Messenger Analogs Research Articles

Differential Ras-dependence of gene induction by nerve growth factor and second messenger analogs in PC12 cells.

Induction of neurite formation by nerve growth factor (NGF) in PC12 pheochromocytoma cells can be efficiently inhibited by expressing a dominant negative mutant form of the small guanine nucleotide binding Ha-Ras protein in these cells. The block in NGF-induced neuritogenesis caused by inhibition of endogenous Ras proteins was found to be partially relieved by simultaneous stimulation of cAMP- or Ca++-dependent signaling pathways. Since expression of certain genes is believed to be involved in NGF-signaling leading to morphological differentiation, we decided to study the combined effects of NGF and second messenger analogs on gene expression in PC12 cell lines expressing different levels of the interfering Ras protein. We found NGF-second messenger combinations that induced normal c-fos, zif268 and nur77 early-response gene expression without neuritogenesis, and, conversely, cell lines in which certain combination treatments caused partial neuronal differentiation in the absence of substantial activation of these genes. Similarly, neurite outgrowth induced by combination treatments does not seem to require the activation of the late-response transin gene. Our results thus suggest a lack of strong correlation between NGF-stimulated early- and secondary-response gene induction and morphological differentiation.

Dynamics of salivary secretion studied by confocal laser and scanning electron microscopy.

Plasma membrane events during secretion of the parotid and submandibular glands of humans and rats were observed in living cells by confocal microscopy and from the cytoplasmic side by scanning electron microscopy after removal of the cell organelles by the OsO4 maceration method. These new microscopic techniques revealed in acinar cells two distinct exocytosis-endocytosis coupling mechanisms elicited in response to different secretory stimuli. Beta-adrenergic stimulation with isoproterenol and its second messenger analog dibutyryl cyclic AMP evoked exocytosis after which fused granule membranes were individually removed from the luminal plasma membrane within several minutes. On the fused membrane area, small endocytotic vesicles about 100-150 nm in diameter were abundant. Muscarinic stimulation with carbachol also induced exocytosis, but in this case the fused granule membranes coalesced to form enlarged invaginations which stayed on the luminal membrane for more than 30 min. These invaginations, almost devoid of small endocytotic vesicles, were then pinched off from the luminal membrane and dispersed into the cytoplasm in the form of light microscopically detectable vesicles. After treatment with isoproterenol and carbachol, acinar cells exhibited different distributional changes of F-actin around the fused membrane area, suggesting the involvement of microfilaments in regulating the membrane events following exocytosis.

Prostanoid-induced expression of matrix metalloproteinase-1 messenger ribonucleic acid in rat osteosarcoma cells.

Individual prostanoids have distinct potencies in activating intracellular signaling pathways and regulating gene expression in osteoblastic cells. The E-series prostaglandins (PGs) are known to stimulate matrix metalloproteinase-1 (MMP-1) synthesis and secretion in certain rodent and human osteoblastic cells, yet the intracellular events involved remain unclear. To further characterize this response and its signal transduction pathway(s), we examined prostanoid-induced expression of the MMP-1 gene in the rat osteoblastic osteosarcoma cell line UMR 106-01. Northern blot analysis demonstrated that prostaglandin E2 (PGE2) and PGE1 were very potent stimulators (40-fold) of MMP-1 transcript abundance, PGF2 alpha and prostacyclin were weak stimulators (4-fold), and thromboxane-B2 had no effect. The marked increase in MMP-1 transcript abundance after PGE2 treatment was first detected at 2 h, became maximal at 4 h, and persisted beyond 24 h. This response was dose dependent and elicited maximal and half-maximal effects with concentrations of 10(-6) and 0.6 x 10(-7) M, respectively. Cycloheximide, a protein synthesis inhibitor, completely blocked this effect of PGE2, suggesting that the expression of other genes is required. Nuclear run-on experiments demonstrated that PGE2 rapidly activates MMP-1 gene transcription, with a maximal increase at 2-4 h. The second messenger analog, 8-bromo-cAMP, mimicked the effects of PGE2 by stimulating a dose-dependent increase in MMP-1 messenger RNA (mRNA) levels, with a maximal effect quantitatively similar to that observed with PGE2. Thus, in UMR 106-01 cells, different prostanoids have distinct potencies in stimulating MMP-1 mRNA abundance. Our data suggest that PGE2 stimulation of MMP-1 synthesis is due to activation of MMP-1 gene transcription and a subsequent marked increase in MMP-1 mRNA abundance. This effect is dependent on de novo protein synthesis and is mimicked by protein kinase-A activation.

