and a well-defined culture system will provide an opportunity to characterize the processes involved in malignant transformation of prostatic cells.

Hepatocyte growth factor region-specifically stimulates gastro-intestinal epithelial growth in primary culture

Hepatocyte growth factor region-specifically stimulates gastro-intestinal epithelial growth in primary culture

Modulators of growth in primary culture of rat proximal tubular cells

Summary: The effects of known growth factors and circulating serum factors in animals following unilateral nephrectomy on cellular hypertrophy and hyperplasia were studied in primary culture of rat proximal tubular cells. the cultured cells were derived from normal rat kidney, as well as from rat kidneys which had undergone compensatory renal hypertrophy. Cellular thymidine incorporation was stimulated with insulin‐like growth factor 1 (IGF‐1; P&lt;0.001), epidermal growth factor (EGF; P&lt;0.01) and additively by the combination of IGF‐1+EGF (P&lt;0.0001). Transforming growth factor β1 (TGF β1) potently inhibited thymidine incorporation in the presence or absence of these growth factors. Cellular protein content was increased in the presence of IGF‐1 (P&lt;0.01). the addition of 10% serum harvested from sham operated (Sx) animals to cell cultures derived from Sx animals significantly increased cell protein content (P&lt;0.02), and stimulated cellular thymidine incorporation (P&lt;0.005). the addition of 10% serum harvested from unilaterally nephrectomized (Nx) animals further enhanced the growth response. In cell cultures derived from Nx rats, a further marked stimulation of thymidine incorporation and cellular protein was observed in the presence of both Sx and Nx serum, indicating an increased sensitivity of these cells to serum‐derived factors. In summary, the growth factors tested have direct effects in regulating rat renal tubular cell growth. Circulating factors stimulating proximal tubular cell growth are present in animals which have undergone nephrectomy. Renal tubular cells derived from animals with compensatory renal hypertrophy are sensitized to the effects of the manipulations which enhance proximal tubular cell growth.

Relationship between epidermal growth factor receptor levels, autophosphorylation and mitogenic‐responsiveness in normal mouse mammary epithelial cells in vitro

Mammary epithelial cells were isolated from mid-pregnant BALB/c mice, grown within collagen gels and maintained on DME/F12 (1:1) media containing 10% bovine calf serum and 10 micrograms/ml insulin. Initial time-course and dose-response studies showed that epidermal growth factor (EGF)-induced autophosphorylation of the EGF-receptor (EGF-R) in these cells was maximal 5 min after exposure to 75 ng/ml EGF. Mammary epithelial cells displaying little or no growth during their first 2 days in primary culture cells were found to contain low levels of EGF-R. However, EGF-induced autophosphorylation of the EGF-R in these cells was extremely intense. Subsequent studies demonstrated that during the proliferative and plateau phases of growth, EGF-R levels progressively increased, while conversely EGF-induced autophosphorylation of the EGF-R decreased over time in primary culture. These results demonstrate that EGF-R levels and autophosphorylation do not show a direct correlation with mammary epithelial cell mitogen-responsiveness. Intense EGF-R autophosphorylation appears to be required for initiating growth, but sustained mammary epithelial cell proliferation occurs when EGF-R autophosphorylation is low. This inverse relationship between EGF-R levels and autophosphorylation may reflect changes in receptor affinity and function during the various phases of mammary epithelial cell growth in primary culture.

Hepatocyte Growth Factor Region Specifically Stimulates Gastrointestinal Epithelial Growth in Primary Culture

Hepatocyte growth factor (HGF) stimulated the growth of fetal rat gastrointestinal epithelial cells in primary culture with a clear dose-response relationship The epithelial response to the mitogenic activity of HGF was different among the region of the gastro-intestinal tract; glandular stomach most responsive, followed by intestine and forestomach. The interaction of HGF with other growth factors in inducing the epithelial growth was also different depending on the type of the epithelial cells, indicating a region-specific growth regulation in the gastro-intestinal tract. Analyses using Northern blot and RT-PCR revealed that HGF mRNA was expressed only in mesenchymes but not in epithelia of the gastro-intestinal tract while expression of c-met (HGF receptor) gene was observed in both tissues. These results suggest that gastro-intestinal mesenchymes secrete HGF which stimulates the growth of attaching epithelial cells by a paracrine mechanism and that the epithelial response to HGF is controlled by a region-specific growth regulatory mechanism.

Phenotype related changes of intimal smooth muscle cells from human aorta in primary culture.

