Interleukin 1 interacts synergistically with forskolin and isobutylmethylxanthine in stimulating bone resorption in organ culture

Summary This study examined the interaction of interleukin 1 (IL-1) with forskolin and isobutyl-methylxanthine (3-isobutyl-1-methyl-xanthine) (IBMX) in stimulating bone resorption in 5-day fetal rat long bone organ culture. Forskolin and IBMX are pharmocologic agents that elevate cyclic adenosine monophosphate (AMP) levels in many cell types, including osteoblasts and osteoclasts. The interaction of IL-1 with forskolin and IBMX are synergistic when submaximal resorptive concentrations of agonists were examined. Stimulated resorption was 2 to 5 times that expected for an additive response. When maximally resorptive concentrations of agonists were examined, the interaction between IL-1 and the other agents was, at most, additive. We have previously reported that parathyroid hormone and prostaglandin E, agents that also activate the cyclic AMP pathway in bone cells, interact synergistically with IL-1 in stimulating bone resorption. The results of this study, together with our previous studies, suggest that activation of the cyclic AMP pathway is a sufficient signal for an agent to interact synergistically with IL-1 in stimulating bone resorption.     

Phorbol esters stimulate bone resorption in fetal rat long-bone cultures by mechanisms independent of prostaglandin synthesis

The phorbol esters, 12-O-tetradecanoylphorbol-13-acetate (TPA) and phorbol-12,13-didecanoate, which activate the enzyme protein kinase C, stimulated resorption in fetal rat long-bone cultures at concentrations of 1 and 10 microM. This effect appeared specific for active phorbol esters, since the inactive analogue 4-alpha-phorbol-12,13-didecanoate was without effect. The resorptive responses of fetal rat long-bone cultures to active phorbol esters differed from those previously described in newborn mouse calvaria cultures, since resorption stimulated by TPA in the rat long bones was not inhibited by either indomethacin (10 microM) or flufenamic acid (10 microM). However, calcitonin, an inhibitor of osteoclastic resorption, did decrease the response to TPA. There were some similarities between the response of fetal rat long-bone cultures to TPA and their response to epidermal growth factor (EGF). Like EGF, TPA stimulated DNA synthesis in the bones (measured as the incorporation of [3H]-thymidine) at concentrations below those necessary to stimulate resorption. TPA also did not stimulate resorption in the presence of aphidicolin (10 microM), an inhibitor of DNA synthesis that has been previously shown to block the resorptive response of these cultures to EGF. However, the responses of the cultures to TPA and EGF were not identical, since, unlike the effects of EGF, the stimulatory effects of TPA on DNA synthesis were biphasic. These results demonstrate that active phorbol esters stimulate bone resorption in fetal rat long-bone cultures through mechanisms that do not require prostaglandin synthesis but do appear to be mediated by osteoclasts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for dual modulation of pepsinogen secretion using isoproterenol, carbachol, CCK-8, forskolin, 8 bromo-cAMP, and A23187 probes

The cellular mechanisms by which pepsinogen (PNG) secretion is controlled are not understood. The aim of this study was to explore whether modulation of PNG secretion is mediated by cAMP or calcium-calmodulin (C-C). PNG secretion in isolated rabbit gastric fundic glands (IGG) was tested, using agents believed to act via cAMP or C-C. IGG were stimulated for 30 minutes with histamine (H) 10(-5) M, isoproterenol (I) 10(-5) M, carbachol (C) 10(-5) M, cholecystokinin-octapeptide (CCK-8) 10(-7) M, forskolin (F) 10(-5) M, 8 bromo-cAMP (8B) 10(-3) M, and A23187 (A) 10(-6) M. PNG levels were determined by spectrophotometric assay of hemoglobin digestion products. PNG amounts secreted were (mean per cent above basal levels of total IGG PNG units +/- SEM): H, -0.02 +/- 0.30%; I, 3.5 +/- 0.9%; C, 5.1 +/- 2.2%; CCK-8, 5.3 +/- 1.5%; F, 10.6 +/- 3.8%; 8B, 13.8 +/- 4.5%; A, 2.1 +/- 1.1%. All secretagogues except H stimulated PNG release significantly above basal levels (p less than 0.05). A primary histaminergic mechanism for pepsinogen secretion is unlikely. Since two other adenylate cyclase activators, isoproterenol and forskolin and the 3':5'-cyclic adenosine monophosphate analog 8-bromo cAMP stimulated pepsinogen secretion, cAMP-dependence is probable. Since carbachol, CCK-8, and A23187, which are believed to act via calcium-calmodulin, also stimulated pepsinogen secretion, this system, too, presumably plays a substantial role. Thus the data support a dual 3':5'-cyclic adenosine monophosphate/calcium-calmodulin modulation of pepsinogen secretion.     