Endocrine and Neurogenic Regulation of the Orphan Nuclear Receptors Nur77 and Nurr-1 in the Adrenal Glands

nurr77 and nurr-1 are growth factor-inducible members of the steroid/thyroid hormone receptor gene superfamily. In order to gain insight into the potential roles of nur77 in the living organism, we used pharmacologic treatments to examine the expression of nur77 in the mouse adrenal gland. We found that nur77 and nurr-1 are induced in the adrenal gland upon treatment with pentylene tetrazole (Ptz; Metrazole). This induction is separable into distinct endocrine and neurogenic mechanisms. In situ hybridization analysis demonstrates that nur77 expression upon Ptz treatment in the adrenal cortex is localized primarily to the inner cortical region, the zona fasciculata-reticularis, with minimal induction in the zona glomerulosa. This induction is inhibitable by pretreatment with dexamethasone, indicating involvement of the hypothalamic-pituitary-adrenal axis in the activation of adrenal cortical expression. When mice were injected with adrenocorticotrophic hormone (ACTH), nur77 expression in the adrenal gland spanned all cortical layers including the zona glomerulosa, but medullary expression was not induced. Ptz also induces expression of both nur77 and nurr-1 in the adrenal medulla. Medullary induction is likely to have a neurogenic origin, as nur77 expression was not inhibitable by dexamethasone pretreatment and induction was seen after treatment with the cholinergic neurotransmitter nicotine. nur77 is also inducible by ACTH, forskolin, and the second messenger analog dibutyryl cyclic AMP in the ACTH-responsive adrenal cortical cell line Y-1. Significantly, Nur77 isolated from ACTH-stimulated Y-1 cells bound to its response element whereas Nur77 present in unstimulated cells did not. Moreover, Nur77 in ACTH-treated Y-1 cells was hypophosphorylated at serine 354 compared with that in untreated cells. These results, taken together with the previous observation that dephosphorylation of serine 354 affects DNA binding affinity in vitro, show for the first time that phosphorylation of Nur77 at serine 354 is under hormonal regulation, modulating its DNA binding affinity. Thus, ACTH regulates Nur77 in two ways: activation of its gene and posttranslational modification. A promoter analysis of nur77 induction in Y-1 cells indicates that the regulatory elements mediating ACTH induction differ from those required for induction in the adrenal medullary tumor cell line PC12 and in 3T3 fibroblasts.

Endocrine and neurogenic regulation of the orphan nuclear receptors Nur77 and Nurr-1 in the adrenal glands.

nurr77 and nurr-1 are growth factor-inducible members of the steroid/thyroid hormone receptor gene superfamily. In order to gain insight into the potential roles of nur77 in the living organism, we used pharmacologic treatments to examine the expression of nur77 in the mouse adrenal gland. We found that nur77 and nurr-1 are induced in the adrenal gland upon treatment with pentylene tetrazole (Ptz; Metrazole). This induction is separable into distinct endocrine and neurogenic mechanisms. In situ hybridization analysis demonstrates that nur77 expression upon Ptz treatment in the adrenal cortex is localized primarily to the inner cortical region, the zona fasciculata-reticularis, with minimal induction in the zona glomerulosa. This induction is inhibitable by pretreatment with dexamethasone, indicating involvement of the hypothalamic-pituitary-adrenal axis in the activation of adrenal cortical expression. When mice were injected with adrenocorticotrophic hormone (ACTH), nur77 expression in the adrenal gland spanned all cortical layers including the zona glomerulosa, but medullary expression was not induced. Ptz also induces expression of both nur77 and nurr-1 in the adrenal medulla. Medullary induction is likely to have a neurogenic origin, as nur77 expression was not inhibitable by dexamethasone pretreatment and induction was seen after treatment with the cholinergic neurotransmitter nicotine. nur77 is also inducible by ACTH, forskolin, and the second messenger analog dibutyryl cyclic AMP in the ACTH-responsive adrenal cortical cell line Y-1. Significantly, Nur77 isolated from ACTH-stimulated Y-1 cells bound to its response element whereas Nur77 present in unstimulated cells did not. Moreover, Nur77 in ACTH-treated Y-1 cells was hypophosphorylated at serine 354 compared with that in untreated cells. These results, taken together with the previous observation that dephosphorylation of serine 354 affects DNA binding affinity in vitro, show for the first time that phosphorylation of Nur77 at serine 354 is under hormonal regulation, modulating its DNA binding affinity. Thus, ACTH regulates Nur77 in two ways: activation of its gene and posttranslational modification. A promoter analysis of nur77 induction in Y-1 cells indicates that the regulatory elements mediating ACTH induction differ from those required for induction in the adrenal medullary tumor cell line PC12 and in 3T3 fibroblasts.

