Presence Of dbcAMP Research Articles

Effect of dibutyryl cyclic adenosine 3',5'-monophosphate on morphological features and biological markers of human salivary myoepithelial cell line in culture.

We have found the emergence of myoepithelial cells (HSG-AZA1) in neoplastic human salivary intercalated duct cell line HSG in culture after treatment with 5-azacytidine. When HSG-ZAZ1 cells were cultured in the presence of N6,O2-dibutyryl cyclic adenosine 3',5'-monophosphate (dB-cAMP), they formed long cytoplasmic processes which were densely packed with ample microfibrils in addition to microtubule bundles. The expression of neuron-specific enolase, synaptophysin, and catecholamine as well as neurofilaments in the treated HSG-AZA1 cells was found by the immunofluorescence staining technique, immunoblotting, immunoelectron microscopy, or catecholamine fluorescence. Both the anchorage-independent and anchorage-dependent growths of HSG-AZA1 cells were suppressed in the presence of dB-cAMP. After the removal of dB-cAMP from the culture, the treated cells returned rapidly to the phenotype and growth rate of the untreated cells. These findings indicate that reversible differentiation into the neuron-like cells of HSG-AZA1 cells occurs in growth medium containing dB-cAMP.

Inhibition of proximal tubule Na(+)-K(+)-ATPase activity requires simultaneous activation of DA1 and DA2 receptors

This study examines the receptor mechanisms by which dopamine (DA) inhibits Na(+)-K(+)-adenosinetriphosphatase (ATPase) activity in single permeabilized proximal tubule (PCT). Na(+)-K(+)-ATPase activity was inhibited in the presence of both DA1- and DA2-specific agonists but not by either agonist alone. The inhibition induced by DA (10(-6) M) was attenuated in the presence of either of the two DA2-specific antagonists S-sulpiride and YM 09151 at 10(-5) M and in the presence of the DA1 antagonist SCH 23390 (10(-5) M). PCT adenosine 3',5'-cyclic monophosphate (cAMP) levels were significantly increased in the presence of DA and DA1 agonist, but DA2 agonist had no effect on cell cAMP levels. Na(+)-K(+)-ATPase activity was significantly inhibited in PCT incubated with DA2 agonist (10(-5) M) and dibutyryl (DB)-cAMP (10(-6) M) but not with DA2 agonist (10(-5) M) only. PCT Na(+)-K(+)-ATPase activity was also significantly inhibited in the presence of both DA2 agonist (10(-5) M) and forskolin (10(-6) M). Neither DBcAMP (10(-6) M) nor forskolin (10(-6) M) alone inhibited Na(+)-K(+)-ATPase activity. In tubules incubated with DA (10(-8) to 10(-9) M), the presence of DBcAMP (10(-6) M) enhanced the sensitivity by which DA inhibited Na(+)-K(+)-ATPase activity. We conclude that PCT Na(+)-K(+)-ATPase activity is inhibited by a synergistic action of the DA1 and DA2 receptors, with the DA1 receptor acting to increase cell cAMP levels.

Effects of methotrexate on astrocytes in primary culture: light and electron microscopic studies

Recent studies on the animal model suggest that astrocytes may be a primary target for methotrexate (MTX) toxicity. To establish whether the astroglial alterations are due to a direct toxic effect of the drug, we studied the morphologic alterations, mitotic index, viability and growth rate of astrocytes in primary culture after exposure to varying concentrations of MTX in the absence or presence of dibutyryl cyclic AMP (dBcAMP). Dense bodies and cellular debris were noted by light and electron microscopy, and became more prominent with increasing doses and greater frequency of treatment. Degenerating cells and areas of necrosis were seen at higher concentrations. These changes became less conspicuous when MTX was given concurrently with dBcAMP. Large reactive-like astrocytes were also seen after MTX administration both in the absence or presence of dBcAMP. Mitotic rate inhibition was noted at all concentrations but was not dose-related. Cell viability was reduced and remained low up to 48 h after withdrawal of MTX and correlated well with drug concentration, although growth rate did not vary significantly from the control. Our findings show that pure populations of astrocytes can be adversely affected by MTX especially in the absence of bBcAMP, while also causing reactive-like changes in some cells. This report provides further evidence that astrocytes may be a primary target for MTX toxicity and suggests that the gliosis seen in MTX encephalopathy may in part be related to MTX-induced astrocytic injury.

