8-bromo Cyclic Adenosine Monophosphate Research Articles

Schwann cell differentiation inhibits interferon-gamma induction of expression of major histocompatibility complex class II and intercellular adhesion molecule-1

Interferon-gamma (IFN-γ) upregulates major histocompatibility complex class II (MHC class II) antigens and intercellular adhesion molecule-1 (ICAM-1) on Schwann cells (SC) in vitro, but in nerves of animals and patients MHC class II is primarily expressed on inflammatory cells. We investigated whether SC maturation influences their expression. IFN-γ induced MHC class II and upregulated ICAM-1; the axolemma-like signal 8-bromo cyclic adenosine monophosphate (8 Br cAMP) with IFN-γ inhibited expression. Delaying addition of 8 Br cAMP to SC already exposed to IFN-γ inhibited ongoing expression; addition of IFN-γ to SC already exposed to 8 Br cAMP resulted in minimal expression. Variability of cytokine-induced MHC class II and ICAM-1 expression by SC in vivo may represent the variability of signals from axolemma.

Identification and characterization of Runx2 phosphorylation sites involved in matrix metalloproteinase-13 promoter activation

Matrix metalloproteinase-13 (MMP-13) plays a critical role in parathyroid hormone (PTH)-induced bone resorption. PTH acts via protein kinase A (PKA) to phosphorylate and stimulate the transactivation of Runx2 for MMP-13 promoter activation. We show here that PTH stimulated Runx2 phosphorylation in rat osteoblastic cells. Runx2 was phosphorylated on serine 28 and threonine 340 after 8-bromo cyclic adenosine mono phosphate (8-Br-cAMP) treatment. We further demonstrate that in the presence of 8-Br-cAMP, the wild-type Runx2 construct stimulated MMP-13 promoter activity, while the Runx2 construct having mutations at three phosphorylation sites (S28, S347 and T340) was unable to stimulate MMP-13 promoter activity. Thus, we have identified the Runx2 phosphorylation sites necessary for PKA stimulated MMP-13 promoter activation and this event may be critical for bone remodeling.

Inflammatory cytokines inhibit upregulation of glycolipid expression by Schwann cells in vitro.

To determine whether products of inflammatory cells can inhibit differentiation and synthesis of myelin glycolipids by Schwann cells. Infiltration of the peripheral nervous system by inflammatory cells is a feature of acquired demyelinating neuropathies. It is not clear what role these cells have in causing demyelination or inhibiting myelin synthesis. Nonmyelinating rat Schwann cells were incubated with 1) different concentrations of activated supernatants (AS) from mitogen-activated inflammatory cells; 2) 8-bromo cyclic adenosine monophosphate (8Br cAMP), known to induce Schwann cell differentiation and synthesis of glycolipids; 3) 8Br cAMP and varying concentrations of AS; 4) 8Br cAMP and cytosine arabinoside (Ara C), which inhibits Schwann cell proliferation; 5) 8Br cAMP, AS, and Ara C; or 6) additional medium. AS inhibits the capacity of cAMP to induce Schwann cell expression of myelin-associated glycolipids. Inhibition of glycolipid expression was independent of the capacity of these AS to induce Schwann cell proliferation. These data suggest that inflammatory mediators are capable of inhibition of Schwann cell differentiation and synthesis of myelin.

Analogs of cyclic nucleotides in rat liver preservation.