Structure and conformation of the cyclic phosphate of Ganciclovir, a broad-spectrum antiviral agent

The title compound, the cyclic phosphate of the antiviral acyclonucleoside Ganciclovir (2′-NDG, DHPG), is itself a potent broad-spectrum antiviral agent, but with a different mechanism of action. The cyclic phosphate, 9-[[[(2-hydroxyl-1,3,2-dioxophosphorinan-5-yl)oxy] methyl]-P-oxide]guanine (2′-nor-cGMP, DHPG-cMP), crystallizes in the monoclinic space group P2 1/n with until cell dimensions a = 6.612(1) A ̊ , b = 11.562(4) A ̊ , c = 19.231(5) A ̊ and β = 91.786(2)° at −165° C . The N7 of the guanine base is protonated, so that the molecule is in a zwitterionic form, with two water molecules in the asymmetric unit. The principal conformational features of DHPG-cMP in the crystal are as follows: the acyclic chain is partially folded; the six-membered cyclic phosphate ring is in a chair form with C3′, O3′, C5′ and O5′ in a plane; P and C4′ are displaced in diametrically opposite directions from this plane; the O4′ is in the axial orientation with respect to this ring; and the aglycon is in the high syn conformation about the glycosidic bond. The conformation of the cyclic phosphate ring in aqueous medium, determined by means of 1H-NMR spectroscopy, is similar to that in the crystalline form. The conformational features of DHPG-cMP were compared with those of the parent DHPG and other related compounds and, in particular, with those of the second messenger 3′:5′-cGMP, of which it is a close structural analogue. Previously reported substrate/inhibitor properties of these compounds in several enzyme systems are examined in relation to the possible mechanism of antiviral activity of DHPG-cMP as a second messenger analogue of cGMP.

Active calcium absorption in primary cultures of cortical collecting duct cells

Primary cultures of rabbit cortical collecting duct (CCD) cells demonstrated accumulation of Ca at the basolateral (BL) side when cultured on either impermeable or permeable supports. Cell monolayers cultured on impermeable plastic surfaces absorbed Ca with such avidity that hydroxyapatite crystals formed. When cultured on a permeable difference. A steady-state BL/A [Ca] ratio of 120 developed across monolayers in 24 h on days 6 through 8 postseeding. Initial rates of unidirectional 45Ca fluxes on days 6 through 8 indicated a negligible BL to A flux (5.4 +/- 2.6 nmol.h-1 x cm-2) compared with A to BL 45Ca flux (99.4 +/- 19.4 nmol.h-1 x cm-2). Parathyroid hormone applied to the BL side had no significant effect on either unidirectional 45Ca flux, but the second messenger analog, 8-bromoadenosine cyclic monophosphate, increased the A to BL flux by 65%. Inhibiting the Na(+)-K+ ATPase with ouabain (10(-4) M) reduced the A to BL flux by 77%; however, a significant net A to BL flux still remained. Apical addition of amiloride (2 x 10(-5) M) did not affect either unidirectional 45Ca flux. In addition, the inorganic Ca channel blockers Ni2+ (100 microM and 1 mM), La3+ (100 microM and 1 mM), and Cd2+ (20 and 50 microM) did not significantly inhibit either unidirectional 45Ca flux. These results demonstrate that CCD monolayers actively absorb Ca and this can be stimulated by cyclic AMP, raising the possibility that apical Ca entry does not involve amiloride-sensitive channels, or typical Ca channels.