Calcium signalling

In a previous study we observed that parotid glands from rats with experimental periodontitis showed an increase in basal amylase release as a result of an increase in cAMP accumulation induced by PGE2 production. The aim of this work was to study whether this change in amylase release influences the secretory effect of carbachol.Experimental periodontitis was induced through placing a black thread around the cervix of the two lower first molars. Experiments were done 22 days after ligature induced periodontitis. Amylase release was evaluated in vitro and determined using a colorimetric method which uses starch as substrate.The effect of carbachol was increased in parotid glands from periodontitis rats. The effect of 10−6 M carbachol was inhibited by 4-DAMP (10−6 M), U-73122 (5 × 10−6 M) and trifluoperazine (5 × 10−6 M) in both groups. No changes were observed in the binding sites and affinity in parotid membranes from rats with experimental periodontitis. The inhibition of the adenylyl cyclase and the cyclooxygenase induced a right shift of the carbachol concentration–response curve in periodontitis group whilst the opposite effect was observed in control group in the presence of db-cAMP and PGE2.Parotid glands from rats with experimental periodontitis release more amylase in response to carbachol suggesting an interaction between Ca2+ and cAMP in the fusion/exocytosis step of secretory vesicles.

Modulation of levels of messenger RNA for tissue-type plasminogen activator in rat Sertoli cells, and levels of messenger RNA for plasminogen activator inhibitor in testis peritubular cells

Messenger RNA for tissue-type plasminogen activator has been detected in RNA extracts from rat Sertoli cells in culture. Relative levels are increased in Sertoli cells stimulated by follicle-stimulating hormone or by dibutyryl cyclic AMP (dbcAMP) and decreased in cells maintained in the presence of transforming growth factor beta, type l (TGF-β 1). Messenger RNA for plasminogen activator inhibitor, type 1 (PAI-1) has been detected in RNA extracts from rat peritubular myoid cells. Relative levels are increased in peritubular cells stimulated by TGF-β 1, and decreased by the presence of dbcAMP in the medium. Data are interpreted to indicate that net protease activities in the seminiferous tubule are regulated at transcriptional levels by endocrine and paracrine agents.

Dibutyryl Cyclic AMP Induces Vimentin and GFAP Expression in Cultured Medulloblastoma Cells

Evidence for the astrocytic lineage in medulloblastomas rests largely on the detection of the glial fibrillary acidic protein (GFAP) from which intermediate filaments (IF) specific for astrocytes are assembled. Astrocyte progenitor cells from the mouse neopallium however express another IF protein, vimentin, before they acquire GFAP in vivo and in vitro. The purpose of the current study was to determine if cells obtained from a focally GFAP-positive posterior fossa medulloblastoma previously shown to acquire GFAP in response to dibutyryl cyclic AMP (dBcAMP), also express vimentin before expressing GFAP. More than 80% of cells in the tumor section contained vimentin while fewer than 1% of cells contained GFAP; occasional clusters of malignant GFAP-positive cells and clusters of cells negative for both vimentin and GFAP were also identified in the tumor. One hundred per cent of cultured cells in the first 10 passages from the tumor contained vimentin and no cells expressed GFAP. When cells were cultured in the presence of dBcAMP. Western immunoblotting showed an increase in vimentin which reached maximal values within 24 h followed by an increase in GFAP which reached maximal values at 72 h. The increase in vimentin followed by that of GFAP in cultured medulloblastoma cells has not previously been reported and suggests that most astrocyte progenitors which are derived from medulloblastoma and cultured in vitro may be at a developmental stage which corresponds to the proastroblast stage in the developing mouse brain.(ABSTRACT TRUNCATED AT 250 WORDS)

Contribution Of Stored Rat Growth Hormone To Restoration Of Depleted Rat Pituitary Immediate Release Pools