The effects of stimulators of intracellular cyclic AMP on rat and chick osteoclasts in vitro: validation of a simplified light microscope assay of bone resorption.

The aim of this study was to investigate whether a cyclic AMP-mediated inhibitory mechanism is present in embryonic chick osteoclasts and to extend data implicating cyclic AMP in the inhibition of neonatal rat osteoclasts. Dibutyryl cyclic AMP ((Bu)2cAMP) (5 x 10(-4) M and above) and isobutylmethylxanthine (IBMX) (10(-4) M and above) reduced the number of pits made in slices of devitalized bovine cortical bone by chick osteoclasts over 24 h. The effect of forskolin (FSK) on chick osteoclasts was biphasic, 10(-5) M producing a weak and variable reduction in pit number while 10(-6) M and 10(-7) M stimulated resorption. Doses of FSK (10(-5) M) and (Bu)2cAMP (3 x 10(-4) M), which individually produced no consistent significant effect, produced a synergistic and highly significant reduction in pit number when used in combination, implying that these agents were acting through a common mechanism, presumably cyclic AMP. Stimulatory doses of FSK were associated with increased osteoclast numbers, implicating cyclic AMP in the formation of osteoclasts. In comparative experiments using neonatal rat osteoclasts, (Bu)2cAMP (10(-4) M and above), IBMX (10(-3) M) and FSK (10(-7) M and above) all reduced the number of pits excavated. Strongly inhibitory doses of these agents caused contraction of chick osteoclasts into a hemispherical shape; contraction of rat osteoclasts into a stellate shape occurred with (Bu)2cAMP and FSK, but not with IBMX. Our results implicate cyclic AMP in the inhibition of both rat and chick osteoclasts, and show that pit counting in the light microscope is a valid method of analyzing the disaggregated osteoclast resorption assay.     

Prostaglandins promote osteoclastlike cell formation by a mechanism involving cyclic adenosine 3',5'-monophosphate in mouse bone marrow cell cultures

We have developed a mouse bone marrow culture system in which multinucleated osteoclast (OC)-like cells are formed within 8 days. Using this culture system, we examined the effect of prostaglandins (PGs), potent bone-resorbing agents, on OC-like cell formation. Four PGs (PGE1 and PGE2 at 10(-8)-10(-5) M, 6-keto-PGF1 alpha at 10(-5) M, and PGF2 alpha at 10(-6)-10(-5) M) significantly stimulated the formation of OC-like cells. The potency of the PGs in inducing OC-like cell formation was the highest in PGE1 and PGE2, followed by PGF2 alpha and 6-keto-PGF1 alpha in that order, and the order was highly correlated with the order of the potency in increasing the production of cyclic adenosine 3',5'-monophosphate (cAMP) in bone marrow cells. Addition of dibutyryl-cAMP also induced OC-like cell formation. Moreover, isobutylmethylxanthine (IBMX), a potent inhibitor of phosphodiesterase, potentiated the OC-like cell formation induced by PGE2, whereas salmon calcitonin greatly inhibited it. Calcitonin induced cAMP production in cultures treated with PGE2, but not in cultures with vehicle. When bone marrow mononuclear cells were cultured on dentine slices in the presence of PGE2, multinucleated OC-like cells were similarly formed and they resorbed calcified dentine, resulting in so-called Howship's lacunae. These results suggest that PGs stimulate resorption of calcified tissues by promoting osteoclast formation. The activity of PGs in inducing OC-like cell formation is considered mediated mainly by a mechanism involving cAMP.     

