Heterogeneity and contact-dependent regulation of amylase release by individual acinar cells

We have used a reverse hemolytic plaque assay to investigate the amylase release of single and aggregated pancreatic acinar cells. We have found that a minority of single acinar cells released detectable amounts of amylase under basal conditions and were modestly stimulated, in a dose-dependent manner, during a 30-min exposure to concentrations of carbamylcholine (CCh) ranging from 10^?8 to 10^?5 M. This stimulation was largely accounted for by the recruitment of additional secreting cells, rather than by a significant increase in their individual secretory output. We have also observed that aggregates comprising two to five acinar cells secreted more frequently and released more amylase than single acinar cells in the presence of each of the CCh concentrations tested. Under both basal conditions and following CCh stimulation, the proportion of secreting aggregates and their amylase output increased linearly with the aggregate size. Under basal conditions as well as in the presence of secretagogue concentrations in the 10^?8?10^?7 M range, individual cells contributed similarly to amylase secretion whether they were single or part of aggregates. By contrast, following stimulation by 10^?6?10^?5 M CCh, aggregated cells showed a much higher average secretion than single cells. Investigating the mechanism of this contact-dependent effect, we found that 10^?3 M heptanol did not significantly modify the secretion of single cells and markedly promoted the basal amylase release of acinar cell pairs. This effect was associated with a marked reduction in gap junctional communication between acinar cells, as evaluated by microinjection of Lucifer yellow, and was not observed during exposure to high concentrations of CCh, which also reduced junctional communication. These data show that pancreatic acinar cells are intrinsically heterogeneous in their ability to release amylase and that their basal as well as stimulated secretion are promoted by the establishment of direct intercellular contacts. Our experiments also suggest that junctional coupling contributes to the contact-dependent mechanism which enhances the recruitment of secreting cells and their individual output. These observations strengthen the view that direct interactions between acinar cells are essential in the control of pancreatic secretion. © 1994 Wiley-Liss, Inc.     

Simultaneous monitoring of pituitary hormone secretion and gene expression within individual cells.

We have recently developed a method to simultaneously quantitate the level of gene expression and the level of secretion of a peptide from individual cells. Our approach has been to combine the reverse hemolytic plaque assay sequentially with in situ hybridization. We present data to show how we have used the pituitary lactotroph as a model to demonstrate the power of this technique. However, we are particularly excited about the potential application of this strategy to approach a broad spectrum of questions regarding the cellular and molecular mechanisms that regulate the coupling of peptide secretion and gene expression at the single cell level. The method can be used in any system in which an appropriate antibody for the reverse hemolytic plaque assay and probes complementary to the mRNA of interest are available.     

内分泌细胞中溶酶体对激素分泌调节的作用

Enzyme cytochemistry and immunocytochemistry were utilized to study the morphological alterations of the lysosomes and associated crinophagic and autophagic structures in the hypo-secreting pituitary gonadotrophin and Leydig cells induced by exogenous androgen. The lysosomes and autophagic vacuoles in the electron micrographs were quantitatively analysed. The morphological and quantitative data led to the following conclusions: 1) The hypo-secreting gonadotrophin showed an increase in the number of lysosomes and an enhancement of crinophagy. It demonstrated once again that the lysosomes in the protein and polypeptide hormone secreting cells play a role in the regulation of secretion process by means of the crinophagy. 2) The hypo-secreting Leydig cells showed an increase in the number of lysosomes and an enhancement of autophagic activity. This indicated that the lysosomes in the steroid hormone secreting cells also function in the regulation of hormone secretion but by means of autophagy which scavenge a part of steroid-producing apparatus and hormone. The autophagy might have similar effect in regulation of steroid secretion to the crinophagy in regulation of protein secretion.     

Thyroid hormone and apotransferrin regulation of growth hormone secretion by GH1 rat pituitary tumor cells in iron restricted serum-free defined medium

Summary Growth hormone (GH) production by GH₁ rat pituitary tumor cells in iron restricted serum-free defined medium requires apotransferrin (apoTf) and triiodothyronine (T₃). As measured by radioimmunoassay, apoTf plus T₃ induced GH levels 2 to 4-fold above controls. Deletion of either apoTf or T₃ arrested GH secretion. ApoTf/T₃ defined medium regulated GH production as effectively as whole serum. Because glucocorticoids enhance GH secretion in serum containing cultures, the effects of dexamethasone were evaluated in apoTf/T₃ defined medium. The steroid hormone showed no enhancing effects unless the cells were exposed to serum prior to incubation in apoTf/T₃ defined medium. Even under these conditions, the response to dexamethasone remained T₃ dependent. These observations indicate that a yet to be characterized serum factor(s), other than apoTf, regulates the reponse to the steroid hormone. This is the first report of thyroid hormone regulation of GH secretion by rat pituitary tumor cells under completely serum-free chemically defined conditions.     