Cyclic nucleotides mediate intracellular signal transduction of several vasodilators. In addition to its vascular relaxant effects, cAMP is known to protect endothelial cells and to suppress Kupffer cell activation. On the other hand, cGMP potently ameliorates adhesion of leukocytes and platelets. We tested the effects of two analogs of cyclic nucleotides (8bromo cyclic adenosine monophosphate [8br-cAMP] and 8bromo cyclic guanosine monophosphate [8br-cGMP]) in rat liver preservation. In experiment 1, either analog (0.1-1.0 mM) alone was added to University of Wisconsin (UW) solution in a survival study. In experiment 2, donors and recipients were also treated with 8br-cAMP or 8br-cGMP, with the following three groups tested: group 1=control; group 2=administration of 8br-cAMP to donors, UW solution, and recipients; group 3=administration of 8br-cGMP to donors, UW solution, and recipients. Experiment 3 tested combined treatments: group 4=administration of 8br-cGMP to donors and UW solution, and cAMP to recipients; group 5=administration of 8br-cAMP to donors and UW solution, and 8br-cGMP to recipients. To elucidate the roles of each nucleotide, two further groups were tested: group 6=administration of 8br-cAMP to donors and UW solution; group 7=administration of 8br-cGMP to recipients. In experiment 4, rats in groups 1, 5, 6, and 7 were killed at several time points after reperfusion, and percent graft blood flow (%BF), number of accumulated neutrophils, plasma levels of tumor necrosis factor-alpha and interleukin-1, and serum alanine aminotransferase levels were examined. In experiments 1 and 2, no significant effect was observed on animal survival. In experiment 3, a significant increase in animal survival was observed only in group 5 (100%, 7/7, P=0.0004 vs. group 1: 16.7%, 2/12). In group 5, no improvement of %BF was observed during the early phase of reperfusion (15 and 30 min) compared with that in group 1. On the other hand, the %BF of group 5 was significantly higher in the later phase (6 hr), consistent with the decrease in accumulation of neutrophils observed then. Production of tumor necrosis factor-alpha and serum alanine aminotransferase levels were also reduced with this treatment. Histologically, the bleeding and segmental necrosis, observed in group 1, were completely prevented in group 5. We conclude that restoration of grafts with cAMP and administration of cGMP to recipients led to successful transplantation, and that the two analogs acted synergistically in opposing preservation and reperfusion injury without improvement of graft blood flow during the early phase of reperfusion. The effect was due to their regulation of neutrophil activation and sequestration.

Polar solute transport across the pigmented rabbit conjunctiva: size dependence and the influence of 8-bromo cyclic adenosine monophosphate.

To characterize the conjunctival permeability to polar solutes ranging from 182 to 167,000 daltons in molecular weight (m.w.). Solute transport across the excised pigmented rabbit conjunctiva with a baseline transepithelial electrical resistance (TEER) of 1,285 +/- 46 ohm.cm2 was evaluated in the modified Ussing chamber under open-circuit conditions. The model solutes were mannitol (m.w. 182), 6-carboxyfluorescein (m.w. 376), and fluorescein isothiocyanate-labeled dextrans (FD4, m.w. 4,400-FD150, m.w. 167,000). For a given solute, the apparent permeability coefficient (Papp) was independent of solute concentration and direction of transport. As expected, the Papp decreased with solute size, from 27.7 x 10(-8) cm/sec for mannitol to 0.31 x 10(-8) cm/sec for FD150. When the experimental temperature was lowered from 37 degrees C to 4 degrees C. Papp decreased by approximately 50% for FD4 through FD40 and by > 80% for both FD70 and FD150. Equivalent pore analysis, assuming restricted solute diffusion via cylindrical, water-filled pores across the isolated tissue, revealed a radius of 5.5 nm at a pore density of 1.9 x 10(8) pores per cm2. The addition of 1 mM 8-bromo cyclic adenosine monophosphate (8-BrcAMP), known to stimulate Cl- secretion and decrease TEER, to the mucosal side of the conjunctiva increased the transport of mannitol, FD4, and FD40 by 28%, while not affecting FD150 transport. Our findings suggest that polar solutes up to FD40 traverse the conjunctival epithelial barrier primarily by restricted diffusion through equivalent pores of 5.5 nm radius and that solute movement is affected by reduction of TEER. On the other hand, polar solutes of the FD70 or larger may cross the barrier primarily via non-diffusional pathways such as non-specific endocytosis.

Expression of the nucleoside triphosphate pyrophosphohydrolase PC-1 is induced by basic fibroblast growth factor (bFGF) and modulated by activation of the protein kinase A and C pathways in osteoblast-like osteosarcoma cells.

