Alton L. Steiner

Temporal sequence of cell shape changes in cultured rat sertoli cells after experimental elevation of intracellular cAMP.

Abstract The ability of FSH and pharmacological agents to induce changes in the shape of cultured rat Sertoli cells has been studied by using time-lapse phase-contrast microscopy and scanning electron microscopy (SEM). Morphological studies were combined with an immunocytochemical method for the localization of cAMP in Sertoli cells and the results correlated with determinations of protein-bound cAMP in Sertoli cells. A variable number of Sertoli cells were converted from a flat, epithelial-like morphology into a stellate morphology after exposure to FSH, isobutyl-methylxanthine (MIX), dibutyryl cyclic AMP (db-cAMP) and an FSH-MIX mixture. The morphological changes followed a time- and biological agent-dependent alteration and recovery pattern. While a 120 min exposure to FSH induced shape changes in 38% of the cells, MIX, db-cAMP and FSH-MIX effected shape changes in 75 % of cells. The morphological conversion induced by MIX, db-cAMP and FSH-MIX persisted as long as these biological agents were present in the medium, whereas the effects induced by FSH alone were transient. The flat-to-stellate transition was preceded by an increase in intracellular protein-bound cAMP, a form of cyclic nucleotide which may account for cAMP immunoreactivity observed in morphologically responsive and non-responsive Sertoli cells. From these data and from previous experimental findings of androgen-binding protein (ABP) immunoreactivity in the cytoplasm of responsive and non-responsive Sertoli cells, we conclude that a surge of cAMP triggers a still undefined mechanism by which Sertoli cells modify their shape in coincidence with a progressive depletion of cytoplasmic secretory granules.

The immunofluorescent localization of regulatory and catalytic subunits of cyclic AMP-dependent protein kinase in neuronal and glial cell types of the central nervous system

Abstract The cellular localization of the regulatory (RI and RII) and catalytic components of the cyclic AMP-dependent protein kinase in selected areas of the central nervous system was determined using a selective and sensitive immunofluorescent technique. The neuronal and glial distribution was similar for the three subunits, however, each unit exhibited differences in the pattern of subcellular localization. Whereas the catalytic unit was confined to intranuclear loci, RI and RII were identified within both the cytoplasm and nucleus, with RII in addition showing distinct staining of the nuclear membrane. A granular distribution of RI in the cell soma further distinguished the cytological localization of these two regulatory components in this cellular compartment. The contrasting distribution of the subunits suggests possible functional differences between the regulatory and catalytic components of cyclic AMP-dependent protein kinase, which are discussed with regard to the role of this enzyme in mediating the physiological effects of cyclic AMP in the central nervous system.

Particulate guanylate cyclase of skeletal muscle. Effects of Ca2+ and other divalent cations on enzyme activity

The properties of particulate guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2) from purified rabbit skeletal muscle membrane fragments were studied. Four membrane fractions were prepared by sucrose gradient centrifugation and the fractions characterized by analysis of marker enzymes. Guanylate cyclase activity was highest in the fraction possessing enzymatic properties typical of sarcolemma, while fractions enriched with sarcoplasmic reticulum had lower activities. In the presence of suboptimal Mn2+ concentrations, Mg2+ stimulated particulate guanylate cyclase activity both before and after solubilization in 1% Triton X-100. Guanylate cyclase activity was biphasic in the presence of Ca2+. Increasing the Ca2+ concentration from 10(-8) to 10(-5) M decreased the specific activity. As the Ca2+ concentration was further increased to 5 . 10(-4) M enzyme activity again increased. After solubilization of the membranes in 1% Triton X-100, Ca2+ suppressed enzyme activity. Studies utilizing ionophore X537A indicated that the altered effect of Ca2+ upon the solubilized membranes was independent of asymmetric distribution of Ca2+ and Mg2+.

Cyclic AMP and cyclic GMP: studies utilizing immunohistochemical techniques for the localization of the nucleotides in tissue.

