Cyclic Adenosine Monophosphate Release Research Articles

Enhanced oral absorption of teriparatide with therapeutic potential for management of osteoporosis

In this study, a system for oral delivery of recombinant human parathyroid hormone [rhPTH(1-34); teriparatide (TRP)] was developed to enhance oral absorption and to demonstrate an equivalent therapeutic effect to that of subcutaneous (SC) TRP injection. The solid oral formulation of TRP was prepared by electrostatic complexation with l-lysine-linked deoxycholic acid (LDA) and deoxycholic acid (DA) at a molar ratio of 1:5:7 in the aqueous dispersion of non-ionic n-dodecyl-β-d-maltoside (DM) at a 1:15 weight ratio, followed by freeze-drying the dispersal, yielding TRP(1:5:7)-15. As expected, TRP(1:5:7)-15 showed a 414% increase in permeability across the Caco-2/HT29-MTX-E12 cell monolayer, resulting in a 13.0-fold greater oral bioavailability compared with free TRP. In addition, the intestinal transport mechanisms in the presence of specific inhibitors of clathrin-mediated endocytosis, macropinocytosis, and bile acid transporters revealed 44.4%, 28.7%, and 51.2% decreases in transport, respectively, confirming that these routes play crucial roles in the permeation of TRP in TRP(1:5:7)-15. Notably, this formulation showed similar activation of the release of cyclic adenosine monophosphate (cAMP) compared with TRP, suggesting equivalent efficacy in the parathyroid hormone receptor-adenylate cyclase system of osteosarcoma cells. Furthermore, oral TRP(1:5:7)-15 (equivalent to 0.4mg/kg TRP) demonstrated increases in bone mineral density (36.9%) and trabecular thickness (31.3%) compared with untreated glucocorticoid-induced osteoporotic mice. Moreover, the elevated levels of biomarkers of bone formation, including osteocalcin, were also comparable with those after SC injection of TRP (0.02mg/kg). These findings suggest that TRP(1:5:7)-15 can be used as an effective oral therapy for the management of osteoporosis.

Ethanolic Extract of Hippocampus abdominalis Exerts Anti-Melanogenic Effects in B16F10 Melanoma Cells and Zebrafish Larvae by Activating the ERK Signaling Pathway

The big belly seahorse (Hippocampus abdominalis), a well-known ingredient of traditional medicine, possesses anti-inflammatory, anti-aging, anti-fatigue, and anti-thrombotic properties, and also increases male fertility. This study demonstrates that the ethanolic extract of dried H. abdominalis (EEHA) has anti-melanogenic effects in B16F10 melanoma cells and zebrafish larvae. EEHA significantly reduced the α-melanocyte-stimulating hormone (α-MSH)-induced melanogenesis in B16F10 melanoma cells without causing cytotoxicity. At a concentration of 200 µg/mL, EEHA had significant anti-melanogenic activity in zebrafish larvae, accompanied by a severe reduction in the heart rate (118 ± 17 heartbeats/min) compared to that of the untreated group (185 ± 8 heartbeats/min), indicating that EEHA induces cardiotoxicity at high concentrations. Below 100 µg/mL, EEHA significantly reduced melanogenesis in zebrafish larvae in the presence or absence of α-MSH, while the heart rate remained unaltered. Additionally, EEHA downregulated the release of cyclic adenosine monophosphate (cAMP) and the phosphorylation of cAMP response element-binding protein (CREB) in B16F10 melanoma cells, which inhibited microphthalmia-associated transcription factor (MITF), leading to the inhibition of tyrosinase activity. EEHA also increased the phosphorylation of extracellular-signal regulated kinase (ERK). The ERK inhibitor PD98059 interfered with the anti-melanogenic activity of EEHA in B16F10 melanoma cells and zebrafish larvae, indicating that the ERK signaling pathway might regulate the anti-melanogenic properties of EEHA. Altogether, we conclude that EEHA represses the cAMP–CREB–MITF axis, which consequently inhibits tyrosinase-mediated melanogenesis. We propose that at low concentrations, EEHA can serve as a promising anti-melanogenic agent that could be used to prepare whitening cosmetics and for treating melanogenic disorders.

Physiological and Functional Basis of Dopamine Receptors and Their Role in Neurogenesis: Possible Implication for Parkinson's disease.