The closely related cytokines bFGF and aFGF regulate the function of bone cells and mineralization. Osteoblasts express PPi-generating nucleoside triphosphate pyrophosphohydrolase (NTPPPH)/nucleotide phosphodiesterase I activity. bFGF and aFGF (10 ng/ml) up-regulated NTPPPH in human SaOS-2 and U2OS osteosarcoma cells, which express osteoblast-like features in culture. The induction was selective as alkaline phosphatase activity was down-regulated and specific as insulin-like growth factor-1 (IGF-1) and interleukin-1 beta (IL-1 beta) were not active. Furthermore, IL-1 beta but not IGF-1 inhibited bFGF-induced up-regulation of NTPPPH. The induced NTPPPH remained predominantly associated with cells. bFGF can induce signaling through pathways including protein kinase A (PKA) and protein kinase C (PKC)-mediated transduction. An activator of the PKA pathway (8-bromo cyclic adenosine monophosphate [cAMP]) induced NTPPPH. Furthermore, pretreatment with the PKC activator phorbol myristate acetate (PMA) (80 nM) markedly increased subsequent NTPPPH induction by both bFGF and cAMP. The PMA effect was associated with morphologic changes characterized by long, thin intercellular extensions. PKC desensitization also potentially contributed to this effect because the PKC inhibitors staurosporine and H-7 enhanced bFGF-induced and cAMP-induced NTPPPH expression in the absence of morphologic changes. We observed that bFGF induced expression of PC-1, a member of the NTPPPH gene family. The majority of NTPPPH activity was depleted by immunoadsorption using a monoclonal antibody to native human PC-1. bFGF- and aFGF-induced production of PC-1/NTPPPH in osteoblastoid cells may contribute to the effects of FGFs on bone metabolism.

Regulation of the promoter of the human corticotropin-releasing hormone gene in transfected human endometrial cells.

Corticotropin-releasing hormone (CRH) is expressed in several peripheral tissues, including normal epithelial cells of the human and rodent uterus. However, the biological role of endometrial CRH is known in neither species. As a first step to clarify this role, we studied the regulation of CRH promoter in endometrial cells. We performed homologous transfection experiments in Ishikawa cells, a human endometrial cell line, using a 0.9-kb fragment of the 5'-flanking region of the human CRH gene coupled to luciferase. Transfected cells were exposed for 18 h to 8-bromo cyclic adenosine monophosphate, forskolin, epidermal growth factor, steroids (estradiol, progesterone, and the synthetic glucocorticoid dexamethasone and their antagonists), and prostaglandin E2; then the activity of the luciferase reporter was determined in the cell lysates. We found that the activity of the 5'-flanking region of the CRH gene was stimulated by cyclic adenosine monophosphate and epidermal growth factor and inhibited in a receptor-mediated, dose-dependent fashion by estradiol and dexamethasone. The antiglucocorticoid RU 486 acted as a glucocorticoid agonist, suppressing the CRH gene activation, while progesterone was devoid of any activity. Prostaglandin E2 stimulated the CRH activation, and the prostanoid inhibitor indomethacin suppressed it, most probably by inhibiting endogenous prostaglandins. These findings suggest that endometrial CRH gene expression may be under the negative control of estrogens and glucocorticoids and under the positive control of prostaglandin E2.

Role of cyclic adenosine monophosphate in prostaglandin E1-induced penile erection in rabbits.

To investigate the role of cyclic adenosine monophosphate (cAMP) in penile erection in relation to the effect of prostaglandin E1 (PGE1) male adult New Zealand white rabbits were utilized as a model to study intracavernous pressure (ICP) in vivo. After intracavernous injection of PGE1 (0.2-1.6 micrograms/kg) and 8-bromocyclic adenosine monophosphate (8-Br-cAMP, 0.5-1.5 mg/kg), both drugs raised the ICP in a dose-dependent manner. The increased ICPs induced by PGE1 and 8-Br-cAMP were 33.4 +/- 8.12 and 24.1 +/- 4.9 mm Hg, respectively (p < 0.05, paired Student's t test). Administrations of cyclic adenosine monophosphothioate, Rp-isomer (cAMP antagonist, 0.02-0.08 mumol/kg) prior to PGE1 injections inhibited the effect of PGE1 in vivo in a dose-dependent manner. The systemic blood pressures and heart rates in rabbits were unchanged during all the intracavernous injections. The corpus cavernosal tissues isolated from rabbits were studied for the cAMP contents after incubation of different doses of PGE1 in vitro. The cAMP contents were also elevated in a manner parallel with the increases in PGE1 concentrations (3-9 microM). We conclude: (1) the feasibility of intracavernous injection of vasoactive drugs is similar to that in man, thus the rabbit can be used as a suitable alternative for the studies of penile erection, and (2) cAMP is mediated in PGE1-induced relaxation of the rabbit corpus cavernosum, and the cAMP system only participates partially in penile erection.