Different roles for calcium and cyclic AMP in the action of PTH: studies in bone explants and isolated bone cells

In fetal mouse calvaria forskolin (0.1-100 microM), like PTH, stimulated cyclic AMP production in a dose-dependent way. The dose-response curve for forskolin-induced bone mineral release (24 hrs), however, demonstrated a biphasic character, showing stimulation at 0.1 microM and inhibition at 5 and 10 microM. In addition, forskolin-stimulated bone resorption reached a plateau after 48 hrs of incubation, a phenomenon which did not occur with PTH. Forskolin (0.1 microM) strongly stimulated PTH-induced cyclic AMP production in fetal mouse calvaria. However, PTH-stimulated bone resorption and PTH-induced increase in cytosolic free Ca2+ in bone fetal rat cells were not stimulated by forskolin (0.1 microM). 9-(Tetrahydro-2-furyl) adenine (100 microM) completely blunted PTH-stimulated cyclic AMP response in fetal mouse calvaria. PTH-stimulated bone resorption was also completely inhibited, but only after 6 hrs and not after 24 hrs of incubation. With nifedipine and varabamil PTH-stimulated bone resorption was significantly inhibited after 24 hrs of incubation and not significantly after 6 hrs of incubation. A23187 (1 microM) significantly stimulated PTH-stimulated cyclic AMP level and increased basal cytosolic free Ca2+ concentration in cultured rat bone cells. In calvaria, however, it had no effect on either basal and PTH-stimulated cyclic AMP production or on basal and PTH-stimulated bone resorption (6 and 24 hrs). From these observations it follows that in calvaria manipulation of intracellular cyclic AMP only (partially) affects bone resorption. This observation points to a role for an additional second messenger in establishing full blown bone resorption. Some of the results are published in short elsewhere.     

Inhibition of cyclic-3',5'-nucleotide phosphodiesterase abrogates the synergism of hypoxia with lipopolysaccharide in the induction of macrophage TNF-alpha production.

BACKGROUND: Local tumor necrosis factor (TNF)-alpha production by resident macrophages (M phi) contributes to posttraumatic tissue injury. Hypoxia decreases cellular cyclic adenosine monophosphate (cAMP) levels and enhances M phi secretion of TNF-alpha following lipopolysaccharide (LPS) stimulation. Thus, tissue hypoxia associated with trauma likely synergizes with proinflammatory mediators in the induction of M phi TNF-alpha production through an influence on cAMP generation or degradation. It is unclear whether elevation of cellular cAMP inhibits LPS-stimulated TNF-alpha production by hypoxic M phi. Moreover, it is unknown whether the synergism of hypoxia with LPS can be abrogated by promotion of cAMP generation or inhibition of cAMP degradation. METHODS: Rat peritoneal M phi were stimulated with Escherichia coli LPS (20 ng/ml) in a normoxic (room air with 5% CO(2)) or hypoxic (95% N(2) with 5% CO(2)) condition. TNF-alpha levels in cell-free supernatants were measured by enzyme-linked immunoassay. The beta-adrenoceptor agonist isoproterenol (ISP; 5.0 microM) and the adenylate cyclase activator forskolin (FSK; 50 microM) were applied to promote cAMP generation. The nonselective cyclic-3',5'-nucleotide phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX; 1.0 mM) and the PDE III-specific inhibitor milrinone (200 microM) were used to inhibit cAMP degradation. The nondegradable cAMP analogue dibutyryl cAMP (dbcAMP; 100 microM) was applied to further determine the role of PDE. RESULTS. Although hypoxia alone had a minimal effect on TNF-alpha production, it dramatically enhanced LPS-stimulated TNF-alpha production (4.08 +/- 0.28 ng/10(6) cells in hypoxia plus LPS vs 1.63 +/- 0.26 ng/10(6) cells in LPS, 2.5-fold, P < 0.01). Promotion of cAMP generation by either ISP or FSK reduced TNF-alpha production by hypoxic cells. However, neither of these two agents abolished the synergism of hypoxia with LPS (1.68 +/- 0.13 ng/10(6) cells in ISP plus hypoxia plus LPS vs 0.55 +/- 0.04 ng/10(6) cells in ISP plus LPS, threefold; 1.17 +/- 0.03 ng/10(6) cells in FSK plus hypoxia plus LPS vs 0.33 +/- 0.02 ng/10(6) cells in FSK plus LPS, 3.5-fold; both P < 0.01). Inhibition of cAMP degradation with IBMX reduced TNF-alpha production in hypoxic cells and abrogated the synergism (0.31 +/- 0.11 ng/10(6) cells in IBMX plus hypoxia plus LPS vs 0.27 +/- 0.04 ng/10(6) cells in IBMX plus LPS, P > 0.05), and the PDE III inhibitor milrinone had a comparable effect. Moreover, dbcAMP also attenuated TNF-alpha production with abrogation of the synergistic effect of hypoxia (0.56 +/- 0.08 ng/10(6) cells in dbcAMP plus hypoxia plus LPS vs 0.46 +/- 0.04 ng/10(6) cells in dbcAMP plus LPS, P > 0.05). CONCLUSIONS: The results show that elevation of cellular cAMP, either by promotion of generation or by inhibition of degradation, suppresses LPS-stimulated TNF-alpha production in hypoxic M phi. It appears that hypoxia synergizes with LPS in the induction of M phi TNF-alpha production through PDE-mediated cAMP degradation. Inhibition of PDE may be a therapeutic approach for suppression of synergistic induction of M phi TNF-alpha production by hypoxia and LPS in posttraumatic tissue.     