To study the functional characteristics of smooth muscle cell (SMC) phenotypes, we have investigated myosin expression, cell proliferation, collagen production and low-density lipoprotein (LDL) receptor activity in intimal SMCs of normal human aorta during their growth in primary culture. By staining with rabbit antibodies to smooth muscle myosin (ASMM) 3 cell types could be distinguished in culture: homogeneously stained cells, cells with discontinuous myosin fibrils and myosin-negative cells. The ratio of cell types greatly changed with culture growth: on days 5, 7 and 14 it was 82:1:17%, 70:5:25% and 10:30:60%, respectively. After 5-6 days of culture intimal SMCs began to proliferate and DNA-synthesizing nuclei were seen 1.5-4.3 times more frequently in myosin-negative cells than in cells with homogeneous myosin distribution. At that time the number of cells reacted with monoclonal antibody (MAb) to an epitope shared collagen types I and III started to increase. By double immunofluorescence staining it was shown that the cultured cells containing both ASMM and MAb markers were found 2.0-4.8 times more rarely than MAb-positive staining in myosin-negative cells. During the first 5 days in culture LDL binding and uptake were diminished in intimal cells with intercellular lipid inclusions independently of their myosin staining pattern, but their activity increased with culture growth. Thus, SMCs from human aortic intima change their phenotype on days 6 and 7 in primary culture as manifested by alteration of myosin expression, increased cell proliferation, collagen production and LDL receptor activity. Changes in myosin expression, however, are not an essential prerequisite for cell proliferation and collagen production.

Morphine alters astrocyte growth in primary cultures of mouse glial cells: evidence for a direct effect of opiates on neural maturation

To determine whether exogenous opiate drugs with abuse liability directly modify neural growth, the present study investigated the effects of morphine on astrocyte proliferation and differentiation in primary cultures of murine glial cells. The results indicate that morphine decreases glial cell production in a dose-dependent, naloxone-reversible manner. Most notably, gliogenesis virtually ceased in the presence of 10 −6 M morphine during the first week in cultures, whereas 10 −8 M or 10 −10 M morphine caused an intermediate suppression of growth compared to control or 10 6 M morphine treated cultures. Moreover, morphine treatment inhibited [ 3H]thymidine incorporation by glial fibrillary acidic protein (GFAP) immunoreactive, flat (type 1) astrocytes, suggesting that the decrease in glial cell production was due in part to an inhibition of astrocyte proliferation. Morphine also caused significant increases in both cytoplasmic area and process elaboration in flat (type 1) astrocytes indicating greater morphologic differentiation. In the above experiments, morphine-dependent alterations in astrocyte growth were antagonized by naloxone, indicating that morphine action was mediated by specific opioid receptors. These observations suggest that opiate drugs can directly modify neural growth by influencing two critical developmental events in astrocytes, i.e., inhibiting proliferation and inducing morphologic differentiation.

Parameters of growth in primary cultures and cell lines established from Drosophila imaginal discs

We have further characterised our tissue culture system for the growth in vitro of Drosophila imaginal disc cells, including the culture medium requirements for optimum growth and we have adjusted the protocol recommended for the initiation of cultures. Many imaginal disc fragments become organised into vesicles, and some of these secrete extracellular material into the lumen. Sensory axons differentiate in primary disc cultures, in the absence of bristle formation. The early stages of cell division to form a cell line are recorded.

Adenosine Triphosphate Stimulates Thymidine Incorporation but Does Not Promote Cell Growth in Primary Cultures of Renal Proximal Tubule Cells

Acute addition of adenosine triphosphate (ATP) stimulated thymidine incorporation in confluent, quiescent primary cultures of rabbit renal proximal tubule cells in a dose-responsive manner. Similar increases in thymidine incorporation was observed with adenosine diphosphate and adenosine monophosphate but not with adenosine. The effect of chronic administration of ATP, however, suppressed cell growth. This suppression appears to be due to an effect of ATP to cause detachment of cells from culture plates, resulting in an increase in thymidine incorporation acutely but in suppression of cell growth chronically. ATP is, therefore, not a direct growth promoter of renal proximal tubule cells in primary culture.

The influence of growth factors on the proliferative potential of normal and primary breast cancer-derived human breast epithelial cells