Dopamine controls various physiological functions in the brain and periphery by acting on its receptors D1, D2, D3, D4, and D5. Dopamine receptors are G protein–coupled receptors involved in the regulation of motor activity and several neurological disorders such as schizophrenia, bipolar disorder, Parkinson’s disease (PD), Alzheimer’s disease, and attention-deficit/hyperactivity disorder. Reduction in dopamine content in the nigrostriatal pathway is associated with the development of PD, along with the degeneration of dopaminergic neurons in the substantia nigra region. Dopamine receptors directly regulate neurotransmission of other neurotransmitters, release of cyclic adenosine monophosphate, cell proliferation, and differentiation. Here, we provide an update on recent knowledge about the signalling mechanism, mode of action, and the evidence for the physiological and functional basis of dopamine receptors. We also highlight the pivotal role of these receptors in the modulation of neurogenesis, a possible therapeutic target that might help to slow down the process of neurodegeneration.

Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays.

Dictyostelium discoideum amoeba are found in soil, feeding on bacteria. When food sources become scarce, they secrete factors to initiate a multicellular development program, during which single cells chemotax towards aggregation centers(1-4). This process is dependent on the release of cyclic adenosine monophosphate (cAMP)(5). cAMP is produced in waves through the concerted action of adenylate cyclase and phosphodiesterases, and binds to G protein-coupled cAMP receptors(6,7). A widely used assay to analyze the mechanisms involved in the developmental cycle of the lower eukaryote Dictyostelium discoideum is based on the observation of cell aggregation in submerged conditions(8,9). This protocol describes the analysis of the role of coronin A in the developmental cycle by starvation in tissue-culture plates submerged in balanced salt solution (BSS)(10). Coronin A is a member of the widely conserved protein family of coronins that have been implicated in a wide variety of activities(11,12). Dictyostelium cells lacking coronin A are unable to form multicellular aggregates, and this defect can be rescued by supplying pulses of cAMP, suggesting that coronin A acts upstream of the cAMP cascade(10). The techniques described in these studies provide robust tools to investigate functions of proteins during the initial stages of the developmental cycle of Dictyostelium discoideum upstream of the cAMP cascade. Therefore, utilizing this aggregation assay may allow the further study of coronin A function and advance our understanding of coronin biology.

Plasma Membrane Mechanisms in a Preclinical Rat Model of Chronic Pain

We have recently shown that the prolongation of prostaglandin E2 hyperalgesia in a preclinical model of chronic pain—hyperalgesic priming—is mediated by release of cyclic adenosine monophosphate from isolectin B4–positive nociceptors and its metabolism by ectonucleotidases to produce adenosine. The adenosine, in turn, acts in an autocrine mechanism at an A1 adenosine receptor whose downstream signaling mechanisms in the nociceptor are altered to produce nociceptor sensitization. We previously showed that antisense against an extracellular matrix molecule, versican, which defines the population of nociceptors involved in hyperalgesic priming, eliminated the prolongation of prostaglandin E2 hyperalgesia. To further evaluate the mechanisms at the interface between the extracellular matrix and the nociceptor's plasma membrane involved in hyperalgesia prolongation, we interrupted a plasma membrane molecule involved in versican signaling, integrin β1, with an antisense oligodeoxynucleotide. Integrin β1 antisense eliminated mechanical hyperalgesia induced by an adenosine A1 receptor agonist, cyclopentyladenosine, in the primed rat. We also disrupted a molecular complex of signaling molecules that contains integrin β1, lipid rafts, with methyl-β-cyclodextrin, which attenuated the prolongation without affecting the acute phase of prostaglandin E2 hyperalgesia, while having no effect on cyclopentyladenosine hyperalgesia. Our findings help to define the plasma membrane mechanisms involved in a preclinical model of chronic pain. PerspectiveThe present study contributes to a further understanding of mechanisms involved in the organization of messengers at the plasma membrane that participate in the transition from acute to chronic pain.

Isoproterenol inhibits transcription of cardiac cytokine genes induced by reactive oxygen intermediates.