Antibodies to the cyclic nucleotides initially were utilized in radioimmunoassays for cyclic AMP and cyclic GMP which might be present in mammalian tissues. allowed measurement of the nucleotides on small amounts of tissue in physiologic studies. To gain further insight into the relative roles of cyclic AMP and cyclic GMP in cell function, these antibodies have been applied to immunohistochemical studies for the localization of the cyclic nucleotides in tissues and cells. This methodology is useful for determining in which cell type in a heterogeneous tissue increases in cyclic nucleotide concentrations occur. In addition, within individual cells, staining patterns for cyclic AMP and cyclic GMP are usually quite distinct. Cyclic GMP in canine thyroid is located to the follicular cell membrane while cyclic AMP is ubiquitously distributed in follicular cell cytoplasm. In both rat adrenal cortex and testis, there is prominent nuclear localization of cyclic GMP, suggesting a role for the nucleotide in growth regulation. These studies provide histologic evidence suggesting diverse roles for cyclic AMP and cyclic GMP in mammalian physiology. It is anticipated that this technique will also be useful in the ultrastructural localization of the cyclic nucleotides and for the identification of other cyclic nucleotides which might be present in mammalian tissues.

Immunofluorescent localization of cyclic GMP, calmodulin and cyclic GMP-dependent protein kinase in the choroid plexus

An immunofluorescent technique has demonstrated that tissue-bound pools of cyclic GMP, calmodulin and cyclic GMP-dependent protein kinase are localized within the cytoplasm of the epithelial cells of the choroid plexus. In all other cell types and regions of the central nervous system previously examined, however, these molecules have shown contrasting immunofluorescent localization. These results suggest that the interaction in the choroid plexus may be related to the specialized physiological functions of the neuroglial epithelial cell.

Immunohistochemical localization of cyclic AMP, cyclic GMP and calmodulin in Dictyostelium discoideum

Using an indirect immunofluorescence technique cyclic AMP, cyclic GMP and calmodulin have been localized in Dictyostelium discoideum cells. The bound cyclic AMP is localized throughout the cell. Cyclic GMP and calmodulin are markedly localized in the nuclear area of the cell. No changes in the pattern of staining was observed during differentiation. Chemotactic stimulation induced a transient increase of cyclic GMP staining.

Immunofluorescent localization of cGMP, cGMP-dependent protein kinase, calmodulin and cAMP in the rat uterus

SummaryCyclic guanosine 3′, 5′ monophosphate (cGMP), cGMP-dependent protein kinase, calmodulin and cyclic adenosine 3′, 5′ monophosphate (cAMP) were localized in the uterus of the immature rat by an indirect immunofluorescence technique. cGMP, cGMP-dependent protein kinase and calmodulin were detected predominantly along epithelial and myometrial plasma membranes and in the adjacent cytoplasm. In contrast, cAMP immunoreactive material was found principally in the cytoplasm of connective tissue. After administration of 17 β estradiol, similar time-dependent changes were observed in the localization of cGMP, cGMP-dependent protein kinase and calmodulin in all uterine cell types. For the three compounds, nucleolar-like distribution of the immunofluorescence appeared approximately 12 h after treatment. A more dispersed, reticular distribution of the nuclear fluorescent staining was observed 20–24 h after hormonal treatment. Estrogen did not affect the localization of cAMP.The simultaneous mobilization of cGMP, cGMP-dependent protein kinase and calmodulin towards the same nuclear loci suggests concerted roles for these three molecules in nuclear metabolic processes during the development of the uterotrophic action of estrogens.