Stored rat pituitary growth hormone (GH) is functionally divided into immediately releasable and more stable compartments. These observations are consistent with either intracellular hormone compartmentalization within cells of a functionally homogeneous somatotroph population or summed responses from a heterogeneous population of functionally specialized cell subgroups. We investigated the pituitary's ability to recruit stored rGH to replenish depleted immediate release pools. We used perifused pituitary fragments whose stored rGH was labeled during pre-incubation in the presence of [3H]leucine. Initial immediate release pool depletion was accomplished by continuous exposure to combined 21 mM potassium ion (K+) and 1 mM dibutyryl cyclic AMP (dbcAMP). During a subsequent perifusion period in the presence of either no secretagogue, continuing 21 mM K+, or continuing 1 mM dbcAMP, we examined release of stored [3H]rGH in response to repetitive 15 min pulses of 21 mM K+ or 1 mM dbcAMP. Analysis was by specific immunoprecipitation. We had demonstrated that K+ and dbcAMP pulses can stimulate repetitive, albeit diminishing, stored [3H]rGH release responses. However, following pre-treatment with combined 21 mM K+ and 1 mM dbcAMP: (i) pulses of dbcAMP stimulated almost no stored [3H]rGH release in the presence of 21 mM K+ or in the absence of a background secretagogue; and (ii) stored [3H]rGH release in response to pulses of K+ was attenuated in the absence of background secretagogue but fully restored in the presence of dbcAMP. (i) at least some individual somatotrophs can compartmentalize stored hormone; and (ii) an active transport system facilitates restoration of somatotroph immediate release pools using stored rGH.

Reversible cAMP-induced translocation of cytoskeleton-associated 300- to 350-kDa proteins from nucleus to cytoplasm

We previously reported that treatment of SV-3Y1 cells in an exponential growth state with 1 m M db-cAMP plus 1 m M theophylline induced reversible disappearance of nuclear dots stained by monoclonal anti-microtubule-associated protein (MAP)-1 antibody [T. Nakayama, K. Nishizawa, G. Kimura, and C. Sato (1986) Exp. Cell Res. 163, 246]. In the present study, we examined the relation between the intracellular localization and phosphorylation of 300- to 350-kDa proteins that are intracellular antigens for our anti-MAP-1 and -2 antibodies. Treatment with 1 m M db-cAMP plus 1 m M theophylline was found to result in a reversible decrease in immunofluorescent staining of the nucleus with polyclonal MAP-1 or -2 antibody, and a reversible increase in that of the cytoplasm. Simultaneous treatment with 2.5 μ M colchicine, 2.5 μ M colcemid, 20 μ M putrescine, or 3 m M α-naphthyl phosphate in the presence of db-cAMP plus theophylline almost prevented this effect of db-cAMP plus theophylline. We examined the cytoplasmic and nuclear fractions by immunoperoxidase staining, immunoprecipitation, and 125I-protein A with anti-MAP-1 and -2 antibodies. Treatment with db-cAMP plus theophylline resulted in the increase of 300- to 350-kDa proteins in the cytoplasm and a decrease in the nucleus. This treatment also caused the dephosphorylation of 300- to 350-kDa proteins. The present research indicated that treatment with db-cAMP plus theophylline resulted in the reversible translocation of 300- to 350-kDa proteins from the nucleus to the cytoplasm accompanied by the dephosphorylation of these proteins.

Enhancement of cis-diamminedichloroplatinum and mitomycin C-induced sister chromatid exchanges by dibutyryl cyclic AMP in a human salivary gland adenocarcinoma cell line in culture.

A human salivary gland adenocarcinoma cell line was cultivated in the presence of dibutyryl cyclic AMP (dB-cAMP), cis-diammine dichloroplatinum (cisplatin) or mitomycin C (MMC) only, or of the combination of dB-cAMP and each of the antineoplastic drugs. Then the treated cells were examined for the induction of sister chromatid exchanges (SCEs), colony-forming efficiency (CFE) in semisolid agar medium and cell survival as measured by counting for viable cells. It has been found that the frequency of SCE induction is significantly increased by treatment of cells with cisplatin or MMC under the presence of dB-cAMP, when compared to that in the cells treated with cisplatin or MMC only. Moreover, marked reduction in CFE and cell survival was found to occur in the cells treated with the combination of dB-cAMP and cisplatin or MMC. These finding indicate that treatment of a human salivary gland adenocarcinoma cell line with dB-cAMP results in modification of SCE induction, CFE and cell survival in the cells exposed to cisplatin or MMC.