Mono-(2-ethylhexyl) Phthalate Suppresses Estradiol Production Independent of FSH-cAMP Stimulation in Rat Granulosa Cells

Di-(2-ethylhexyl) phthalate (DEHP) exposure suppressed preovulatory granulosa cell estradiol production in adult cycling rats. The active metabolite of DEHP, mono-(2-ethylhexyl) phthalate (MEHP), suppressed follicle-stimulating hormone (FSH)-stimulated cAMP and progesterone production in cultured rat granulosa cells. To examine how DEHP altered granulosa cell estradiol production, the effects of MEHP were studied in cultures of rat granulosa cells. Granulosa cells were obtained from DES-implanted 25-day-old female Fisher 344 rats and exposed in culture to various concentrations of MEHP (0 to 400 μm) in DMSO. Granulosa cells were stimulated with FSH, 8-bromo cyclic adenosine monophosphate (8br-cAMP), a stable cAMP analog, and various concentrations of testosterone. Estradiol production was measured by standard radioimmunoassays and normalized to cell protein. MEHP suppressed estradiol in a concentration-dependent manner whether granulosa cells were stimulated by FSH or 8-br cAMP. Therefore, MEHP suppressed estradiol independent of its suppression of the FSH-cAMP pathway and, thus, suppressed aromatase conversion of testosterone to estradiol. MEHP (100 μm) decreased the maximum velocity of aromatase in cells supplied with increasing concentrations of testosterone. However, MEHP did not alter the velocity or affinity of microsomal aromatase isolated from adult virgin Sprague-Dawley rat ovaries. Therefore, MEHP altered the absolute amount or availability of aromatase in granulosa cells. Decreased aromatase in granulosa cells would explain decreased estradiol concentrations from DEHP exposure in vivo.

CAMP and/or acetylcholine permit an insulin response to fuel nutrients in chicken

1. 1. The possibility that 8-bromo cyclic adenosine monophosphate (8-Br-cAMP) or acetylcholine (ACh) potentiates insulin release in chicken pancreas in response to d-glyceraldehyde (D-GA, a weak insulinotropic fuel), and permits an insulin release in response to D-mannose or α-ketoisocaproic acid (α-KIC) (two non-insulinotropic fuels in chicken pancreas) is examined. 2. 2.8-Br-cAMP (1 mM) or ACh (1 μM) permitted a sustained although delayed insulin release in response to D-GA (5 and 15mM). 3. 3. The resistance to D-mannose (50 mM) or α-KIC (10 mM) persisted in the presence of 8-Br-cAMP. 4. 4. At 1 or 100 μM, ACh permitted a slight, immediate and transient insulin output in response to α-KIC but not to d-mannose (with one unexplained exception). 5. 5. The simultaneous perfusion of 8-Br-cAMP + ACh increased the basal rate of insulin release, and permitted a large and sustained response to D-mannose. It also greatly increased the immediate response to α-KIC + ACh. 6. 6. In conclusion, in chicken pancreas fuel nutrients require the activation of cAMP- and/or ACh-dependent pathways to induce insulin release. Whether this peculiarity is related to the high glycemia of chickens awaits further investigation.