Cytokines such as tumor necrosis factor alpha (TNF-alpha) are produced by the myocardium in heart disease and might be stimulated by reactive oxygen. In some cell types, cyclic adenosine monophosphate (AMP) inhibits TNF-alpha production. The authors tested the hypothesis that stimulation of cardiac beta-adrenergic receptors would inhibit cytokine gene transcription induced by reactive oxygen. Rat hearts were perfused with buffer containing hypoxanthine. Reactive oxygen intermediates were generated by infusion of xanthine oxidase. Myocardial mRNA encoding 11 cytokines was determined. TNF-alpha, interleukin-6, and cyclic AMP were measured in the coronary effluent. In control hearts, of the screened RNA, only mRNA encoding interleukin-1beta, -4, and -6 was detected. Stimulation with hypoxanthine-xanthine oxidase (HX-XO) induced detectable mRNA for TNF-alpha and interleukin-5 and increased mRNA band density for interleukin-1beta, -4, and -6. Simultaneous infusion of isoproterenol inhibited HX-XO-stimulated cytokine gene expression and caused release of cyclic AMP into the coronary effluent. In control hearts, TNF-alpha was not detected in the coronary effluent. After HX-XO administration, TNF-alpha was reliably detected at 60 min and interleukin-6 at 90 min. Simultaneous infusion of isoproterenol inhibited TNF-alpha and interleukin-6 release. Inclusion of propranolol in the perfusion buffer blocked the isoproterenol-induced inhibition of HX-XO-stimulated TNF-alpha release and release of cyclic AMP into the coronary effluent. In addition, elevating myocardial cyclic AMP with forskolin also blocked release of TNF-alpha stimulated by HX-XO. Finally, delaying infusion of isoproterenol until 30 min after HX-XO administration still suppressed release of TNF-alpha. Reactive oxygen species activate cytokine gene transcription in the myocardium. The sympathetic nervous system, acting through beta-receptors to elevate myocardial cyclic AMP, regulates cardiac cytokine production by inhibition of transcription.

Stimulation of beta-adrenergic receptors inhibits the release of tumor necrosis factor-alpha from the isolated rat heart.

Beta-adrenergic receptor agonists such as isoproterenol inhibit production of tumor necrosis factor (TNF)-alpha in a number of cell types. Because the heart is a source of TNF-alpha, we hypothesized that isoproterenol would inhibit cardiac production of the cytokine. Analysis of cardiac release of TNF-alpha. Medical research laboratory. Rats. None. With the approval of the Institutional Animal Care and Use Committee, rats were anesthetized and hearts were removed and perfused. After 30 mins, bacterial lipopolysaccharide (LPS) with or without isoproterenol was infused for 60 mins. At 30, 60, 90, 120, and 150 mins, coronary flow was measured and coronary effluent was analyzed for TNF-alpha. Cardiac production of TNF-alpha was expressed as pg/min. Cyclic adenosine monophosphate (AMP) in the coronary effluent was measured. TNF-alpha messenger RNA was determined in ventricular tissue. After 30 mins, TNF-alpha was undetectable in the coronary effluent However, 60 mins after the initiation of LPS infusion, TNF-alpha release was 875+/-255 pg/min and increased to 2164+/-721 pg/min at 150 mins. Simultaneous infusion of isoproterenol with LPS stimulated cyclic AMP release and inhibited TNF-alpha production. For instance, at 60 and 150 mins, TNF-alpha release was 75+/-38 and 58+/-29 pg/min, respectively (p < .05 vs. LPS alone). Simultaneous infusion of isoproterenol with LPS blocked the induction of TNF-alpha messenger RNA by LPS. Isoproterenol, begun 30 mins after the initiation of LPS infusion, still suppressed LPS-stimulated TNF-alpha release by 95% at 150 mins. Similar results were obtained with norepinephrine. Activation of beta-adrenergic receptors inhibits cardiac TNF-alpha release. This implies that cytokine production by the heart is inhibited by the sympathetic nervous system. In heart failure, the cardiac response to the sympathetic nervous system is impaired. This impairment may play a role in the high plasma levels of TNF-alpha found in heart failure.