38-Amino acid form of pituitary adenylate cyclase activating peptide induces process outgrowth in human neuroblastoma cells.

Permanent cell lines from human neuroblastoma, a sympathoadrenal malignancy, are known to exhibit a more neuronal phenotype characterized by outgrowth of long processes in response to multiple second messenger analogs. In this report we demonstrate that the 38-amino acid form of a peptide homologous to vasoactive intestinal peptide (VIP), pituitary adenylate cyclase activating peptide (PACAP), as well as the 27-amino acid form of PACAP, induce NB-OK human neuroblastoma cells to extrude cellular processes within 5 hr of treatment with either peptide at 10(-8) M. Treatment of NB-OK cells with PACAP38 or PACAP27 at 10(-8) M for 1 hr also elevates cAMP content greater than 100-fold and inositol lipid turnover 11- to 12-fold. VIP acutely induces process outgrowth and elevates intracellular second messenger levels in NB-OK cells only at higher concentrations, 10(-6) M or greater. In contrast to the equipotency of PACAP27 and PACAP38 in stimulating the outgrowth of processes observed after 5 hr of treatment, PACAP38 is much more potent than PACAP27 when NB-OK cells are scored for process outgrowth after 72 hr of treatment. Correlating with the extended time course over which morphologic changes are seen with PACAP38, cAMP levels remain elevated for a more prolonged time span during treatment with PACAP38 than PACAP27. After 72 hr of treatment with PACAP38 versus treatment with PACAP27, cAMP levels are elevated 10-fold versus 3-fold, respectively. PACAP38 at 10(-8) M also induces process outgrowth in two additional human neuroblastoma lines tested, SMS-KAN and LA-N-1, whereas PACAP27 and VIP at the same concentration are less effective.(ABSTRACT TRUNCATED AT 250 WORDS)

ChemInform Abstract: Total Synthesis of the Second Messenger Analogue D‐myo‐Inositol 1‐ Phosphorothioate 4,5‐Biphosphate: Optical Resolution of DL‐1‐O‐Allyl‐2, 3,6‐tri‐O‐Benzyl‐myo‐inositol and Fluorescent Labeling of myo‐Inositol 1,4,5‐Triphosphate.

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Parathyroid hormone induces c-fos and c-jun messenger RNA in rat osteoblastic cells.

PTH is a potent regulator of osteoblast gene expression, yet the nuclear events that mediate PTH action are poorly understood. We were interested in identifying immediate early genes which may regulate PTH-altered gene expression in the osteoblast. Therefore, we examined the effects of PTH on c-fos and c-jun gene expression in a rat osteoblastic cell line (UMR 106-01). Under control conditions, c-fos and c-jun mRNAs were present at low basal levels. After PTH treatment, c-fos mRNA abundance dramatically increased, with a maximal and transient response at 30 min. PTH also stimulated an increase in c-jun mRNA, but in a biphasic manner, with maximal levels at 30 min and 2 h. These responses were dose dependent, not altered by cotreatment with the protein synthesis inhibitor cycloheximide, and preceded PTH-induced expression of matrix metallo-proteinase-1 mRNA. Nuclear run-on assays demonstrated an increased rate of c-fos and c-jun transcription after PTH exposure. To determine the signal transduction pathways involved, second messenger analogs were tested for their ability to mimic the effects of PTH. 8-Bromo-cAMP and phorbol 12-myristate 13-acetate (PMA) caused increases in the abundance of c-fos and c-jun transcripts. Ionomycin had no effect on the expression of these genes. Pretreatment of the cells with PMA resulted in a decrease in basal c-jun expression, but did not alter the PTH-mediated increase in c-fos, c-jun, or matrix metalloproteinase-1 mRNAs.(ABSTRACT TRUNCATED AT 250 WORDS)

The 5'-flanking region of the rat luteinizing hormone/chorionic gonadotropin receptor gene confers Leydig cell expression and negative regulation of gene transcription by 3',5'-cyclic adenosine monophosphate.