Cyclic AMP-calcium interaction in the transmission of tubuloglomerular feedback signals

Recent studies have suggested that cytosolic calcium serves in the transmission of signals between distal tubular fluid and glomerular vascular elements. Since cAMP can modify calcium-mediated events, it was of interest to determine if agents that elevate intracellular cAMP could influence feedback responses and to assess the interaction of cAMP and intracellular calcium in the transmission of feedback signals. Stop flow pressure (SFP) was measured in the rat during retrograde microperfusion into the distal tubule at 15 nl/min. SFP averaged 37.5 +/- 0.5 mm Hg and decreased by 12.8 +/- 0.8 mm Hg (N = 44) during perfusion with an isotonic Ringer's solution. Addition of 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor, decreased the magnitude of SFP feedback responses; 89 +/- 4.5% (N = 16) inhibition was observed at an IBMX concentration of 500 microM. In addition, SFP decreased by only 3 +/- 1 mm Hg (N = 8) during perfusion with 10 mM dibutyryl cAMP in the presence of a low concentration of IBMX and by 1.6 +/- 0.7 mm Hg (N = 12) during perfusion with forskolin, an agent that stimulates adenylate cyclase activity. Calcium ionophore (A23187) significantly increased the magnitude of SFP feedback responses obtained with IBMX alone from 5 +/- 1.0 mm Hg (N = 9; 250 micron IBMX) to 10 +/- 1.2 mm Hg (N = 11). These results indicate that agents which elevate intracellular cAMP markedly attenuate tubuloglomerular feedback responses and that calcium ionophore can reverse the inhibitory effects of increased cAMP.     

Divergent effects of forskolin on 3′,5′ cyclic adenosine monophosphate production and parathyroid hormone secretion