Thyroid stimulating hormone upregulates secretion of cathepsin B from thyroid epithelial cells

Constant levels of thyroid hormones in the blood are principal requirements for normal vertebrate development. Their release depends on the regulated proteolysis of thyroglobulin which is extracellularly stored in the follicle lumen under resting conditions. Thyroglobulin is proteolytically degraded to a major part in lysosomes, but in part also extracellularly leading to the release of thyroxine. Extracellularly occurring lysosomal enzymes are most probably involved in the proteolytic release of thyroxine. In this study we have analyzed the secretion of cathepsin B by thyroid follicle cells (primary cells as well as FRTL-5 cells) and its regulation by thyroid stimulating hormone, which stimulated the secretory release of the proenzyme as well as of mature cathepsin B. Within one to two hours of stimulation with thyroid stimulating hormone, the cathepsin B activity associated with the plasma membrane increased significantly. This increase correlated closely with the localization of lysosomes in close proximity to the plasma membrane of cultured thyrocytes as well as with the thyroxine liberating activity of thyrocyte secretion media. These observations indicate that thyroid stimulating hormone induces the secretion of cathepsin B, which contributes to the extracellular release of thyroxine by thyrocytes.     

Hypothalamic regulation of pituitary secretion of luteinizing hormone

Luteinizing hormone (LH) is secreted continuously from the anterior pituitary gland. The concentration in the blood of this gonadotropic hormone plays a regulatory role in the development of puberty in both sexes, in the induction of ovulation in females, and in the production of testosterone in males. The secretion of LH is in turn controlled by luteinizing hormone releasing hormone (LHRH) secreted by the hypothalamus. LH and LHRH are removed from the blood by degradation and excretion. This hormonal system is modelled by a system of ordinary differential equations based upon specific physiological and biochemical assumptions current among experimentalists in this field. The one exception is the assumption that LHRH may bind reversibly to a serum protein; an analysis of the data shows that this or a similar mechanism is a crucial specification. Data on the serum levels of LH and LHRH in two human subjects were fitted using the model. The data consist of the transients and subsequent decays created by a bolus intravenous injection of LHRH. Primary appointment: Chemistry Dept., Dalhousie University. Primary appointment: Mathematics Dept., Dalhousie University.     

Heterogeneity of glycolytic oscillatory behaviour in individual yeast cells

There are many examples of oscillations in biological systems and one of the most investigated is glycolytic oscillations in yeast. These oscillations have been studied since the 1950s in dense, synchronized populations and in cell-free extracts, but it has for long been unknown whether a high cell density is a requirement for oscillations to be induced, or if individual cells can oscillate also in isolation without synchronization. Here we present an experimental method and a detailed kinetic model for studying glycolytic oscillations in individual, isolated yeast cells and compare them to previously reported studies of single-cell oscillations. The importance of single-cell studies of this phenomenon and relevant future research questions are also discussed.     

Dynamics and regulation of TSH secretion by superfused anterior pituitary cells

Superfused dispersed cells respond rapidly to 2- to 10-min pulses of TRH (10(-10) to 10(-7) M) in a dose-dependent manner. The effects of decreasing the stimulus duration can be overcome by a proportional increase in concentration of TRH. A TRH stimulus of 10 min or greater duration results in a sharp peak in TSH secretion followed by a lower plateau. Somatostatin (10(-8) M inhibits the response to TRH (t X 10(-9) M). T3 (2.0 microgram/dl) inhibits TRH-induced TSH secretion by superfused pituitary fragments, but not by dispersed cells. Corticosterone (50 microgram/dl), however, inhibits crude CRF-induced ACTH secretion by such cells.     

