Formation of electrical connections between cultured identified neurons and muscle fibers of the snail Helisoma.

We studied the formation of connections between identified neurons removed from the buccal ganglion of the snail Helisoma and muscle fibers dissociated from the buccal mass. Three types of identified neurons--B19, B5, and B4--were placed into cell culture and muscle fibers from the supralateral tensor muscle (SLT), normally innervated by B19, were subsequently plated adjacent to the neuronal cell bodies. Growth cones from the neurons contacted the muscle fibers within 6-12 h after isolation. Simultaneous intracellular recordings from the neuronal cell bodies and muscle fibers after 4 days in culture indicated that the neurons had formed electrical connections with the fibers. All 3 types of neurons coupled to the muscle fibers but displayed differing probabilities and strengths of connections. The role of growth cone contact in the formation of these connections was tested by plating muscle fibers onto fields of neurites after neuronal growth had stopped. Under these conditions, neurons still became electrically coupled to the muscle fibers, but the strength of these connections differed from those formed by neurons and fibers that were plated simultaneously. Thus, quantitative characteristics of electrical connections formed between cultured Helisoma neurons and dissociated muscle fibers are influenced by neuronal identity and the timing of neuronal contacts.

Human thyroid cells transfected with SV40 DNA retain the TSH receptor.

Human thyroid cells were transfected with SV40 DNA using the calcium phosphate co-precipitation technique. The transfected cells grew rapidly and could be passaged readily. TSH and the immunoglobulin fraction from a patient with hyperthyroid Graves' disease stimulated adenylate cyclase activity in the transfected cells. These cells could potentially be used in clinical assays of patients' immunoglobulins without concern for species incompatibilities. On immunoblot analysis, extracts from SV40-transfected human thyroid cells, from sheep thyroid cells and from rat FRTL5 cells reacted with the immunoglobulin fraction from the patient with hyperthyroid Graves' disease but not with the immunoglobulin fraction from a pool of serum from normal subjects. Proteins of molecular weights 77,000, 70,000 and 54,000 were identified by the patient's immunoglobulin fraction in the three thyroid-derived cell types.

Thyrotropin-induced formation of functional follicles in primary cultures of ovine thyroid cells.

In primary cultures of ovine thyroid cells, TSH induced the expression of several differentiated functions including the formation of follicles, and synthesis and storage of iodinated thyroglobulin in the follicular lumen. In the present report, these follicles were shown by transmission (TEM) and scanning electron microscopy (SEM) to be intact, comprised of two or more cells and to possess numerous microvilli on the inner cell membranes facing the follicular lumen. The TSH-induced formation of follicles was reversible and dynamic, with the kinetics of formation preceding that of iodination. The follicles were further demonstrated to be functional in terms of thyroglobulin storage and iodination.

Growth and differentiation in cultured human thyroid cells: effects of epidermal growth factor and thyrotropin.

Human thyroid cells were grown and subcultured in vitro to examine their responses to known hormones and growth factors, and to serum. The cells were obtained from surgical specimens and were either neoplastic or nonneoplastic. The effects of culture conditions on cell growth were measured by changes in cell numbers and by stimulation of [3H]thymidine incorporation. The results showed that serum (0.5%) was essential for cell proliferation, and that a mixture of insulin (10 micrograms/ml), transferrin (5 micrograms/ml), hydrocortisone (10 micrograms/ml), somatostatin (10 ng/ml), and glycyl-histidyl-lysine (10 ng/ml) enhanced the effect of serum. Maximum growth of the cells was obtained when epidermal growth factor was present at 10(-9) M. Differentiation was measured by production of thyroglobulin, which was found to be stimulated by thyrotropin. This system provides a means to study the hormonal control of growth and differentiation in human thyroid cells.

Epidermal growth factor inhibits thyrotropin-mediated synthesis of tissue-specific proteins in cultured ovine thyroid cells.

Primary cultures of ovine thyroid cells were induced to differentiate by addition of thyrotropin (TSH). This was demonstrated as an accumulation of 2 thyroid-specific proteins, thyroglobulin and thyroid peroxidase, using immunofluorescent staining methods and immunoprecipitation of biosynthetically labeled cultures. As an additional measure of differentiation, cells exhibited a morphological response to TSH and regained the ability to incorporate radioactive iodide. Epidermal growth factor (EGF) markedly inhibited differentiation when added together with TSH. Thyroglobulin synthesis was reduced to low levels and peroxidase synthesis was reduced to levels that were undetectable by the methods used. Morphological changes in response to TSH were also diminished by EGF. The antagonistic interaction between TSH and EGF in regulating differentiation in cultured thyroid cells may reflect the type of control that exists in vivo.

Phosphatidylethanolamine synthesis in ethanolamine-responsive and -nonresponsive cells in culture.

Mammalian cells can be classified into two types based upon whether or not they show growth response to ethanolamine (Etn) in culture. The content of phosphatidylethanolamine (PE) in phospholipid and incorporation of radioactive Etn into the cells were examined in the Etn-responsive and -nonresponsive cells in order to elucidate the mechanisms of growth stimulation by Etn. In all Etn-responsive cells tested, 5 microM Etn significantly altered the composition of cellular phospholipid compared to that grown without Etn, while Etn-nonresponsive cells had a similar phospholipid composition whether the growth medium contained Etn or not. Using two rat mammary carcinoma cell lines, 64-24 (responsive type) and 22-1 (nonresponsive type), further studies were carried out. In 64-24 cells there was a proportional increase in PE content as the dosage of Etn in the medium was increased. The increase in PE content leveled off at 10 microM. Further, the increase in PE content was correlated with increased rate of growth. In contrast, PE content or growth rate did not change at all in 22-1 cells. In 64-24 cells radioactive Etn (0.1-50 microM) was incorporated four- to five-fold more efficiently into phospholipid, and the aqueous pool of precursors of PE was ten times less as compared to 22-1 cells, indicating that Etn-responsive cells utilize Etn supplied in the medium to synthesize PE far more efficiently than Etn-nonresponsive cells. De novo synthesis of PE must not be sufficient to support optimum growth in Etn-responsive cells.

Growth responsiveness to prolactin and its loss in normal rat mammary cells in culture.

Growth response of mammary epithelial cells to hormones, particularly to prolactin, was studied by using a primary culture of rat mammary gland organoids. After allowing the cells to attach and spread on the surface of plastic culture dishes, the effect of hormones was tested by means of [3H]-thymidine incorporation and autoradiography in a medium containing 1% fetal calf serum. In this system, prolactin showed a modest but significant growth-stimulatory activity (50% over control), and addition of insulin or hydrocortisone or both enhanced the growth stimulation of prolactin to a large extent. Growth stimulation caused by these hormones without prolactin was always significantly lower than that caused with prolactin. A dose-response study indicated that prolactin can stimulate growth at physiological concentrations. The maximum stimulation was observed at 1-5 micrograms/ml. The growth-stimulatory effect of prolactin was decreased as the culture period was prolonged, and by the 4th day in culture the effect was no longer observed. In contrast, the stimulatory effect of insulin was constant over the 5-day culture period. Phosphoethanolamine, which has been shown to be a growth factor for some rat and human mammary carcinoma cells, showed 2-fold growth stimulation when added with prolactin, insulin or hydrocortisone. The stimulatory effect was again not observed in older cultures, as in the case of prolactin.