Summary Forskolin, a diterpene which directly stimulates adenylate cyclase, markedly stimulated cAMP production in intact rat parathyroid glands and dispersed cells from hyperplastic and adenomatous human parathyroid tissues. Stimulation of cAMP production in human parathyroid adenomas occurred as early as 2 min and continued for at least 2 h; furthermore, a dose-response relationship was observed, with a maximal 80-fold cAMP response occurring at 100 μM forskolin. When PTH secretion by rat or human parathyroid tissues was studied at low (0.5 mM) and high (2.5 mM) extracellular Ca²⁺ in either the presence or absence of forskolin, no significant stimulation by forskolin was observed at 15 min, 1 h, and 2 h. When 10 human parathyroid specimens were studied with varying concentrations of forskolin at 1 mM Ca²⁺, 6 failed to show stimulation of PTH secretion and 4 showed modest but detectable increases in PTH that did not appear dose-related. We conclude that (1) at low and high Ca²⁺ levels, marked stimulation of cAMP production by forskolin can occur without a corresponding increase in PTH secretion; (2) inhibition of PTH secretion by high extracellular Ca²⁺ levels continues unchanged despite stimulation of cAMP production by forskolin; and (3) at intermediate Ca²⁺ levels (1.0 mM), PTH secretion is affected either minimally or not at all by forskolin in human hyperparathyroid tissue preparations. The marked stimulation of parathyroid adenylate cyclase by forskolin without concomitant increases in PTH secretion in the majority of tissues suggests that the level of cAMP production is not a primary or sufficient determinant of hormone secretion.     

Endothelin-1 affects AVP-stimulated cAMP but not ANP-induced cGMP productions in cultured rat IMCD cells

Summary: The present study was undertaken to explore a potential interaction of endothelin-1 (ET-1) on vasopressin (AVP)-dependent cyclic 3',5'-adenosine monophosphate (cAMP) and atrial natriuretic peptide (ANP)-dependent cyclic 3',5'-guanosine monophosphate (cGMP) metabolisms in primary cultured rat inner medullary collecting duct (IMCD) cells. Endothelin-1 did not affect ANP-stimulated cGMP accumulation in the presence or absence of 3-isobutyl-1-methylxanthine (IBMX). Levels of 10⁻¹⁰ to 10⁻⁷ mol/LET-1 showed a dose-dependent inhibition of the AVP-dependent cAMP accumulation in the presence of IBMX and 10⁻⁷ mol/LET-1 inhibited the cAMP generation by 34 ± 5.3% ( n = 5, n = 20, P <0.01). Endothelin-1 did not affect the cAMP generation either by 100 ng/mL cholera toxin or by 10⁻⁴ mol/L forskolin. Endothelin-1 failed to inhibit the cAMP generation in the presence of 100 ng/mL pertussis toxin (PTX). the inhibitory effect of ET-1 was reversed by 10⁻⁸ mol/L staurosporin (SSP), a protein kinase C (PKC) inhibitor. Furthermore, this inhibitory effect of ET-1 was mimicked by 10⁻⁸ mol/L phorbol 12-myristate 13-acetate (PMA), an activator of PKC. A dose of 5 × 10⁻⁶ mol/L indomethacin did not affect this inhibitory effect of ET-1. From these results, we suggest that the effect of ET-1 on cyclic nucleotides metabolism seem to be selective. Endothelin-1 did not affect the cGMP generation by ANP, whereas it inhibited cAMP production by AVP via PTX and SSP sensitive pathway in cultured rat IMCD cells.     

The effect of thyrotropin and cAMP on DNA synthesis and cell growth of human thyrocytes in monolayer culture

Monolayer cultures of human thyrocytes from normal tissue (n = 10), and adenomas (n = 7), differentiated (n = 4), poorly differentiated (n = 2), and undifferentiated (n = 3) thyroid cancers were established to assess the significance of thyrotropin (TSH) and cAMP (adenosine 3',5'-cyclic monophosphate) on cell growth and DNA (deoxyribonucleic acid) synthesis. Cell growth of thyrocytes from normal and adenomatous tissues increased more rapidly (p less than 0.01) after TSH (0.1 IU/ml) was added but was unaffected by cAMP (10(-4) mol/L). In these cells, TSH also enhanced DNA synthesis twofold to twelvefold (p less than 0.01). The adenylate cyclase (AC) inhibitor, 2',3' dideoxyadenosine (ddA), increased DNA synthesis 1.3 to 6 times at a concentration of 2 X 10(-4) mol, whereas the membrane/passable cAMP analogue, dibutyryl-cAMP, and the AC stimulator, forskolin, failed to show any effect on DNA synthesis up to a concentration of 10(-5) mol/L (p less than NS). When administered simultaneously, TSH (1/2 maximum) and ddA (20 mumol) had no cumulative effect on DNA synthesis (p = NS). TSH stimulation in cancerous thyroid tissue (n = 11) demonstrated a lack of TSH response in seven of 11 monolayer cultures with no apparent correlation to cancer differentiation, patient age, or sex. Thus TSH was demonstrated to stimulate DNA synthesis and cell growth of human thyrocytes in monolayer cultures independent of the AC system. However, the TSH effect on cell growth and DNA synthesis was unpredictable in thyrocytes from cancerous tissues.     

