Quantification of morphologic, cytologic, and kinetic parameters of unirradiated swine skin: a histologic model

The gross changes of erythema and desquamation produced by irradiation indicate population and functional variations occurring in the epidermis, microvasculature, and dermis. However, the parameters do not distinguish the individual population kinetics. This study determines multiple histologic and cell kinetic parameters of unirradiated swine skin. The prickle cell layer exists as a shell three to six cells thick. The proliferative basal layer exists as a confluent monolayer with 2031 +/- 48 cells/cm; it has a growth fraction of 1, an average cell generation time of 12.3 +/- 2.4 days, a TS of 9.4 +/- 2.9 hr, and a TS + T1/2M of 17 to 19 hr. The labeling index is 3.9 +/- 0.1% with a diurnal variation having a 5% peak at 1800 hr and a 2.5% nadir at 0900-1100 hr. The mitotic index varies from 1.7 to 3.2% and has no clear-cut diurnal variation. These values are similar to those available for man. These data and those published previously are utilized to define a histologic model of the irradiated epidermal cell renewal system. These measured values are compared with those derived from analysis of available time-dose isoeffect data using a single-hit multitarget and a linear-quadratic model. The derived dose-survival curve is steeper with a D0 between 97 and 255 rad compared to the measured value of 337 rad.     

Analysis of cell differentiation by division tracking cytometry.

We propose a quantitative method to characterize growth and differentiation dynamics of multipotent cells from time series carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) division tracking data. The dynamics of cell proliferation and differentiation was measured by combining (CFDA-SE) division tracking with phenotypic analysis. We define division tracking population statistics such as precursor cell frequency, generation time and renewal rate that characterize growth of various phenotypes in a heterogeneous culture system. This method is illustrated by study of the divisional recruitment of cord blood CD34(+) cells by hematopoietic growth factors. The technical issue of assigning the correct generation number to cells was addressed by employing high-resolution division tracking methodology and daily histogram analysis. We also quantified division-tracking artifacts such as CFDA-SE degeneration and cellular auto-fluorescence. Mitotic activation of cord blood CD34(+) cells by cytokines commenced after 2 days of cytokine stimulation. Mean generation number increased linearly thereafter, and it was conclusively shown that CD34(+) cells cycle slower than CD34(-) cells. Generation times for CD34(+) and CD34(-) cells were 24.7 +/- 0.8 h and 15.1 +/- 0.9 h (+/-SD, n = 5), respectively. The 20-fold increase in CD34(+) cell numbers at Day 6 could be attributed to a high CD34(+) cell renewal rate (91% +/- 2% per division). Although cultures were initiated with highly purified CD34(+) cells (approximately 96%), CD34(-) numbers had expanded rapidly by Day 6. This rapid expansion could be explained by their short generation time as well as a small fraction of CD34(+) cells (approximately 5%) that differentiated into CD34(-) cells. Multitype division tracking provides a detailed analysis of multipotent cell differentiation dynamics.     

Circadian variation in the mitotic rate of the rat corneal epithelium. Cell divisions and migration are analyzed by a mathematical model

A stathmokinetic method was used to study the diurnal variation in the mitotic rate (MR) of the rat corneal epithelium, and in the adjacent conjunctival epithelium. A prominent circadian variation in cell proliferation was observed in both epithelia, both showing almost the same pattern, which may indicate that both tissues are submitted to the same regulatory mechanisms. The average rate of cell renewal during a 24 h period indicated a mean cell renewal time of 12.3 days. This is longer than previously assumed. The MR declined toward the central cornea. Based on the above observations and the known centripetal migration of cells in the corneal epithelium, we have developed a mathematical model showing isomorphism with the renewal of the corneal epithelium.     