Effect of polylysine-bound laminin on human retinoblastoma cell lines.

We have studied the responses of three human retinoblastoma cell lines (GM1232, Y79, and WERI-Rb1) to substratum-bound laminin (LN), fibronectin (FN), or collagen (CN) in serum-containing medium. About 95% of the cells attached to poly-D-lysine (PN)-pretreated plastic surfaces either unbound or protein-bound within 1 h after plating. With PN-bound LN, GM1232 cells showed an outgrowth of processes and cell spread within 1 d, but very little was seen on PN-bound FN, CN, or unbound PN even by Day 4. Both the percentage of cells with processes and the number of processes/cell were dependent on the amount of LN bound to the surfaces at Day 4. On LN surfaces without PN pretreatment, by Day 2 most cells had formed floating aggregates and were not attached to the surfaces. By Day 4 only a part of the peripheral cells of attached aggregates displayed process outgrowth and cell spreading. Dibutyryl cyclic AMP (dbcAMP) maintained these effects of PN-bound LN, and promoted process branching, cell spreading, and elongation with concomitant inhibition of cell growth. The percentage of cells with processes was 83%. Y79 and WERI-Rb1 cells, passed for several years, showed little and weak response to PN-bound LN either in the absence or presence of dbcAMP. These results indicate that the morphologic differentiation of GM1232 cells is elicited specifically by PN-bound LN, and that dbcAMP maintains and promotes this differentiated status.

Cyclic AMP functions as a primary sexual signal in gametes of Chlamydomonas reinhardtii.

When Chlamydomonas reinhardtii gametes of opposite mating type are mixed together, they adhere by a flagella-mediated agglutination that triggers three rapid mating responses: flagellar tip activation, cell wall loss, and mating structure activation accompanied by actin polymerization. Here we show that a transient 10-fold elevation of intracellular cAMP levels is also triggered by sexual agglutination. We further show that gametes of a single mating type can be induced to undergo all three mating responses when presented with exogenous dibutyryl-cAMP (db-cAMP). These events are also induced by cyclic nucleotide phosphodiesterase inhibitors, which elevate endogenous cAMP levels and act synergistically with db-cAMP. Non-agglutinating mutants of opposite mating type will fuse efficiently in the presence of db-cAMP. No activation of mating events is induced by calcium plus ionophores, 8-bromo-cGMP, dibutyryl-cGMP, nigericin at alkaline pH, phorbol esters, or forskolin. H-8, an inhibitor of cyclic nucleotide-dependent protein kinase, inhibits mating events in agglutinating cells and antagonizes the effects of cAMP on non-agglutinating cells. Adenylate cyclase activity was detected in both the gamete cell body and flagella, with the highest specific activity displayed in flagellar membrane fractions. The flagellar membrane adenylate cyclase is preferentially stimulated by Mn++, unresponsive to NaF, GTP, GTP gamma S, AlF4-, and forskolin, and is inhibited by trifluoperazine. Cyclic nucleotide phosphodiesterase activity is also present in flagella. Our observations indicate that cAMP is a sufficient initial signal for all of the known mating reaction events in C. reinhardtii, and suggest that the flagellar cyclase and/or phosphodiesterase may be important loci of control for the agglutination-stimulated production of this signal.

Morphogenetic restructuring and formation of basement membranes by Sertoli cells and testis peritubular cells in co-culture: Inhibition of the morphogenetic cascade by cyclic AMP derivatives and by blocking direct cell contact