Cyclic nucleotides and the rapid inhibitions of bone45Ca uptake in response to bovine parathyroid hormone and 16,16-dimethyl prostaglandin E2 in chicks

Summary Intravenous injection of chicks with bovine parathyroid hormone (1–34) (3.3 μg/100 g body wt.) or 16,16-dimethyl PGE₂ (5 μg/100 g body wt.) caused rapid (3 minute) net inhibition of⁴⁵Ca uptake into femur and calvarium. These agents also elevated bone adenosine 3′,5′-cyclic monophosphate (cAMP) but not guanosine 3′,5′-cyclic monophosphate (cGMP) levels at this time. Methylxanthine phosphodiesterase inhibitors (MXPI), caffeine, theophylline, and 3-isobutyl-1-methylxanthine (IBMX) (0.3–5 mg/100 g body wt.) similarly inhibited net⁴⁵Ca uptake into femur and to a lesser extent calvarium. Plasma⁴⁵Ca and total Ca levels were unaltered or showed a slight tendency to be increased over control values 3 minutes after injection. However, the effects of the non-MXPI, dibutyryl-cAMP (0.5–5 mg/100 g body wt.) on bone⁴⁵Ca uptake were negligible. Of the MXPI, only IBMX elevated total cAMP levels in chick bone at 3 minutes. These data implicate but do not confirm a mediatory role for cAMP in the rapid inhibitory actions of PTH and PGEs on bone net⁴⁵Ca uptake in chicks.     

Intracellular cyclic AMP levels in endothelial cells subjected to cyclic strain in vitro.

Human saphenous vein endothelial cells (EC) were grown to confluence in fibronectin-coated culture plates with flexible membrane bottoms and maintained in M-199 supplemented with substrates. One hour prior to experimentation 5 mM IBMX, a phosphodiesterase inhibitor, was added. Vacuum was used to deform the membrane bottoms to 24% strain at 60 cycles/min (0.5 sec elongation alternating with 0.5 sec relaxation). After 10-60 min of cyclic strain, or upon exposure of EC to 100 microM forskolin or 0.1 microM galanin, intracellular cyclic AMP (cAMP) was measured by radioimmunoassay. In parallel experiments, tissue plasminogen activator (tPA) secretion was determined after 24 hr of cyclic strain in the absence or presence of forskolin or galanin. The results demonstrate that exposure of EC to cyclic strain led to no change in cAMP levels and confirmed our previous observation that tPA secretion was enhanced with cyclic strain. Addition of forskolin, which led to an almost 10-fold increase in cAMP levels, or galanin, which led to a 34% decrease in cAMP levels, did not significantly alter the rise in tPA induced by cyclic strain.     

On the role of cyclic AMP as a mediator of bone resorption: gamma-interferon completely inhibits cholera toxin- and forskolin-induced but only partially inhibits parathyroid hormone-stimulated 45Ca release from mouse calvarial bones.