The basalis of the primate endometrium: a bifunctional germinal compartment

Radioautographic analysis of epithelial and stromal cell proliferation in the primate endometrial functionalis and basalis (rhesus monkey) has identified horizontal zonal patterns of mitotic activation and inhibition during natural menstrual cycles. At 1 h after a single i.v. injection of [3H]thymidine, mitotic activity in endometrial biopsies (hysterotomy) was determined on 9 days from the late proliferative to the late luteal phase (-2 days to + 14 days relative to the estrogen [E2]peak). Labeling indices (LIs) were determined within glandular segments of the 4 horizontal endometrial zones: Transient functionalis Zone I (luminal epithelium) and Zone II (uppermost gland); Germinal basalis: Zone III (middle gland) and Zone IV (basal gland). The size of the dividing epithelial populations (LI) differed zonally. During E2 dominance (-2 days to +3 days), the epithelial LIs of functionalis I (10 +/- 0.3%) and II (9.8 +/- 1.0%) were greater than those of basalis III (5.8 +/- 0.2%) and basalis IV (3.7 +/- 0.8%). During progesterone (P) dominance (+5 days to +14 days), epithelial mitosis was strongly inhibited in functionalis I (4.3 +/- 1.9%), functionalis II (0.8 +/- 0.2%), and basalis III (1.4 +/- 0.5%). Thus germinal basalis III was linked functionally with transient functionalis I and II by periovulatory uniformity in epithelial proliferation and postovulatory mitotic inhibition. A unique mitotic pattern set basalis IV apart from other zones by a steady rise in LI from 1% (-2 days) to 11% (+10 days). The LIs for stromal fibroblasts remained quite uniform in basalis IV but varied in other zones. Thus the postovulatory primate basalis was a distinct bipartite compartment in which the mitotic rate in basalis IV glandular epithelium increased steadily whereas that of basalis III was strongly inhibited. The remarkable enhancement of epithelial mitotic activity in basalis IV may reflect expansion of the stem-progenitor cell population for gestational growth or for post-menstrual regeneration.     

Growth regulation in X-irradiated mouse skin; the possible role of chalones.

Extracts of hairless mouse skin were tested for their content of epidermal G1 inhibitor and G2 inhibitor at daily intervals after X-irradiation with 4 500 or 2 250 rad. After either dose the skin extracts lacked G1 inhibitory activity on days 5 and 6 respectively after irradiation. This coincided with the time when the epidermal mitotic rate again became normal and started a period of over-shoot. The time interval of 5-6 days corresponds to the turnover time of the differentiating cells in hairless mouse back epidermis. The findings indicate that the proliferating cells in epidermis can respond to changes in local chalone concentration, even after X-irradiation at the tested doses, and that the irradiated epidermal cell population still retains some important properties inherent in a cybernetically regulated system. The local G2-inhibitory activity also varied after irradiation, but these variations could not be directly related to the corresponding mitotic rates.     

Promoting effect of bombesin on the cell proliferation in the rat endocrine pancreas during the early postnatal period

The present work investigated whether orally administered bombesin influences cell proliferation in the endocrine pancreatic islets of rats during the suckling period and after weaning. Four series of pups were given bombesin diluted in milk (20 micrograms/kg, 3 times daily) or milk alone for 5 days during either the first, second, third or fourth postnatal week of life. Oral administration was used because bombesin-like peptides have been identified in the breast milk of mammals. 45 min before death, animals were given a single [3H]thymidine pulse injection. Tissue sections were processed for radioautography; DNA labeling and mitotic indices were estimated after counting at least 1000 endocrine cells per rat pancreas. In control rats, the labeling and mitotic indices in pancreatic islets dropped regularly from the first week to the fourth week of life (3.6% +/- 0.2% versus 1.9% +/- 0.1% and 0.46% +/- 0.09% versus 0.08% +/- 0.02%, respectively). Orogastric bombesin administration significantly increased the DNA labeling and mitotic indices at the end of the first week (+20% and +62%, respectively) and second week of life (+37.5% and +49%, respectively) (P less than 0.05 to P less than 0.005), but did not modify these parameters for the third and subsequent weeks of life. Therefore, this study provides evidence that bombesin stimulates DNA synthesis and cell division in pancreatic endocrine cells during the developmental period.     

Restorative processes in the parathyroid glands in experimental hypothyroidism

By means of morphological, morphometrical and autoradiographical methods restorative processes in the parathyroid glands in 41 euthyroid and in 41 hypothyroid rats have been studied during 1-24 days after mechanical trauma of the glands or after hemithyroparathyroidectomy. Seven hypothyroid and 7 euthyroid rats serve as a control. Hypothyroidism is produced with daily injection of mercazolil (6 mg/kg) 3 weeks before the operation and during the time of the experiment. In nonoperated hypothyroid rats development of hypertrophy in parathyrocytes is noted. Prolonged injection of mercazolil weakens (posttraumatic regeneration) or completely suppresses (compensatory hypertrophy) mitotic activity of the glandular cells (in comparison with the euthyroid animals). Manifestation of hypertrophy in parathyrocytes of the hypothyroid rats in comparison with the corresponding control is also less, than against the background of euthyreosis.     