Addition of dibutyryl cyclic AMP (dbcAMP), methylisobutylxanthine (MIX), or cytochalasin D to co-cultures of Sertoli cells and testicular peritubular myoid cells blocks a series of morphogenetic changes which otherwise occur during culture. When Sertoli cells are plated directly onto preexisting layers of peritubular cells maintained under basal conditions, structures form which display many of the characteristics of germ cell-depleted seminiferous tubules. The presence of dbcAMP, MIX, or cytochalasin D, added at varying times after plating Sertoli cells, results in the inhibition of each successive stage of in vitro remodeling: the inhibition of migration of Sertoli cells, the inhibition of initial ridge formation, the blockage of subsequent formation of mounds and nodules of compacted Sertoli cell aggregates, the prevention of the formation of basal lamina and associated layers of extracellular matrix between Sertoli cell aggregates and surrounding peritubular cells, and the inhibition of tubule formation. The presence of dbcAMP also inhibits the migration of peritubular cells, contractions by these cells, and compaction of Sertoli cell aggregates. When intimate cell apposition is prevented by plating the two cell types on either side of a membrane filter, the morphogenetic cascade is blocked, and no formation of a germ cell-depleted seminiferous tubule-like structure occurs. Other effects of dbcAMP on cell shape, cell movement, and cell association patterns during co-culture are described. Possible mechanisms by which dbcAMP, MIX, or cytochalasin D blocks restructuring are discussed. Since each elicits perturbations of the cytoskeleton, we offer the interpretation that cytoskeletal changes may be correlated with the prevention of closely apposing cell compact and the inhibition of basement membrane formation. Interactions observed between Sertoli cells and peritubular cells during co-culture are postulated to be analogous to those occurring in other types of mesenchymal cell-epithelial cell interactions during organogenesis and during tubulogenesis in the fetal testis. Speculatively, the blockage by dbcAMP of the morphogenetic cascade in the co-cultured system may be related to the inhibition by dbcAMP of testis cord formation in organ cultures of fetal gonads reported by others.

Involvement of cAMP-dependent protein kinase and protein phosphorylation in regulation of mouse oocyte maturation

We report the results of experiments which support the hypothesis that, in mouse oocytes, a decrease in intraoocyte cyclic AMP (cAMP) initiates meiotic maturation; oocytes microinjected with cyclic nucleotide phosphodiesterase (PDE) underwent germinal vesicle breakdown (GVBD) in the presence of 3-isobutyl-1-methylxanthine (IBMX), which inhibited GVBD both in oocytes not injected with PDE and in oocytes injected with heat-inactivated PDE. Cyclic AMP-dependent protein kinase (PK) has been proposed to mediate maintenance of meiotic arrest by cAMP. In support of this hypothesis is the observation that 2′-deoxy cAMP, which does not activate PK, did not maintain meiotic arrest as did cAMP; this result was obtained both by microinjection of these compounds and by incubating oocytes in the presence of their membrane-permeable N 6-monobutyryl derivatives. Furthermore, microinjection into oocytes of the heat-stable inhibitor of PK, PKI, induced GVBD in the presence of either dibutyryl cAMP (dbcAMP) or IBMX. Meiotic arrest was maintained in the absence of dbcAMP or IBMX, however, by microinjected catalytic subunit of PK, but not by catalytic subunit coinjected with PKI. In addition, specific changes in oocyte phosphoproteins that preceded resumption of meiosis were induced, in the presence of dbcAMP, by microinjected PKI; these changes were also tightly coupled with commitment of oocytes to resume meiosis. These results are discussed in terms of our model for regulation of meiotic arrest and maturation.

Differentiation of a cell line from olfactory epithelium is induced by dibutyryladenosine 3′, 5′-cyclic monophosphate and12-O-tetradecanoylphorbol-13-acetate

Olfactory receptor neurons undergo continual replacement with new cells differentiating from a population of basal cells located in the olfactory epithelium. We have previously described the isolation of a cell line from murine olfactory epithelium (MOE CL1) which is now shown to be sensitive to two differentiation promotion agents, dibutyryladenosine 3′, 5′-cyclic monophosphate (db-cAMP) and12-O-tetradecanoylphorbol-13-acetate(TPA). Cells grown in the presence of db-cAMP or TPA, after a lag phase of 72–96 h, became bipolar, with long neurite-like processes. Cells conditioned for at least one passage in db-cAMP or TPA, or cells exposed to a mixture of db-cAMP and TPA, attained a bipolar morphology within 24 h. Cultures grown with db-cAMP, TPA or db-cAMP + TPA showed significant increases in the percentage of process-bearing cells. An increase in population doubling time and decrease in saturation density were also observed in cultures exposed to db-cAMP. Cells exposed to TPA continued to divide at the same rate as control cells, while those treated with db-cAMP + TPA underwent terminal differentiation. Peripheral-type benzodiazepine binding sites, which within the olfactory epithelium are localized on the receptor neurons, were present in homogenate membranes of undifferentiated cells. Cells differentiated by exposure to db-cAMP + TPA for 7 days showed a 65% increase in the density (B max) of peripheral-type benzodiazepine binding sites. Undifferentiated cells in control cultures showed no spontaneous changes in membrane potential or regenerative responses during current injection. Small, bipolar cells in morphologically differentiated cultures, however, were capable of generating several types of spontaneous changes in membrane potential including slow, regular oscillations; irregular, slow and fast depolarizations; and trains of action potentials.