In previous studies, we developed serum-free, bovine pituitary extract (BPE)-free culture conditions for the growth of normal and neoplastic rat mammary epithelial cells. The present studies were aimed at determining if these culture methods could be used to study the influence of specific growth factors on the proliferative potential of normal human mammary epithelial (HME) cells and cells derived from human breast cancer (HBC) specimens. Our results indicate that normal HME cells in primary culture express stringent requirements for insulin (IN), epidermal growth factor (EGF), and cholera toxin (CT). Of these factors, EGF is most important, with essentially no proliferation taking place in the absence of this factor. By contrast, when cells are grown in serum-free primary culture in the presence of a full complement of growth factors and then subcultured, growth in secondary culture is not influenced by the removal of individual growth factors. Growth in secondary culture in the absence of EGF is mediated by autocrine factors secreted by the cells. However, there is no evidence for autocrine activity that mediates growth in the absence of IN in secondary cultures. Primary culture of HBC cells in serum-free, BPE-free medium revealed two patterns of growth factor requirements. One set of HBC cells expressed identical requirements for IN and EGF in primary culture as normal cells. Likewise, these cells grew in secondary culture in the absence of either factor. The second set of tumors expressed independence of IN for growth in primary culture. These cells grew to confluence in primary culture in the absence of IN and could be subcultured in this medium. All tumor cells examined expressed a requirement for EGF for primary culture growth, whereas none of the HBC cells examined expressed a significant CT requirement. In many cases, growth in the absence of CT exceeded that observed in its presence. Thus, our culture system allows analysis of the growth factor requirements of HME and HBC cells in primary culture. Our results indicate significant differences between HME and HBC cells in this regard. However, the results of secondary culture experiments indicate that the growth factor milieu from which cells are taken can have a profound effect on the requirements for growth factors in culture.

Insulin Enhances Epidermal Growth Factor- and Transforming Growth Factor- α-Stimulated Growth in Primary Cultures of Guinea Pig Gastric Mucous Epithelial Cells

The effect of insulin on epidermal growth factor (EGF)- and transforming growth factor-alpha (TGF alpha)-induced cell proliferation was examined in primary cultures of gastric surface mucous epithelial cells by using changes in cell counts and DNA as indices of cell growth. With 1.0% fetal bovine serum and Dulbecco's modified Eagle medium (low-serum DMEM) there was no effect of bovine insulin (0.1-10.0 micrograms/ml) on cell growth. Higher doses of insulin (20.0 micrograms/ml) in low-serum DMEM were able to stimulate growth of these cells compared with controls. In low-serum DMEM with no insulin the lowest effective dose of EGF or TGF alpha for stimulating cell growth was 10 nM and 5 nM, respectively. In low-serum DMEM containing 2.5 micrograms/ml insulin there was a dose-dependent increase in cell growth with EGF and TFG alpha at concentrations of 0.5-10.0 nM. The addition of insulin alone at 2.5 micrograms/ml, EGF alone at 2 nM, or TGF alpha alone at 2 nM in serum-free DMEM media had no effect on gastric surface mucous cell DNA. In serum-free DMEM media with 2.5 micrograms/ml insulin, the addition of 2 nM EGF or 2 nM TGF alpha caused an increase in both cell counts and DNA of 1.80- and 3.50-fold, respectively. The present study demonstrates that insulin, at concentrations that are not mitogenic, will enhance the proliferative effects of EGF and TGF alpha on the growth of primary cultures of guinea pig gastric surface mucous epithelial cells.

Urea transport in freshly isolated and cultured cells from rat inner medullary collecting duct

Regulation of urea transport by vasopressin in inner medullary collecting duct (IMCD) cells is thought to be important for the urinary concentrating mechanism. Isolated tubule perfusion studies suggest the existence of a saturable urea carrier. We have measured 14C-urea efflux in IMCD cells which were freshly isolated and grown in primary culture. Cells were isolated from rat papilla by collagenase digestion and hypotonic shock. In suspended cells, 14C-urea efflux (Jurea) from loaded cells was exponential with time constant 59 +/- 3 sec (SEM, n = 6, 23 degrees C). Jurea had an activation energy of 4.1 kcal/mole and was inhibited 42 +/- 7% by 0.25 mM phloretin and 30-40% by the high affinity urea analogues dimethylurea and phenylurea. Jurea was increased 40-60% by addition of vasopressin (10(-8) M) or 8-bromo-cAMP (1 mM); stimulated Jurea was inhibited 55 +/- 8% by the kinase A inhibitor H-8. Phorbol esters and epidermal growth factor did not alter Jurea. IMCD cells grown in primary culture were homogeneous in appearance with greater than fivefold stimulation of cAMP by vasopressin. The exponential time constant for urea efflux was 610 +/- 20 sec (n = 3). Jurea was not altered by vasopressin, cAMP or phloretin. Another function of in vivo IMCD cells, vasopressin-dependent formation of endosomes containing water channels, was absent in the cultured cells. These results demonstrate presence of a urea transporter on suspended IMCD cells which is activated by cAMP and inhibited by phloretin and urea analogues. The urea transporter and its regulation by cAMP, and cAMP-dependent apical membrane endocytosis, are lost after growth in primary culture.