Cytological mechanisms of the growth and regeneration of the parathyroid glands

Contribution of proliferation and hypertrophy of the epitheliocytes to the growth and regeneration of the rat parathyroid glands was estimated using organo- and cytometry, cytophotometry of DNA content in the nuclei and determination of mitotic index. During postnatal development and in the case of hypertrophy of the adult glands following a moderate resection (50%), the gland growth is provided by mitotic divisions of the parathyroid cells, rather than by the increase in cell size. When up to 75 and 90% of the gland volume is removed, cell hyperplasia is accompanied by stable hypertrophy of the parathyroid cells unrelated to their polyploidization. The contribution of nonmitotic cell hypertrophy to the total increment of the organ volume amounts to 40-50%.     

Origin and Kinetics of Resident Tissue Macrophages

Abstract To elucidate the origin and renewal kinetics of peritoneal macrophages, as a typical example of the mononuclear phagocytic system, syngeneic rats were treated with tritiated thymidine [³H]TdR and leucocytes were transferred to unlabelled recipients over a bilateral arteriovenous shunt. Labelled and unlabelled monocytes were evenly distributed in both animals as shown by autoradiography. It was ascertained that no ‘autoradiographically’ detectable reutilization of label occurred and that transferred cells showed undisturbed kinetics. the results imply: 1 resident peritoneal macrophages derive from blood monocytes; 2 peritoneal macrophages represent a homogeneous population in respect to their cellular origin; 3 blood monocytes as a myelogeneous cell line do not represent a generative end cell. They migrate into the tissue (peritoneal cavity) and differentiate into resident macrophages, undergoing on average one mitosis per cell during a period of approximately 7 days. 4 resident peritoneal macrophages are derived 50% from blood monocytes and 50% from division in situ; and 5 under steady-state conditions the renewal rate amounts to 0. 18%/h, which yields a half-life time of 16 days and a renewal time of 23 days.     

Two-stage cultures for the production of astaxanthin from Haematococcus pluvialis

A two-stage culture system was established for the production of astaxanthin from Haematococcus pluvialis. In a first stage green vegetative cells were produced in semicontinuous cultures maintained with daily renewal rates between 10 and 40%. The steady-state cell density decreased with increasing renewal rates. Highest cell productivity, 64 x 10(6) cells l(-1) day(-1) was obtained with a daily renewal rate of 20%. In a second stage the harvested cultures were submitted to high light (240 micromol photon m(-2) s(-1)) under batch conditions for 15 days in order to stimulate the transition to the aplanospore stage and the accumulation of astaxanthin. No decrease in cell density was recorded during the induction period in any of the cultures. Cultures obtained at high renewal rates continued growing during the induction period and no astaxanthin was accumulated until all nitrogen in the media had been consumed. The final concentration of astaxanthin was inversely correlated to the growth rate at which first-stage cultures were maintained. Optimal renewal rate for maximal astaxanthin production depended on the duration of the induction period. After a 12-day induction period the highest astaxanthin production, 5.8 mg l(-1) of semi-continuous culture day -1, was obtained with cultures maintained at a renewal rate of 20%. When the induction period was increased to 15 days maximal astaxanthin productivity, 9.6 mg l(-1) of semi-continuous culture day -1, was obtained from cultures maintained at a renewal rate of 40% despite the much lower astaxanthin concentration achieved in these cultures. Results demonstrate the feasibility of semi-continuous cultivation of H. pluvialis for the two-stage production of astaxanthin.     

A connective tissue tumor in the mussel Mytilus trossulus from a polluted region of Nakhodka Bay, the Sea of Japan

A tumor was found for the first time in a mussel Mytilus trossulus from a heavily polluted area of Nakhodka Bay, Sea of Japan. Tumor cells were found in the connective tissue of different organs and also in gill vessels and hemal sinuses of the visceral mass. They were both attached and diffuse. The tumor was at an advanced stage, replacing the normal connective tissue cells, and formed nodes. The tumor cells were polymorphic, with a high nucleocytoplasmic ratio, and had a prominent nucleolus. The size of their nuclei was three to five times that of the nuclei of agranular hemocytes. The mitotic activity of the tumor cells was more than an order of magnitude higher than in the normal cells: the mean mitotic index was 1.4 +/- 0.5%, ranging from 0.97 to 2.3% in different organs. The mitotic indices in the connective tissue cells of three normal mussels were 0, 0, and 0.12%. A significant proportion (up to 78%) of the mitotic cells were at metaphase. The frequency of abnormal mitoses was 17%. Metaphases with displaced (often multiple) chromosomes constituted 71% of abnormal mitoses; anaphases, 8%; and tri- and tetrapolar mitoses, 11%. The tumor described is similar to diffuse sarcomatoid diseases of mussels from other geographical regions.     