Changes in rat adrenal cyclic nucleotides during normal and neoplastic growth

Abstract In an attempt to correlate changes in cyclic nucleotide levels with in vivo growth of the rat adrenal gland we have measured adrenal cyclic AMP and cyclic GMP in normal, hyperplastic, and neoplastic rat adrenals. The first group of animals were subject to either unilateral adrenalectomy (ADX) or acute hypophysectomy 1 h prior to unilateral adrenalectomy (HADX). Cyclic nucleotides were measured in the contralateral adrenal post-operatively. In HADX rats cyclic GMP rose steadily throughout the 7 day study period, while ADX rats exhibited significant decreases in adrenal cyclic GMP. Cyclic AMP remained approximately 1.5 pm/mg tissue in HADX rats, while in ADX rats there was significant elevation of adrenal cyclic AMP at all time points. Cyclic GMP/cyclic AMP ratios remained constant in HADX animals; however, the growing adrenals of ADX animals exhibited depressed cyclic GMP/cyclic AMP ratios at all time periods. Adrenal hyperplasia was induced in a seond group of animals by a transplantable, corticotropin-secreting, pituitary tumor. Adrenals from age-matched animals served as controls. Adrenal cyclic AMP was significantly elevated in tumor-bearers at a time correspinding to the peak accumulation of adrenal weight, protein and DNA in these animals. In contrast, adrenal cyclic GMP in both tumor-beares and control animals fell steadily throughout the study period. Cyclic GMP/cyclic AMP ratios of control animals decreased from 2 to 3 weeks post-transplant remaining at the 3 week value during the period corresponding to rapid adrenal growth in tumor-bearers. The cyclic GMP/cyclic AMP ratio in the hyperplastic adrenals of tumor-bearers decreased steadily throughout their rapid growth period, suggesting a positive correlation between adrenal growth and depression of the cyclic GMP/cyclic AMP ratio. Cyclic nucleotide levels in neoplastic adrenals of rats bearing the transplantable adrenocortical carcinoma 494 were compared with cyclic nucleotides in normal rat adrenal glands. Cyclic AMP was not different in the two groups. However, the cyclic GMP content of neoplastic adrenals was significantly lower than that of normal adrenal tissue, causing a suppression of the cyclic GMP/cyclic AMP ratio in the neoplastic tissue. Thus, measurement of adrenal cyclic nucleotides in both hyperplastic and neoplastic rat adrenal glands suggests that adrenal growth in vivo may be characterized by a depression of the cyclic GMP/cyclic AMP ratio.

Post-natal ontogenesis of calmodulin and cyclic AMP-dependent protein kinase subunits in the Purkinje cell using immunofluorescence ☆

Specific immunofluorescent techniques were utilized to demonstrate the regulatory (RI and RII) and catalytic (C) subunits of cyclic AMP-dependent protein kinase, and calmodulin, in the rat cerebellar Purkinje cell during post-natal ontogenesis. Whereas these second messenger receptor proteins were not detectable at 5 days, an increase in staining intensity occurred from this time until adult levels and distribution were attained at 25 days. Differences in immunofluorescent staining were noted between these proteins during ontogenesis. The relationship of these immunocytochemical changes to synaptogenesis and cellular maturation are discussed, including possible interactions between cyclic AMP and calcium messenger systems.

Characterization of cyclic AMP-binding proteins in rat Sertoli cells using a photoaffinity ligand

SummaryProtein-bound cyclic AMP (cAMP) levels in cultured rat Sertoli cells have been determined after exposure to follicle-stimulating hormone (FSH) and agents which elevate intracellular cAMP or mimic cAMP action. Changes in the content of protein-bound cAMP were correlated with changes in receptor availability determined by measuring [3H] cAMP binding. Using the photoaffinity analog of cAMP, 8-N3 [32P] cAMP, two major cAMP-binding proteins in Sertoli cell cytosol, with molecular weights of 47 000 and 53 000 daltons, were identified as regulatory subunits of type I and type II cAMP-dependent protein kinases, respectively. Densitometric analysis of autoradiograms demonstrated differential activation of the two isozymes in response to treatment with FSH and other agents. Results of this study demonstrate the value of measuring changes in protein-bound cAMP and the utility of the photoaffinity labeling technique in correlating hormone-dependent processes in which activation of cAMP-dependent protein kinase occurs.