Further confirmation of the role of adenyl cyclase and of cAMP-dependent protein kinase in primary afferent hyperalgesia

Recent evidence has suggested that cAMP plays a role as a second messenger in the decrease in nociceptive threshold (or hyperalgesia) produced by agents acting on primary afferent terminals. In support of this hypothesis we report that intradermal injection of a direct activator of adenyl cyclase, forskolin, produces a dose-dependent hyperalgesia in the rat. The duration of this hyperalgesia was prolonged by the phosphodiesterase inhibitors, isobutylmethylxanthine and rolipram. Forskolin hyperalgesia was antagonized by the Rp isomer of cyclic adenosine-3′5′-monophospnothioate, an analog of cAMP that prevents the phosphorylation of the cAMP protein kinase. The Rp isomer of cyclic adenosine-3′5′-monophosphothioate also inhibited the hyperalgesia induced by a membrane-permeable analogue of cAMP, 8-bromocyclic adenosine monophosphate, as well as the hyperalgesia induced by agents that are presumed to act directly on primary afferent nociceptors: prostaglandin E 2, prostaglandin I 2, (8 R,15 R)-dihydroxyicosa(5 E-9,11,13 Z)tetraenoic acid; and the adenosine A 2-agonist 2-phenylaminoadenosine. Although the cAMP second messenger system contributes to primary afferent hyperalgesia, we found no evidence for a contribution of protein kinase C. Thus, hyperalgesia induced by prostaglandin E 2, prostacyclin (prostaglandin I 2), (8 R,15 S)-dihydroxyicosa(5 E-9,11,13 Z)tetraenoic acid, the adenosine A 2-agonist 2-phenylaminoadenosine, 8-bromocyclic adenosine monophosphate and the direct activator of adenyl cyclase, forskolin, were not significantly attenuated by the selective inhibition of protein kinase C by trie 19–31 fragment of protein kinase C. Two other inhibitors of protein kinase C, sphingosine and staurosporine, also failed to attenuate prostaglandin E 2-induced hyperalgesia.

Interleukin (IL)-8-induced in vitro human lymphocyte migration is inhibited by cholera and pertussis toxins and inhibitors of protein kinase C

To further investigate the intracellular mechanisms involved in IL-8-induced human mixed peripheral blood lymphocyte (PBL) migration, the effects of pertussis toxin (PTX), cholera toxin (CTX), and protein kinase C (pkC) inhibitors were investigated. Potent inhibition of IL-8-induced PBL migration was observed following exposure of PBL to PTX and CTX (1pM to 0.1μM), 8-bromo cyclic adenosine monophosphate (cAMP; 1nM to 1μM), H7 (1 pM to 0.1μM), sphingosine (0.1μM to 100μM) and the novel pkC inhibitors Ro 31-7549 and Ro 31-8220 (10pM to 1μM) for 10 min. Following incubation of the lymphocytes for 30 min in the presence of the direct activators of pkC, 1-oleoyl-2-acetyl-sn-glycerol (OAG) and 1,2-dioctanoyl-sn-glycerol (DOG; 10nM to 100μM), there was a reversal of the effects of a suboptimal dose of the specific pkC inhibitors Ro 31-7549 and Ro 31-8220. These results suggest that intracellular signals transduced during IL-8-induced in vitro PBL migration may involve pertussis and cholera toxin-sensitive G protein subunits and activation of pkC, processes which are characteristically linked to receptor binding.

FSH-induced expansion of the mouse cumulus oophorus in vitro is dependent upon a specific factor(s) secreted by the oocyte