The LH/CG receptor is a G protein-coupled receptor present on gonadal cells whose levels are modulated by a number of hormones, growth factors, and second messenger analogs. With the recently cloned cDNA for the LH/CG receptor, it has been shown that changes in the levels of the cognate mRNA are involved, at least in part, in the observed changes in receptor density. In order to study the transcriptional regulation of the LH/CG receptor we have isolated a 2-kilobase region of the 5'-flanking region of the rat LH/CG receptor gene and subcloned nucleotide -1 (relative to the translational initiation codon) to -1370 into a luciferase reporter plasmid. We show here that this region of the LH/CG receptor gene is able to enhance luciferase activity in MA-10 cells, a line of Leydig tumor cells that normally express LH/CG receptors, as opposed to human kidney 293 cells, which do not. Furthermore, the addition of 8-bromo-cAMP to MA-10 cells, under conditions known to decrease LH/CG receptor numbers and receptor mRNA levels, decreases the relative luciferase activity to about 26% of control. This decrease in reporter gene activity is severely blunted in a subclone of MA-10 cells with a cAMP-resistant phenotype. Our studies show, for the first time, that sequence(s) present with 1370 base pairs of the translational start site of the rat LH/CG receptor gene are sufficient for conferring expression of this gene in Leydig cells and for the negative modulation of LH/CG receptor gene transcription by high concentrations of cAMP.

Total synthesis of the second messenger analogue D-myo-inositol 1-phosphorothioate 4,5-bisphosphate: optical resolution of DL-1-O-allyl-2,3,6-tri-O-benzyl-myo-inositol and fluorescent labelling of myo-inositol 1,4,5-trisphosphate

Two routes for the synthesis of the myo-inositol 1,4,5-triphosphate analogue myo-inositol 1-phosphorothioate 4,5-bisphosphate have been devised. DL-2,3,6-Tri-O-benzyl-1-O-(cis-prop-1-enyl)-myo-inositol was prepared from DL-1-O-allyl-2,3,6-tri-O-benzyl-myo-inositol and phosphorylated to the protected 4,5-biphosphate. Removal of the propenyl group generated DL-1,2,4-tri-O-benzyl-5,6-bis[bis(2-cyanoethoxy)phospho]-myo-inositol which was thiophosphorylated to give DL-2,3,6-tri-O-benzyl-1-O-[bis(2-cyanoethoxy)thiophospho]-4,5-bis[bis(2-cyanoethoxy)phospho]-myo-inositol. Deblocking afforded racemic myo-inositol 1-phosphorothioate 4,5-bisphosphate, which was reacted with the iodoketone of a fluorescent label to generate a fluorescently labelled inositol 1,4,5-trisphosphate analogue. DL-1-O-Allyl-2,3,6-tri-O-benzyl-myo-inositol was resolved into its enantiomers by means of the crystalline 4,5-biscamphanate ester. 1 D-(+)-1-O-Allyl-2,3,6-tri-O-benzyl-myo-inositol was used to prepare D-myo-inositol 1-phosphorothioate 4,5-bisphosphate in a fashion analogous to the racemic modification via1 D-(+)-2,3,6-tri-O-benzyl-1-O-(cis-prop-1-enyl)-4,5-bis[bis(2-cyanoethoxy)phospho]-myo-inositol.

Total synthesis of myo-inositol-1-phosphate-4,5-pyrophosphate, a novel second messenger analogue, via myo-inositol-1-phosphate-4,5-bisphosphorothioate

The synthesis of the novel analogues of myo-inositol 1,4,5-trisphosphate, myo-inositol 1-phosphate 4,5-bisphosphorothioate and myo-inositol 1-phosphate 4,5-pyrophosphate is reported; the latter was prepared via desulphurisation and intramolecular coupling.

Regulation of steroid hormone biosynthesis. Identification of precursors of a phosphoprotein targeted to the mitochondrion in stimulated rat adrenal cortex cells.