The effects of gamma-interferon (gamma-IFN) on bone resorption and cyclic AMP formation stimulated by parathyroid hormone (PTH), forskolin, and cholera toxin have been studied in cultured neonatal mouse calvarial bones. Bone resorption was assessed by the release of 45Ca from prelabeled mouse calvarial bone fragments. Cyclic AMP formation was quantified by analyzing the amount of the nucleotide in calvarial bone tissue. gamma-IFN completely blocked the 45Ca release response to forskolin and cholera toxin in 96 h cultures. In contrast, the 45Ca release response to PTH was only partially inhibited, an effect that was seen over a wide range of PTH concentrations. The inhibitory effect of gamma-IFN was dose dependent, with a threshold for action at 10 U/ml. Forskolin-stimulated 45Ca release could only be inhibited when gamma-IFN was added simultaneously with forskolin; gamma-IFN added to bones prestimulated with forskolin had no effect. The inhibitory effect of gamma-IFN on PTH-stimulated 45Ca release was seen first after a time lag of 48 h. In contrast calcitonin caused an inhibition after only 3 h. PTH and cholera toxin stimulation of radioactive calcium release was also inhibited by gamma-IFN in bones treated with indomethacin. gamma-IFN inhibited forskolin-induced 45Ca release in bones treated with the mitotic inhibitor hydroxyurea. No effect of gamma-IFN on cyclic AMP formation induced by PTH, cholera toxin, or forskolin could be seen. These data show that gamma-IFN inhibits forskolin- and cholera toxin-induced bone resorption by a mechanism unrelated to prostaglandin production or mitotic activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Diazepam enhances inotropic responses to dopamine in rat ventricular myocardium

Diazepam inhibits phosphodiesterase type 4 and enhances the effect of some 3',5'-cyclic adenosine monophosphate (cAMP)-dependent positive inotropic drugs. We sought to determine whether diazepam and the selective phosphodiesterase type 4 inhibitor rolipram enhances the contractile response and cAMP levels induced by dopamine in rat myocardium. Dopamine (3-100 microM) produced concentration-dependent positive inotropic effects (-log EC50 = 5.21 +/- 0.2, n = 5), which were augmented in the presence of 10 microM diazepam (-log EC50 = 5.40 +/- 0.08, n = 6, P < 0.05) or 1 microM rolipram (-log EC50 = 5.41 +/- 0.1, n = 6, P < 0.05). The effect of diazepam was not mimicked by 100 microM gamma-aminobutyric acid nor it was antagonized by a 5 microM concentration of the blockers of central and peripheral type benzodiazepine receptors, flumazenil and PK 11195. cAMP levels (pmol/g) produced by dopamine (744.4 +/- 111.8, n = 5) in this tissue were enhanced by the presence of diazepam (1073 +/- 97.7, n = 6, P < 0.05) or rolipram (1034.0 +/- 245.2, n = 5, P < 0.05). Therefore, diazepam, like rolipram, augments the inotropic and biochemical effects of dopamine in rat myocardium. This effect is not mediated by benzodiazepine receptors but is probably the consequence of the phosphodiesterase type 4 inhibitory activity of diazepam.     

Factors modulating the response of a porcine renal tubular cell line to calcitonin and antidiuretic hormone

Summary A cell line (LLC-PK₁) isolated from porcine kidney increases 3'5'-cyclic adenosine monophosphate (cAMP) content when incubated with salmon calcitonin (SCT) or antidiuretic hormone (ADH). We have examined several factors which modulate the hormone-induced changes in cAMP levels in these cells. Preincubation with increasing concentrations of SCT results in a dose-dependent decrease in cAMP levels in cells retested with this hormone. Addition of 3-isobutyl-1-methylxanthine (IBMX) to cells preincubated with SCT results in 15–30-fold increases in cAMP levels compared to cells preincubated without this hormone. These observations suggest that the decrease in SCT-induced cAMP response in cells pretreated with this hormone is related at least in part to stimulation of phosphodiesterase activity. Preincubation with ADH does not affect subsequent cAMP response to either ADH or SCT, suggesting that these hormones interact with different cell surface receptors. Cell cycle and plating density also affect cAMP levels. cAMP content per cell increases with increasing cell density, which is associated with an increase in the SCT-induced cAMP response. These studies illustrate that factors other than receptor occupancy modulate cAMP responses of these cells to specific hormones.     