Effect of dibutyryl cyclic AMP on morphologic features and biologic markers of a human salivary gland adenocarcinoma cell line in culture.

Dibutyryl cyclic AMP (dB-cAMP) has marked effects on the growth, morphologic features, and biologic markers of a human salivary gland adenocarcinoma cell line in culture. A cell line with ultrastructure and biologic markers specific to the intercalated duct cells of human salivary glands was cultivated in the presence of dB-cAMP. Major alterations, such as process formation and expression of myofilaments and oxytocin receptor in addition to myosin and S-100 protein, were observed in those cells with a phenotype similar to myoepithelial cells. Both the anchorage-independent and anchorage-dependent growths were markedly suppressed in the presence of dB-cAMP. After the removal of dB-cAMP from the culture, the treated cells returned rapidly to the phenotype of the untreated cells. These findings indicate that reversible differentiation into the myoepithelial cells of a human salivary gland adenocarcinoma cell line occurs in growth medium containing dB-cAMP.

Inhibition by dibutyryl cyclic AMP of the transition to metaphase of mouse oocyte nuclei and its reversal by cell fusion to metaphase oocytes

Mouse oocytes at metaphase I of meiotic maturation were treated with puromycin, which caused the condensed chromosomes to become decondensed to form an interphase nucleus. The chromosomes returned to a metaphase state 6.3 hr after the oocytes were transferred to puromycin-free medium [H. J. Clarke and Y. Masui (1983) Dev. Biol. 97, 291–301]. In contrast, the chromosomes of the puromycin-treated oocytes remained decondensed within the nucleus if dibutyryl cyclic AMP (dbcAMP) was included in the puromycin-free medium. This implies that dbcAMP inhibited the development of conditions in the oocytes that were required for the transition to metaphase. The chromosomes of puromycin-treated oocytes that were incubated for 7.5 hr in dbcAMP-containing medium returned to metaphase just 1.9 hr after transfer to dbcAMP-free medium. Therefore, the protein synthesis-dependent process that is required for the transition to metaphase could occur in the presence of dbcAMP. Fusion to metaphase II oocytes, or to puromycin-treated oocytes that had returned to metaphase, rapidly induced transition of the nuclei of dbcAMP-inhibited oocytes to metaphase, despite the presence of the inhibitor. These results suggest that the transition of nuclei to metaphase can be induced by a cytoplasmic factor that is present in metaphase oocytes, and that dbcAMP inhibits the development of this factor.

Inhibition of cell communication between Balb/c 3T3 cells by tumor promoters and protection by cAMP.

The effect of phorbol ester tumor promoters on the communication between individual cells in confluent culture was studied using a fluorescent dye transfer method. Cell-cell communication between mouse Balb/c 3T3 cells and between Chinese hamster V79 cells was inhibited almost completely by tumor-promoting phorbol esters, but not by nonpromoting derivatives; the effect was reversed upon removal of the promoter. Intercellular communication between Balb/c 3T3 cells, but not Chinese hamster V79 cells, was increased significantly in the presence of dbcAMP and caffeine, and these compounds counteracted the effects of tumor promoters. Inhibition of cell communication by phorbol esters appears to be receptor-mediated, since specific binding of 3H-phorbol-12,13-dibutyrate to Balb/c 3T3 cells was inhibited only by compounds that also inhibit intercellular dye transfer. A study with cycloheximide suggests that the reversible inhibition of intercellular communication by phorbol esters may not need de novo protein synthesis, while upregulation of communication by cAMP requires protein synthesis.