Although it has been shown that granulosa cells regulate the growth and meiotic maturation of mammalian oocytes, there is little evidence of a role for the oocyte in the differentiation or function of granulosa cells. To test the hypothesis that the oocyte participates in the regulation of granulosa cell function, oocytes were removed from isolated oocyte-cumulus cell complexes by a microsurgical procedure and oocytectomized complexes were tested for their ability to undergo expansion in response to follicle-stimulating hormone (FSH). FSH increased the levels of intracellular cAMP, the activity of the hyaluronic acid-synthesizing enzyme system, and induced cumulus expansion in intact complexes. In contrast, FSH did not induce increased hyaluronic acid-synthesizing enzyme activity or cumulus expansion in oocytectomized complexes. Therefore, the participation of the oocyte is necessary for the cumulus cells to synthesize hyaluronic acid and undergo cumulus expansion in vitro in response to stimulation with FSH. FSH induced the elevation of intracellular cAMP to the same extent in both intact and oocytectomized complexes and the cAMP analog 8-bromo cyclic adenosine monophosphate (8Br-cAMP) did not stimulate expansion in oocytectomized complexes. Therefore, the influence of the oocyte on cumulus expansion occurs downstream from the elevation of cAMP levels in the cumulus cells. Epidermal growth factor (EGF), a potent stimulator of cumulus expansion in intact complexes, which probably acts by a mechanism at least initially different from FSH, failed to stimulate cumulus expansion after oocytectomy. Next, oocytectomized complexes were either cocultured with germinal vesicle stage denuded oocytes or cultured in medium conditioned by denuded oocytes. In both cases, FSH or EGF stimulated expansion by oocytectomized complexes. The degree of expansion was directly correlated to the number of oocytes used to condition the medium. Contact between the oocyte and the cumulus cells is not necessary for cumulus expansion. Rather, a factor(s) secreted by the oocyte is necessary for the cumulus cells to undergo expansion in response to either FSH or EGF. FSH did not induce expansion of oocytectomized complexes in media conditioned by various somatic cells such as granulosa cells, fibroblasts, and Sertoli cells; by a mixed population of male germ cells; or by spermatozoa. This suggests that the expansion enabling activity is specific to the oocyte. These results demonstrate that the oocyte participates in the regulation of cumulus cell function.

Plasmodium falciparum gametocyte formation in vitro: its stimulation by phorbol diesters and by 8-bromo cyclic adenosine monophosphate.

Phorbol 12-myristate 13-acetate increased the number of gametocytes by 50 to 100% in well or petri dish cultures of the HB-3 clone of Plasmodium falciparum. Phorbol dibutyrate had a similar effect. The optimal concentration for each of these agents was 20 ng/ml or approximately 30 nM. No effect of forskolin was found, other than a general inhibition of growth at concentrations over 10 microM. An inhibitor of phosphodiesterase, 8-bromo cyclic adenosine monophosphate (at concentrations of 0.1 and 1.0 microM) also significantly increased the number of gametocytes formed by this clone.

Involvement of the mu-opiate receptor in peripheral analgesia

The intradermal injection of mu (morphine, Tyr- d-Ala-Gly-NMe-Phe-Gly-ol and morphiceptin), kappa ( trans-3,4-dichloro-N-methyl-N[2-(1-pyrrolidinyl)cyclohexyl]benzeneactemide) and delta ([ d-Pen 2,5]-enkephalin and [ d-Ser 2]-[Leu]enkephalin-Thr) selective opioid-agonists, by themselves, did not significantly affect the mechanical nociceptive threshold in the hindpaw of the rat. Intradermal injection of mu, but not delta or kappa opioid-agonists, however, produced dose-dependent inhibition of prostaglandin E 2-induced hyperalgesia. The analgesic effect of the mu-agonist morphine was dosedependently antagonized by naloxone and prevented by co-injection of pertussis toxin. Morphine did not, however, alter the hyperalgesia induced by 8-bromo cyclic adenosine monophosphate. We conclude that the analgesic action of opioids on the peripheral terminals of primary afferents is via a binding site with charcteristics of the mu-opioid receptor and that this action is mediated by inhibition of the cyclic adenosine monophosphate second messenger system.

Electrophysiological actions of norepinephrine in rat lateral hypothalamus. II. An in vitro study of the effects of iontophoretically applied norepinephrine on LH neuronal responses to γ-aminobutyric acid (GABA)