Fluoro Jade-B Detection of Dying Cells in the SVZ and RMS of Adult Rats After Bilateral Olfactory Bulbectomy

A novel fluorochrome, Fluoro-Jade B, was used to detect dying precursor cells in the subventricular zone (SVZ) and rostral migratory stream (RMS) of adult rats after bilateral olfactory bulbectomy and in control intact rats. The animals in experimental group were left to survive 3 days and from 3 till 16 months after surgical procedure. 1. In the control animals, Fluoro-Jade B positive cells were visible in the SVZ and within the whole extent of the RMS. The number of Fluoro-Jade B positive cells increased in the elbow in comparison to the rest parts of the RMS. 2. In the experimental animals surviving either 3 days or from 3 till 16 months after bilateral olfactory bulbectomy, Fluoro-Jade B positive cells displayed the similar pattern of distribution as in the control animals. However, some quantitative differences in the labeled cells number along the rostral migratory pathway appeared. 3. The average number of degenerating cells within the control SVZ and RMS was 26.24+/- 0.686. In bulbectomized animals, regardless of survival time, an insignificant increase of Fluoro-Jade B positive cells number occurred. We can conclude that dying of precursor cells is a physiological process running within the SVZ/RMS in both control and experimental animals. Moreover, this physiological process is not influenced by survival period after bilateral olfactory bulbectomy. Our results demonstrate Fluoro-Jade B as a useful marker of dying cells.     

Life shortening in mice exposed to fission neutrons and gamma rays. VIII. Exposures to continuous gamma radiation

Data are presented on the mean aftersurvival of male B6CF1 mice exposed for 22 h per day, 5 days per week, to 60Co gamma radiation at dose rates of 1.36 to 12.64 x 10(-3) cGy/min for 23 weeks or 1.36 to 6.32 x 10(-3) cGy/min for 59 weeks. For deaths from all causes, linear dose-response curves were obtained with slopes (days of life lost/cGy) of 0.158 +/- 0.016 and 0.077 +/- 0.002 for 23- and 59-week exposures, respectively. These values were not significantly altered when the analysis was restricted to those mice dying with tumors (92% of the total) or to those presumably dying from tumors (82% of the total). Analysis of mortality rates showed that about 90% of the radiation-specific excess mortality was tumor related. The 59-week exposure series induced only a small increase in the number of days of life lost/cGy/weekly fraction over that induced by 23 weeks of irradiation, 4.53 +/- 0.15 compared to 3.64 +/- 0.36 days lost/cGy/weekly fraction. This lower than expected value for 59 weeks of exposure may signal the approach to the final linear, additive, injury term postulated from earlier studies at this laboratory with low-dose-rate, daily, duration-of-life 60Co gamma irradiation.     

Proliferation kinetics of mouse tongue epithelium under normal conditions and following single dose irradiation.

Epithelial proliferation in the ventral surface of mouse tongue follows a pronounced circadian rhythm with a peak in mitotic activity at 10.00 a.m., preceded by a wave of DNA synthesis 8 h earlier. Nearly all cells (85%) pass through G2 and mitosis immediately after the S-phase; they subsequently divide again, usually after 2 or 3 days, indicating cohorts of cells with different G1-duration. The fraction of all nucleated cells comprised in one daily proliferation wave is about 20%, indicating a turnover time of the nucleated cell compartment of about 5 days. Cytotoxic injury by a single radiation dose of 20 Gy causes a steep decrease in cell counts, leading to complete denudation after 9-13 days. The difference between the latent period before ulceration and the tissue turnover time is explained by a marked proliferative activity of the doomed cells. The mitotic index increases steeply after day 1 to three times the control level, but most mitotic figures display gross abnormalities such as multipolar spindles or chromosome clumping. As a consequence cells with abnormal or multiple nuclei appear in the basal layers 3 days post irradiation and subsequently migrate to the upper layers. After denudation the epithelium rapidly becomes restored, with a phase of transient hyperplasia on days 13-14. Normal architecture is regained by day 15. Over the whole healing period the mitotic index remains at a high level, with most of the mitoses appearing histologically normal.     