Two short-lived precursor proteins, pp37 and pp32, of the mitochondrial phosphoprotein pp30 (formerly denoted as ib) have been detected in Bt2cAMP-stimulated rat adrenal cortex cells, incubated at 25 degrees C or with 1,10-ortho-phenanthroline at 37 degrees C. Subsequently, these two precursor proteins were also identified in cells incubated at 37 degrees C, where they are present only at low levels due to their short half-life. pp30 is produced in several steroidogenic tissues in response to trophic hormone or second messenger analogue. pp37 and pp32 are also phosphoproteins located in the mitochondrion that are produced in response to cAMP analogue and give rise to proteolytic peptide maps similar to that of pp30. As for pp30, inhibition of cytosolic translation prevents the production of pp37 and pp32. The larger precursor protein pp37 has an apparent molecular mass of 37 kDa, an isoelectric point of approximately 7.1, and a half-life at 37 degrees C of 3-4 min. Pulse-chase studies indicate that this protein is processed into the smaller protein, pp32, which has an apparent molecular mass of 32 kDa, an isoelectric point of approximately 6.4, and a half-life at 37 degrees C of 3-4 min. This latter protein is the immediate precursor of pp30. Since ortho-phenanthroline inhibits the mitochondrial processing protease, while the lower incubation temperature slows both protein import and protease processing, the experimental conditions necessary to detect these proteins are consistent with pp37 being a precursor protein that contains two cleavable presequences and is imported into the mitochondrion. The sequential removal of these sequences produces the mature protein pp30.

Changes in the Shape of Leishmania major Promastigotes in Response to Hexoses, Proline, and Hypo‐osmotic Stress

Leishmania major promastigotes in late-log phase are generally long and slender, and remain so during a 1 h incubation in buffer without exogenous substrate. When glucose, 2-deoxyglucose, fructose, mannose, or proline are added, the cells become shorter and more rounded. The shape change in response to glucose is complete within 20 min and is reversible upon incubating the cells without substrate. Galactose, 3-O-methylglucose, 6-deoxyglucose, sucrose, maltose, ribose, glycerol, alanine, glutamate or aspartate do not cause the shape change. Decreasing the osmolarity of the medium causes a rounding of the cells similar to that observed in the presence of glucose, and increasing the osmolarity inhibits the shape change in response to glucose. Inhibitors of glucose transport and 2nd messenger analogs do not affect the shape change.

Ca2+ release by inositol-trisphosphorothioate in isolated triads of rabbit skeletal muscle

The effectiveness of the nonmetabolizable second messenger analogue DL-myo-inositol 1,4,5-trisphosphorothioate (IPS3) described by Cooke, A. M., R. Gigg, and B. V. L. Potter, (1987b. Jour. Chem. Soc. Chem. Commun. 1525-1526.) was examined in triads purified from rabbit skeletal muscle. A Ca2+ electrode uptake-release assay was used to determine the size and sensitivity of the IPS3-releasable pool of Ca2+ in isolated triads. Uptake was initiated by 1 mM MgATP, pCa 5.8, pH 7.5 Release was initiated when the free Ca2+ had lowered to pCa approximately 7. We found that 5-25 microM myo-inositol 1,4,5-trisphosphate (IP3), and separately IPS3, consistently released 5-20% of the Ca2+ pool actively loaded into triads. Single channel recording was used to determine if ryanodine receptor Ca2+ release channels were affected by IPS3 at the same myoplasmic Ca2+ and IPS3 concentrations. Open probability of ryanodine receptor Ca2+ release channels was monitored in triads fused to bilayers over long periods (200 s) in the absence and following addition of 30 microM IPS3 to the same channel. At myoplasmic pCa approximately 7, IPS3 had no effect in the absence of MgATP (Po = 0.0094 +/- 0.001 in control and Po = 0.01 +/- 0.006 after IPS3) and slightly increased activity in the presence of 1 mM MgATP (Po = 0.024 +/- 0.03 in control and Po = 0.05 +/- 0.03 after IPS3). Equally small effects were observed at higher myoplasmic Ca2+. The onset of channel activation by IPS3 or IP3 was slow, on the time scale 20-60 s. We suggest that in isolated triads of rabbit skeletal muscle, IP3-induced release of stored Ca2+ is probably not mediated by the opening of Ca2+ release channels.