The preceding studies demonstrated that norepinephrine (NE) can consistently augment synaptically mediated (70%) and γ-aminobutyric acid (GABA)-induced (69%) inhibitory responses of lateral hypothalamic (LH) neurons in vivo. The present experiments further characterized the interactions of NE with LH neuronal responses to GABA in terms of α- and ß-receptor mechanisms and demonsrated the utility of the in vitro LH tissue slice preparation as a model for future extra- and intracellular studies of NE modulatory phenomena. Extracellular activity of LH cells was recorded from diencephalic slices (450 μm) incubated in artificial cerebrospinal fluid at 33 °C. Interactions between iontophoretically applied NE, isoproterenol (ISO) or phenylephrine (PE) and responses of LH neurons ( n = 64) to GABA microiontophoresis were quantitated and characterized using computer-generated ratemeter and histogram records. This analysis revealed two distinct actions of NE on GABA-induced responses of LH neurons. In 8 of 32 cells tested (25%), locally applied NE markedly enhanced inhibitory responses to GABA iontophoresis in a manner identical to that observed in vivo. However, in 20 cells (62.5%), iontophoretic application of NE produced a clear antagonism of GABA responses. NE also exerted dual effects on the background firing rate of LH neurons, causing both inhibition and excitation. Overall, in those cells where NE administration increased spontaneous discharge, it either antagonized or had no effect on GABA-mediated inhibition. In contrast, spontaneous firing rate was never elevated above control levels in those cases where NE potentiated GABA responses. Additional experiments demonstrated that the GABA potentiating actions of the benzodiazepine, flurazepam, were preserved in LH tissue slice preparations. In addition, iontophoretic application of the ß-agonist, ISO, routinely suppressed the spontaneous activity of LH neurons and mimicked the facilitating action of NE on GABA. Likewise, microiontophoretic application of 8-bromo cyclic adenosine monophosphate (AMP) enhanced GABA-induced inhibition of LH firing rate in each of 11 cells tested. On the other hand, local administration of the alpha agonist, PE, routinely produced NE-like antagonism of GABA inhibition along with increases in spontaneous firing rate. Taken together these findings indicate that the commonly observed in vivo phenomena of NE augmentation of GABA and suppression of LH neuron spontaneous firing can be demonstrated in vitro, and most likely result from activation of beta adrenoceptors and subsequent elevation of cyclic AMP levels. As such these results suggest that the in vitro preparation will be a useful tool for further investigation of the transmembrane and intracellular events associated with noradrenergically mediated enhancement of GABA. However, in contrast to results obtained in vivo, NE antagonism of GABA inhibition and excitatory effects on spontaneous activity were more frequently observed in LH slices and appear to be mediated by alpha receptor activation. The reduced capacity of NE to augment GABA in vitro might be related to changes in the balance between α- and ß-mediated effects rather than deficits in GABA-facilitating mechanisms, since ISO, cyclic AMP and the benzodiazepine were all routinely capable of enhancing GABAergic responses.

Dopamine inhibition of proopiomelanocortin gene expression in the intermediate lobe of the pituitary. Interactions with corticotropin-releasing factor and the beta-adrenergic receptors and the adenylate cyclase system.

The effects of dopamine on proopiomelanocortin (POMC) gene expression were compared in primary cultures of the anterior and intermediate lobes of the rat pituitary. A single-stranded POMC complementary DNA was used to quantitate POMC messenger RNA levels. Treatment with dopamine (1 microM) for 48 h reduced POMC messenger RNA levels in the intermediate lobe by 77%, but had no effect on POMC gene expression in the anterior lobe. Dopamine D2 receptors were implicated in the response, as bromocriptine (100 nM). reproduced the dopamine inhibition. The responses to dopamine and bromocriptine were antagonized by haloperidol (10 microM). The decrease in POMC messenger RNA levels was dose dependent with ED50 values of about 50 and 0.1 nM for dopamine and bromocriptine, respectively. The accumulation of POMC-derived peptides, beta-endorphin and alpha-melanocyte-stimulating hormone, over 2 days was measured by radioimmunoassay and was shown to parallel the changes in POMC synthesis. The dopamine-induced inhibition of intermediate lobe POMC synthesis was unaffected by isoprenalin (5 microM) and corticotropin-releasing factor (10 nM), although these treatments had stimulatory effects when tested alone. Activating adenylate cyclase with forskolin (1 microM) or treatment with 8-bromocyclic adenosine monophosphate (1 mM) doubled POMC messenger RNA levels, and, when tested against these stimuli, bromocriptine still produced a 30% inhibition of POMC gene expression. These observations suggest that D2 receptor induced inhibition of POMC gene expression is not only mediated by a decrease in cyclic adenosine monophosphate levels. When cells were pretreated with pertussis toxin (100 ng/ml), the bromocriptine-induced inhibition was almost completely lost, suggesting that the dopaminergic inhibition is mediated by guanosine triphosphate binding proteins.