Feedback regulation of gonadotropic hormone secretion in neonatal pigs

The effect of castration and of administration of charcoal-treated porcine follicular fluid (pFF) containing inhibin-like activity on plasma concentration of gonadotropic hormones was studied in neonatal pigs. Plasma follicle-stimulating hormone (FSH) concentration averaged 25.1 +/- 1.5 ng/ml (mean +/- SEM) in 1-wk-old females and gradually declined to 20.2 +/- 0.7 ng/ml 6 wk later. Ovariectomy did not significantly influence plasma FSH concentration. In males, concentration averaged 8.0 +/- 0.7 ng/ml before castration but rose significantly within 2 days after castration. Injection of luteinizing hormone-releasing hormone (LHRH) did not influence plasma FSH concentrations in intact males, but did in females and in 7-wk-old males castrated at 1 wk. Plasma luteinizing hormone (LH) concentrations in 1-wk-old females (2.2 +/- 0.4 ng/ml) gradually declined and were not influenced by castration. Concentrations of plasma LH in 1-wk-old male piglets (2.8 +/- 0.7 ng/ml) were not significantly influenced by castration within 2 days but were significantly higher 6 wk later. LHRH induced a significant rise in plasma LH concentrations in all animals. Injection of pFF resulted in a decline of plasma FSH concentrations in intact and castrated males and in intact females, but did not influence plasma LH concentrations. These data demonstrate a sex-specific difference in the control of plasma FSH, but not in plasma LH concentration in the neonatal pig. Plasma FSH concentrations, but not plasma LH concentrations, are suppressed by testicular hormones in 1-wk-old piglets. Plasma FSH concentrations can be suppressed in both neonatal male and female pigs by injections of pFF.     

Hormonal regulation of progesterone secretion by cultured mouse granulosa cells

Secretion of progesterone by granulosa cells from preovulatory follicles of mice was determined during 2 weeks of cell culture in the presence of androgens, estrogen and pituitary gonadotropins. Androstenedione (10(-7) M) and dihydrotestosterone (10(-7) M) stimulated (P less than 0.05) progesterone secretion during the first 11 days of culture. In contrast, 17 beta-estradiol (10(-11)-10(-7) M) did not alter (P greater than 0.10) progesterone secretion throughout the culture period. Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) stimulated (P less than 0.01) the granulosa cells in a dose-dependent manner during the first few days of culture. This luteotropic effect was rapidly lost and at later times when FSH was not effective, LH suppressed (P less than 0.05) progesterone secretion. In the presence of prolactin (Prl) (1 microgram/ml), granulosa cells progressively secreted more progesterone during the first week of culture. After maximal stimulation on Days 7-9, progesterone secretion by Prl-treated cells began to decline, but the amount of steroid produced on Day 13 was still higher (P less than 0.05) than in control cultures. Androstenedione and Prl gave an additive effect on progesterone secretion during Days 3-5 of culture. Thereafter, the androgen, although stimulatory by itself, did not influence the luteotropic action of Prl. Unlike the early effect of androgens, 17 beta-estradiol acted synergistically with Prl to maintain maximal secretion of progesterone during the last 4 days of culture.(ABSTRACT TRUNCATED AT 250 WORDS)     

Age-related changes in the regulation of luteinizing hormone secretion by estrogen in women

Despite the many studies that have been conducted using both primate and human models to understand the control of the menstrual cycle, there are many aspects of the hormonal dynamics of the menstrual cycle that are not understood. This Minireview summarizes the changes in estrogen regulation of luteinizing hormone (LH) secretion that occur throughout life in women from the time of maturation of the hypothalamic-pituitary axis resulting in the occurrence of the LH surge during puberty, through the reproductive years, to the changes in the regulation of the LH surge during premenopause and, subsequently, menopause.     

Heterogeneity among T helper cells. Interleukin 2 secretion and help for immunoglobulin secretion

T lymphocytes are thought to provide "help" for B cells by activating them from the resting state, by secretion of antigen-nonspecific lymphokines that promote B cell differentiation and maturation, and by providing signals that induce isotype switching. To clarify the extent to which these different forms of helper activity could be carried out by individual T cells, we set up cultures in which B cells activated, and were in turn themselves stimulated by, limiting numbers of T cells through differences at the H-2 or Mls loci. At T cell doses at which responses were likely to represent the activity of individual helper T cells (or their immediate clonal progeny), we found that some T cells were able both to produce interleukin 2 (IL-2) and to induce secretion of both IgM and IgG, whereas others induced immunoglobulin (Ig) secretion without detectable IL-2 production, and still others made IL-2 but did not promote antibody secretion. We could not detect B cell stimulatory factor 1 production by alloantigen-stimulated T cells, and the addition of antibodies to B cell stimulatory factor 1 did not prevent Ig production. Two results, however--higher Ig accumulation in those wells that received an IL-2-producing cell, and inhibition by anti-IL-2 receptor antibodies of B cell but not T cell function--are consistent with a direct stimulatory effect of IL-2 on B cells in this system. The pattern of helper functions exhibited by T cells freshly isolated from mice differs from that inferred from studies of cloned lines of T cells in long term cultures.