Insulin receptors in fetal rabbit lung type II cells.

Insulin binding was studied in type II pneumocytes isolated from fetal rabbit lungs (27 days of gestation) and grown in monolayers in tissue culture. The mean high affinity receptor site number was 11.8 +/- 1.4 X 10(3) (+/-SEM)/cell, with a Kd of 0.45 +/- 0.07 nM (n = 6). Low affinity sites averaged 432 +/- 10.7 X 10(3)/cell, with a Kd of 45.6 +/- 11.8 nM. Incubation of the cells with 5 X 10(-10) M (Bu)2cAMP (DBcAMP) and 10(-3) M methylisobutylxanthine (MIX) for 18 h led to significant increases in the number of high affinity receptor sites and Kd (P = 0.025 and 0.05, respectively). Incubation of the cells with insulin (1 microgram/ml) for 18 h led to a significant diminution in the mean number of high affinity sites to 3.23 +/- 0.68 X 10(3)/cell (P = 0.0025). There was no significant change in the Kd of the high affinity sites. There was also no significant change in the number or affinity of the low affinity sites. When the cells were incubated with insulin in the presence of DBcAMP (5 X 10(-4) M) and MIX (10(-3) M), there was a significant increase in high affinity binding sites to a mean of 8.87 +/- 2.18 X 10(3)/cell (n = 4) compared to the value after incubation in the presence of insulin alone. There was no significant increase in the Kd of the high affinity sites. The following conclusions were drawn from these experiments. 1) Fetal type II pneumocytes possess receptors with high affinity for insulin. 2) The up-regulation of insulin receptor binding induced by high ambient concentrations of insulin in vivo in rabbit fetal lungs and circulating human monocytes does not occur in vitro when isolated pneumocytes are grown in tissue culture. 3) Insulin binding to type II pneumocytes is enhanced by DBcAMP and MIX. 4) Insulin down-regulation of receptor binding is significantly counteracted by DBcAMP and MIX.

Differential effects of various cAMP derivatives on the morphological and electrical maturation of neuroblastoma X glioma hybrid cells

The influence of adenosine 3′: 5′-cyclic monophosphate (cAMP) and some of its derivatives on the morphological differentiation and on the expression of electrical activity was investigated in neuroblastoma X glioma hybrid cells. This permanent cell line constitutes a well established culture system for studying neuronal properties in vitro. cAMP (1 mM) caused cell death. With 8-bromo-cAMP (0.1–1 mM) present giant multinuclear cells appeared, which were more obvious at 0.1 than at 1 mM 8-bromo-cAMP. 8- p-chlorophenylthio-cAMP (0.1–1 mM) induced an extension of neurites. These cellular processes were comparable to those elicited in the presence of db-cAMP (1 mM). However, only the cells treated with db-cAMP, but not those exposed to 8- p-chlorophenylthio-cAMP, were found to generate action potentials upon electrical stimulation. Neither dexamethasone nor carboxymethylcellulose, nor 8-bromo-cAMP could elicit the formation of processes in hybrid cells.

Mutants of Mucor racemosus insensitive to dibutyryl cAMP-mediated changes in cellular morphology

Wild-type Mucor racemosus yeasts grown under CO 2 treated with dibutyryl cyclic adenosine 3′,5′-monophosphate (dbcAMP) and glucose do not convert to hyphae during a CO 2-to-air shift. By using procedures which select for hyphal cells, we have isolated a class of mutants of M. racemosus which do convert to hyphae from CO 2 yeast in the presence of dbcAMP and glucose but otherwise respond like wild-type cells to dbcAMP. One of these mutants (MLT-3) has been analyzed and appears to be wild type with respect to two other dbcAMP effects: (1) dbcAMP prevented the appearance of an NAD-dependent glutamate dehydrogenase during a CO 2-to-air shift; (2) dbcAMP inhibited sporangiospore germination. It is probable that the altered gene product in not involved in cAMP metabolism nor does it alter permeability of this organism to dbcAMP, but rather represents an alteration of one of the systems under cAMP control.