Evidence that pulsed electromagnetic fields inhibit coupling of adenylate cyclase by parathyroid hormone in bone cells

To investigate the biochemical effects of pulsed electromagnetic fields (PEMF) on bone in particular and on cell membrane-associated activity in general, we have studied the modification by PEMF of cAMP metabolism in primary calvarial bone cells. We report that PEMF inhibited cAMP accumulation stimulated by bovine PTH(1-34) peptide. After a 1-hr PEMF exposure, the cAMP response to PTH (2-7 min) was decreased in exposed cells to 48-70% (p less than 0.05) of the response of unexposed cells; furthermore, this inhibition disappeared after 10-20 min with PTH. This inhibition occurred at submaximal PTH doses (2.4-7.3 nM) and no effect was observed at maximal PTH doses (24 nM). Thus with PEMF, the dose response curve for PTH became 0.5 log unit less sensitive. PEMF did not affect the cAMP response to cholera toxin and forskolin. However, when submaximal doses of both forskolin (0.5-1.0 microM) and PTH (0.24-2.4 nM) were used, forskolin prevented inhibition of cAMP production by PEMF in the range of fields and stimulus epochs which normally inhibit cAMP production. It is proposed that PEMF inhibits PTH-stimulated coupling of the adenylate cyclase system and that this inhibition does not affect the intrinsic activity of the G-protein and the catalytic subunit.     

Inhibition of bone resorption by alpha-difluoromethylornithine may not be mediated by polyamine depletion

We have examined the effect of alpha-difluoromethylornithine (DFMO) on bone polyamine content and parathyroid hormone (PTH)- and calcitriol-stimulated bone resorption in cultures of neonatal mouse calvaria. Polyamine content in bone homogenates was determined by reverse-phase paired-ion HPLC. Treatment with 5 mM DFMO for 48 h reduced putrescine from 0.4 nmol/bone to nondetectable levels, slightly decreased spermidine, and did not affect spermine. Bone resorption elicited by 48 h of treatment with PTH or calcitriol was inhibited by concentrations of DFMO greater than or equal to 5 mM added 48 h prior to hormone. This observation supported the concept that polyamines may play a role in bone resorption. However, other observations cast uncertainty on this conclusion. Measurement of calvarial polyamine content at 2 h intervals revealed no increase in endogenous polyamines for up to 10.5 h after calcitriol addition. Although addition of putrescine restored bone polyamine content, exogenous polyamines failed to reverse the inhibitory effects of DFMO on calcitriol-stimulated resorption. These results suggest that a mechanism other than depletion of polyamines could be contributing to the inhibitory effect of DFMO on resorption.     

Augmentation of cAMP improves muscle-flap survival and tissue inflammation in response to ischemia/reperfusion injury

The authors hypothesized that augmenting skeletal muscle adenosine 3',5'-cyclic monophosphate (cAMP) levels could reduce tissue inflammation and improve muscle survival in response to ischemia/ reperfusion (I/R) injury. Gracilis muscle flaps in male Wistar rats were subject to 4 hr of ischemia followed by 3 hr of reperfusion, to assess neutrophil accumulation and microvessel tone, or by 24 hr to evaluate percentage of muscle survival. Animals were grouped as follows: positive (saline) or negative (sham) control, or with infused cAMP elevating agents (8 Bromo-cAMP (8 Br-cAMP) or forskolin). Radioimmunoassay demonstrated significant increases in tissue cAMP levels throughout 3 hr of reperfusion with forskolin, while the 8 Br-cAMP-treated group showed only a temporary increase. Compared with vehicle-infused controls, forskolin administered 5 min prior to reperfusion and repeated as an infusion during the first 45 min of reperfusion, resulted in reduced neutrophil adherence and transmigration, and muscle edema with sustained vasodilatation. The percentage of muscle survival using nitro-blue tetrazolium staining demonstrated enhanced muscle-flap preservation with forskolin. There was no beneficial change in the presence of 8 Br-cAMP These observations suggested that sustained elevation of the cAMP pathway may reduce ischemia-reperfusion injury by decreasing neutrophil-mediated injury and improving vessel tone. Elucidation of the cAMP pathway may provide novel opportunities to modulate ischemia/ reperfusion injury.