Growth of Melanocytes in Human Epidermal Cell Cultures

Epidermal cell cultures were grown in keratinocyte-conditioned medium for use as burn wound grafts; the melanocyte composition of the grafts was studied under a variety of conditions. Melanocytes were identified by immunohistochemistry based on a monoclonal antibody (MEL-5) that has previously been shown to react specifically with melanocytes. During the first 7 days of growth in primary culture, the total number of melanocytes in the epidermal cultures decreased to 10% of the number present in normal skin. Beginning on day 2 of culture, bipolar melanocytes were present at a mean cell density of 116 +/- 2/mm2; the keratinocyte to melanocyte ratio was preserved during further primary culture and through three subpassages. Moreover, exposure of cultures to mild UVB irradiation stimulated the melanocytes to proliferate, suggesting that the melanocytes growing in culture maintained their responsiveness to external stimuli. When the sheets of cultured cells were enzymatically detached from the plastic culture flasks before grafting, melanocytes remained in the basal layer of cells as part of the graft applied to the patient.

Effect of pituitary gonadotrophins on proliferation of primary cultures of human ovarian stroma

Proliferation of ovarian stromal cells is a common phenomenon in peri- and post-menopausal ovaries. It is generally assumed to be secondary to the rise in circulating gonadotrophins at the menopause, though the process by which it occurs is poorly understood. This study aimed to examine the effect of menopausal levels of pituitary gonadotrophins on the growth of primary cultures of ovarian stroma. A culture system was developed using primary explants of ovarian stroma on a collagen substrate. The effect of follicle stimulating hormone (FSH; 10(-5) g/l) and luteinizing hormone (LH; 10(-5) g/l) on the proliferation of cultures derived from the cortices and medullae of ten ovaries was evaluated using a dual radiothymidine labelling technique. FSH was stimulatory to cortical cultures from 9/10 ovaries and medullary cultures from 7/10 ovaries, while LH was stimulatory to cortical cultures from 6/9 ovaries and medullary cultures from 5/10 ovaries. The responsiveness of the cultures did not correlate with the degree of hyperplasia in vivo. This study demonstrates that pituitary gonadotrophins may modulate the growth of stromal cells in culture, and thus may play a role in the process whereby stromal proliferation occurs in peri- and post-menopausal ovaries.

Muscarinic Receptor Expression in the Primary Culture of Tracheal Smooth Muscle Cells

Tracheal smooth muscle cells (TSMCs) were isolated from dog trachea in order to analyze the direct effects of growth factors and hormones on cell proliferation and muscarinic receptor (mAchR) expression. Dissection and dissociation of tracheal smooth muscle tissue with a collagenase I, deoxyribonuclease I and elastase IV mixture resulted in high yield and viability of TSMCs. A screen of growth factors, hormones, and serum concentration for the stimulation of cell growth, revealed that insulin-like growth factor, basic fibroblast growth factor, epidermal growth factor, insulin, transferrin, or hydrocortisone alone at the concentration used was not necessary or sufficient to stimulate growth of TSMCs in the primary culture with DMEM/F-12 containing 1% FBS. The regulation of cell surface mAchR expression in response to serum and cell growth in primary culture of TSMCs has been examined. In the presence of 1% serum, TSMCs withdraw from the cell cycle and express high levels of cell surface mAchRs. Exposure of quiescent TSMCs to 10% serum results in a loss of surface mAchRs. In addition, insulin-like growth factor, insulin or transferrin could stimulate the expression of mAchRs on TSMCs cultured in DMEM/F-12 containing 1% FBS. The results demonstrated that low serum concentration culture system may provide a useful model to elucidate the expression of mAchRs in the culture of TSMCs.

Effect of pH on growth of mouse renal cortical tubule cells in primary culture.

We examined the effect of the medium pH on growth of primary cultures of mouse cortical tubule cells grown in defined medium. A significantly higher DNA content was observed within 24 h of lowering medium pH from 7.4 to 6.8 or 7.1 and persisted for the duration of the study. Further studies revealed that either medium acidification or insulin plus prostaglandin E1 nearly doubled uptake of [3H]thymidine in cells deprived of other growth factors for the previous 72-110 h. Moreover, the effects of insulin, prostaglandin E1, and medium acidification on [3H]thymidine uptake of quiescent cells were additive. An alkaline medium pH appeared to have a small but significant effect on cell hypertrophy, since cells exposed to pH 7.4 and 7.7 had a higher protein-to-DNA ratio than cells incubated at a lower pH. Cell pH of monolayers grown on glass slides determined from fluorescence of the carboxyfluorescein analogue 2',7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) was linearly correlated with medium pH, and changes in medium pH resulted in changes in steady-state cell pH of a similar magnitude. Four hours after medium acidification, relative increases in cell Na+ and water content occurred, whereas medium alkalinization led to decreases in cell Na+ and water content. The increases in cell Na+ and cell water content at pH 6.8 could be inhibited by amiloride. We conclude that decreasing the cell pH can be a mitogenic stimulus for renal tubule cells. Medium acidification is accompanied by changes in cell Na+ transport, which may be mediated in part by altered Na+-H+ antiporter activity.