Isolation of BM mesenchymal stem cells by plastic adhesion or negative selection: phenotype, proliferation kinetics and differentiation potential

BACKGROUND: BM mesenchymal stem cells (MSC) have the capacity for renewal and the potential to differentiate into multiple tissues. In this study, we compared different enrichment methods to obtain MSC from BM. METHODS: Three different methods were compared with a view to obtaining MSC more rapidly from BM: negative selection (RosetteSep and MACS) and plastic adhesion. The three cell fractions were grown in complete alpha-minimum essential medium in order to evaluate their proliferative capacity, their phenotype during culture and their potential to differentiate into adipocytes, osteocytes and chondrocytes. Identification of MSC was performed by immunofluorescence with putative mesenchymal markers SH2 and SH3 but also with hematopoietic markers. RESULTS: After negative selection, only 1+/-0.2% and 2.9+/-0.8% of cells were recovered from BM with the RosetteSep and MACS methods, respectively. However, negative depletion permitted a homogeneous population of MSC, with more than 90% SH2+ and SH3+ cells, to be obtained rapidly and in large quantities after 10 days of culture. Similar homogeneity was observed after three passages if the plastic adhesion was used as selection method and after an average of 25-30 days of culture. Different levels of MSC maturity were also suggested by the variable level expression of Stro-1. DISCUSSION: Depleting selection by RosetteSep may represent an easy method of obtaining MSC rapidly from BM with the aim of potential therapeutic use.     

Study of folliculogenesis in vivo in guinea pig

Understanding normal folliculogenesis in guinea pigs is fundamental as a first step towards the development of a guinea pig follicle culture system. The aims of this study were (1) to characterise morphological changes during follicular development in vivo and (2) to describe the growth pattern of follicles. Cycling guinea pigs were infused with 5-bromo-2'-deoxyuridine for 1 or 2 weeks and sacrificed at time points ranging from 0 to 37 days after the infusion. The granulosa cell number in the largest cross-sections increased from 25.0+/-6.1 (mean+/-S.D.) in primary (type 2) to 192.0+/-65.9 in preantral (type 5) and 256.3+/-96.9 in antral (type 6) follicles. The oocyte diameter increased from 44.8+/-6.2 microm (type 2) to 72.8+/-9.1 microm (type 5) and 78.9+/-9.3 microm (type 6) and the follicle diameter from 67.9+/-10.1 microm (type 2) to 188.9+/-29.7 microm (type 5) and 231.0+/-56.1 microm (type 6). After a 1-week labelling period, about 71% of type 2 follicles had at least one labelled granulosa cell, as did 95% of type 3-4, and 100% of type 5 and 6. About 1 week was needed to achieve 95% mitotic activity in granulosa cells (GC) of type 5 and 6 follicles, while about 2 weeks was required to achieve 100% mitotic activity in GC of type 3-4 and more than 2 weeks for GC of type 2 follicles. These data provide some baselines for the examination of a guinea pig follicle culture system.     

Parathyroid hormone secretion does not respond to changes of free calcium in electropermeabilized bovine parathyroid cells, but is stimulated with phorbol ester and cyclic AMP

The secretion of parathyroid hormone (PTH) is suppressed in bovine parathyroid cells by raised extracellular [Ca2+], and 12-0-tetradecanoyl-phorbol-13-acetate (TPA) stimulates the release of PTH from cells suppressed by high extracellular [Ca2+]. Extracellular and cytosolic free [Ca2+] are proportionally related in intact cells. To assess the role of cytosolic free [Ca2+] on PTH secretion, bovine parathyroid cells were rendered permeable by brief exposure to an intense electric field. PTH secretion was comparable at 40 nM, 500 nM, 5 microM, 28 microM, 0.5 mM and 2 mM [Ca2+] (release of total cellular PTH 3.7 +/- 0.5%, 3.9 +/- 0.4%, 3.4% +/- 0.3%, 3.9 +/- 0.4%, 3.1 +/- 0.3%, 3.5 +/- 0.7%, respectively), but the secretion was stimulated twofold (P less than 0.05 vs. control) in a dose and ATP dependent manner with TPA (100 nM) and cyclic AMP (1 mM). As a result, free [Ca2+] in the range of those observed in intact cells during regulation of PTH secretion by changes of extracellular [Ca2+] did not affect the release of PTH in permeabilized cells. The [Ca2+] independent stimulation of PTH release by TPA and cyclic AMP indicates that changes of cytosolic free [Ca2+] may represent a secondary event not related to the regulation of PTH secretion.     