Human chorionic gonadotropin and 8-bromo cyclic adenosine monophosphate promote an acute increase in cytochrome P450scc and adrenodoxin messenger RNAs in cultured human granulosa cells by a cycloheximide-insensitive mechanism.

Treatment of human granulosa cells with human chorionic gonadotropin (hCG) or an analogue of its second messenger, cyclic AMP (cAMP), promotes a rapid accumulation of the messenger RNAs (mRNAs) for cytochrome P450 side-chain cleavage (scc) and adrenodoxin. A twofold increase in the cellular content of these mRNAs was observed within 4 h of exposure to 8-bromo-cAMP, and was maintained for up to 48 h. Inhibition of protein synthesis by cycloheximide did not prevent the hCG- or 8-bromo-cAMP-stimulated accumulation of either cytochrome P450scc or adrenodoxin mRNAs. We conclude that human granulosa cells respond rapidly to hCG and cAMP analogues with a coordinate increase in levels of the mRNAs encoding two key proteins of the steroidogenic machinery, and that this stimulation does not require synthesis of a protein intermediate.

Interactions between oestrogen, progestogen and cytotoxic drugs in human breast cancer cells in culture

To further investigate the intracellular mechanisms involved in IL-8-induced human mixed peripheral blood lymphocyte (PBL) migration, the effects of pertussis toxin (PTX), cholera toxin (CTX), and protein kinase C (pkC) inhibitors were investigated. Potent inhibition of IL-8-induced PBL migration was observed following exposure of PBL to PTX and CTX (1pM to 0.1μM), 8-bromo cyclic adenosine monophosphate (cAMP; 1nM to 1μM), H7 (1 pM to 0.1μM), sphingosine (0.1μM to 100μM) and the novel pkC inhibitors Ro 31-7549 and Ro 31-8220 (10pM to 1μM) for 10 min. Following incubation of the lymphocytes for 30 min in the presence of the direct activators of pkC, 1-oleoyl-2-acetyl-sn-glycerol (OAG) and 1,2-dioctanoyl-sn-glycerol (DOG; 10nM to 100μM), there was a reversal of the effects of a suboptimal dose of the specific pkC inhibitors Ro 31-7549 and Ro 31-8220. These results suggest that intracellular signals transduced during IL-8-induced in vitro PBL migration may involve pertussis and cholera toxin-sensitive G protein subunits and activation of pkC, processes which are characteristically linked to receptor binding.

Effect of ouabain and furosemide on pepsinogen secretion by gastric glands in vitro.

Gastric glands were isolated from rabbit stomach and pepsinogen secretion was measured after stimulation with isoproterenol, forskolin, 8-bromo cyclic adenosine monophosphate (8-bromo cAMP), cholecystokinin octapeptide (CCK-OP), carbachol, and hyperosmolar medium. The responses to these stimuli in medium containing 143 mM Na+ and 5.4 mM K+ (normal medium) were compared with responses to the same stimuli in media containing either 0 Na+ and 5.4 mM K+, or 143 mM Na+ and O K+. In addition, the effects of ouabain and furosemide on secretion elicited by these stimuli were determined. Medium containing 0 Na+ inhibited all stimuli. Medium containing 0 K+ inhibited the action of 8-bromo cAMP and stimuli postulated to be mediated by cAMP. Ouabain inhibited the same stimuli as O K+ medium, and, in addition, inhibited the response to hyperosmolar medium. However, ouabain enhanced the response to CCK-OP. Furosemide inhibited the response to hyperosmolar medium but had no effect on the action of any secretagogue employed. Intraglandular [Na+] increased and [K+] decreased after exposure to K+-free medium or ouabain. cAMP content of the glands was assayed after stimulation with both isoproterenol and hyperosmolar medium. Isoproterenol and hyperosmolar medium significantly increased cAMP levels. The results are discussed in relation to possible involvement of ion transport or intracellular ion concentration in the secretory process.