Cyclic AMP-dependent and calcium-dependent signals in parathyroid hormone function

Previous work demonstrated that parathyroid hormone (PTH) activates the Ca 2+/protein kinase C (PKC) system in addition to cAMP production. Therefore, the authors explored the role of cAMP-dependent and Ca 2+-dependent signals in the regulation of osteoblastic growth and bone resorption. In exponentially growing UMR 106-01 osteogenic sarcoma cells, PTH (10 −7 M) inhibited [ 3H] thymidine incorporation by 80%. This effect was reproduced by maximal doss of both dibutyryl-cAMP (dbcAMP) and forskolin. The Ca 2+ ionophore ionomycin (10 −7 M) had no effect, whereas phorbol 12-myristate 13-acetate (PMA) was slightly mitogenic. The antimitogenic action of dbcAMP was dose-dependent, with ED 0.5 at about 3 × 10 −5 M. Ionomycin enhanced this dbcAMP effect at submaximal doses of the cAMP analog. PMA used in combination with both dbcAMP and ionomycin induced further depression of cell proliferation, indicating synergism with cAMP. Both dbcAMP (10 −4 M) and ionomycin (10 −7 M) stimulated 45Ca release from fetal rat limb bones after five days in culture, although the Ca 2+ ionophore was less potent. 1-Oleoyl 2-acetyl-glycerol (2 × 10 −6 M) was ineffective alone, and slightly inhibited the 45Ca release produced by the other second messenger analogs in all combinations. The combination of dbcAMP and ionomycin showed a synergistic effect, and fully reproduced PTH effect. In conclusion, PTH signal transduction for control of cell proliferation and bone resorption is mediated mainly by cAMP. Activation of the Ca 2+/PKC message system is nevertheless necessary to express a full hormonal response in both cell and organ culture systems.

Second Messenger Signaling in the Regulation of Collagenase Production by Osteogenic Sarcoma Cells*

Recent work indicates that PTH can stimulate osteoblastic cells to secrete neutral collagenase, an enzyme thought to be linked to bone matrix turnover. Since recent studies suggest that the calcium/protein kinase-C (PKC) message system is involved in signal transduction stimulated by PTH, we examined the role of these putative second messengers of PTH in the regulation of collagenase production by the osteoblastic tumor cell line UMR 106-01. Immunohistochemical staining of cells exposed to PTH (10(-7) M) revealed that about 20% of the entire population was positive for collagenase, compared to less than 3% staining positively in control untreated cells. Incubation with the cAMP analog 8-bromo-cAMP (8BrcAMP) increased the number of collagenase-staining cells in a dose-dependent manner (ED0.5 = 2.5 x 10(-4) M), but to a lower level than PTH, with the maximal effect producing about 15% positive cells. The calcium ionophore ionomycin (10(-7) M) was ineffective, whereas phorbol 12-myristate 13-acetate (PMA), a PKC activator, increased collagenase-specific staining to about 5%, but only at high concentrations (10(-5) M). Incubation of UMR 106-01 cells with ionomycin and PMA did not change the effect of the latter. When the three agents were used in combination, an additive effect was observed, which fully reproduced that of PTH. Similarly, the amount of collagenase released into the medium by cells stimulated with maximal concentrations of 8BrcAMP (10(-3) M) was only 80% of that induced by maximal doses of PTH (10(-7) M). PMA (10(-5) M) was slightly stimulatory, and ionomycin was ineffective alone, but they were synergistic with submaximal doses of 8BrcAMP (10(-4) M). In agreement with the immunohistochemical results, the full hormonal effect was reproduced when the three second messenger analogs were used in combination. In conclusion, signal transduction from PTH receptor to collagenase production is mediated mainly by cAMP; the Ca2+/PKC system appears to have a contributory role necessary for the full expression of hormonal response. These results support the hypothesis of a dual pathway of target cell activation by PTH.

High molecular weight polypeptides synthesized using DNA as messenger

Polypeptides ranging in size from 5000 to 40 000 daltons can be synthesized using single-stranded ∅X174 as a messenger analog. At 10 mM-12 mM Mg 2+ most of the product migrates on sodium dodecylsulfate gels as a fairly homogeneous band of molecular weight 20 000.