Cell population kinetics and DNA content during thyroid carcinogenesis

The proliferation kinetics and DNA content of thyroid follicular cells in rats were studied by autoradiography and cytophotometry. Continuous treatment of animals with methylthiouracil (MTU) results in hyperplasia followed by tumour growth in the thyroid gland. The mitotic index (MI) increases from 0.006 +/- 0.002% in controls to 0.13 +/- 0.06% in hyperplasia and to 0.09 +/- 0.03% in malignant cells. The same is true for the labelling index (LI) which rises from 0.08 +/- 0.003% in controls to 1.4 +/- 1.1% in hyperplasia and to 1.0 +/- 0.6% in follicular adenomas. The S-phase duration (TS) is shortened from 8.0 +/- 1.2 hr in controls to 6.0 +/- 1.4 hr in animals treated for 9 months with MTU and prolonged to 15.4 +/- 2.1 hr in papillary carcinomas. In all MTU-treated animals a decrease in the value of the potential population doubling time (TPD) and thyroid weight doubling time (TD) was observed. The cell loss factor (phi) decreases in animals treated for 3 months with MTU and increases during the stage of tumour growth in the gland (animals treated 12-15 months with MTU). DNA measurements in the nuclei of hyperplastic and neoplastic thyroid tissues reveal cells with values exceeding that in control animals. However, no difference was found in the DNA content between thyroid adenomas and carcinomas, nor between thyroid hyperplasia and neoplasia.     

Evidence that chlorophyllin (CHLN) may behave as an inhibitor or a promoter of radiation-induced genetic damage in somatic cells of drosophila

Irradiation of 96h old Drosophila following a 24h pretreatment with 5% chlorophyllin (CHLN) was delayed 0-4 days. The antimutagenic effect of CHLN in somatic cells monitored by the wing spot test persisted for 3 days after completion of the pretreatment and appeared to terminate at a time corresponding to the cessation of mitotic divisions of wing anlagen cells. Within the same population of cells, CHLN demonstrated both an inhibitory effect as measured in mwh single spot classes, and contrarily, a promoting effect in the class of mwh/flr twin spots and to an extent in the class of large flr spots. The reason for the contrasting effects of CHLN remains to be determined.     

Vasopressin: a potent growth factor in adrenal glomerulosa cells in culture

Previous studies have shown that vasopressin stimulates the mitotic activity in adrenal zona glomerulosa cells in intact as well as in hypophysectomized rats. (Payet and Isler, Cell and Tissue Res. 172, 1976; Payet and Lehoux, J. steroid Biochem. 12, 1980). We now report that this effect is direct and specific, since vasopressin stimulates the mitotic activity of rat adrenal zona glomerulosa cells in primary cultures. These cells were prepared by dissociation with collagenase in the culture medium MEM-d-Valine. Isolated cells were placed in 3.5 diameter petri dishes in MEM-d-valine medium containing 15% fetal calf serum and antibiotics for two days and 5% fetal calf serum for subsequent cultures. The medium was changed at 24 hr intervals. The hormones were added 3 days after the culture was started. The mitogenic effect of vasopressin was found to be dependent both on time and hormone concentrations. Vasopressin (10(-11) M) stimulated thymidine incorporation 4.8 +/- 0.6-fold after 2 days of treatment and 5.3 +/- 1.6-fold after 8 days. When ACTH (10(-11) M) was added together with vasopressin (10(-11) M) the mitogenic effect was enhanced at 6.5 +/- 1.9-fold after 2 days and 12.9 +/- 6.9-fold after 8 days of treatment. The aldosterone and corticosterone outputs were also stimulated by the combined presence of vasopressin and ACTH in the incubation medium; a maximal effect was observed between 6 and 8 days of treatment. Vasopressin (10(-11) M) + ACTH (10(-11) M) stimulated the aldosterone output 7-fold and that of corticosterone by 18-fold. When added alone, vasopressin, as well as ACTH alone had only a small effect on the aldosterone output. However, ACTH alone stimulated the corticosterone output 10-fold. In conclusion, vasopressin is an important and specific growth factor of the adrenal zona glomerulosa cells. In addition, together with ACTH vasopressin stimulates the aldosterone and corticosterone output both in vivo and in vitro in primary cell cultures.