Demonstration of a Probenecid-Inhibitable Anion Exchanger Involved in the Release of Cortisol and cAMP and in the Uptake of p-Aminohippurate in Bovine Adrenocortical Cells

Recently we provided evidence for the involvement of a probenecid-inhibitable anion exchanger in cortisol release from primary cultures of bovine adrenocortical cells. In the present study, we further characterized this exchange transporter. Adrenocorticotropic hormone stimulated ³H-p-aminohippurate (³H-PAH) uptake into as well as cortisol release from the cells about two- and tenfold, respectively. Probenecid inhibited both ³H-PAH uptake and cortisol release by about 55 and 63%. Preincubation of the cells with 1 mM PAH trans-stimulated ³H-PAH uptake by 30%, whereas cortisol release was inhibited by 30%. ³H-PAH uptake was cis-inhibited by 1 mM glutarate or by 1 mM cortisol in the medium, while cortisol release was trans-stimulated by glutarate. PAH in the incubation medium showed saturable cis-inhibition of ³H-PAH uptake. The release of cyclic adenosine monophosphate, a substrate of the renal PAH exchanger, was also inhibited by probenecid and trans-stimulated by glutarate. In summary, the trans-stimulation and cis-inhibition experiments support the concept of an anion exchanger involved in cortisol and cyclic adenosine monophosphate release from and PAH uptake into adrenocortical cells.

New cardiotonic agents

D RUGS THAT INCREASE the positive inotropic (ie, cardiotonic) potential of the myocardium are used in a variety of clinical settings. All catecholamines exert their inotropic effects by stimulating adrenergic mediated receptors in the sympathetic nervous system. 1,2 Stimulation of these adrenergic receptors within the cardiovascular system generates predictable organ specific responses (Table 1). Catecholamines can act either directly or indirectly on these adrenergic receptors. Indirect acting catecholamines stimulate the release of stored neurotransmitters from sympathetic nerve terminals, whereas direct acting agents stimulate the adrenergic receptors directly. Director indirect-acting catecholamines exert their positive inotropic action by stimulation of the ill-receptor. This stimulation activates the second messenger pathway, facilitating the release of cyclic adenosine monophosphate (cAMP). 3 The efficacy of any adrenergic agent is influenced by the availability (ie, density) and responsiveness (ie, affinity) of these beta receptors. A reduction in both the number and sensitivity of B-receptors has been noted with chronically elevated levels of plasma catecholamines (such as that occurring in congestive heart failure [CHF] or hypertension). This was described by Bristow et al4 as B-receptor down regulation. Conversely, B-receptor up regulation has been described in patients undergoing long-term fl-adrenergic antagonist therapy, thus resulting in a significant increase in the numbers of B-receptors. Down regulation, initially thought to develop only when plasma catecholamine levels were chronically elevated, has now been observed with short-term exposure. Marty et al5 recently reported a significant increase in B-receptor density

Myocardial adenosine stimulates release of cyclic adenosine monophosphate from capillary endothelial cells in guinea pig heart.

Activation of coronary endothelial cell adenylate cyclase was studied in the isolated guinea pig heart by prelabelling endothelial adenine nucleotides using intracoronary infusion of [3H]-adenosine, and measuring the coronary efflux of [3H]-cyclic adenosine monophosphate (cAMP). Hypoxia (30% O2) caused a 4-fold increase in coronary release of [3H]-cAMP, which was decreased by 63% by infusion of the adenosine receptor antagonist, theophylline (50 microM). During normoxic control conditions, degrading adenosine to non-vasoactive inosine by intracoronary infusion of adenosine deaminase (1.7 U/ml) caused a 20% decrease in the release of [3H]-cAMP. The effect of adenosine deaminase was reversed by a specific enzyme inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride. Coronary efflux of [3H]-cAMP during intracoronary infusion of 1 microM adenosine triphosphate (ATP), adenosine diphosphate or adenosine monophosphate (AMP) (plus adenosine deaminase 8 U/ml) was only 13% of that due to 1 microM adenosine. Adenosine receptor blockers theophylline and CGS 15943A caused equivalent inhibition of the coronary vasodilator actions of adenosine and ATP. Intracoronary infusion of prostaglandin E1 and the beta 2-adrenergic agonist procaterol caused parallel, dose-dependent increases in coronary conductance and the venous release of [3H]-cAMP. It is concluded that (1) under both normoxic and hypoxic conditions, adenosine formed by the heart may activate endothelial cell adenylate cyclase via membrane adenosine receptors, (2) coronary receptors for adenosine and ATP share common ligand affinities but ATP receptors are not coupled to adenylate cyclase, and (3) other vasodilators known to activate endothelial adenylate cyclase in vitro cause parallel increases in coronary conductance and adenylate cyclase activity in the beating heart.

Structural requirements for parathyroid hormone action in mature bone. Effects on release of cyclic adenosine monophosphate and bone gamma-carboxyglutamic acid-containing protein from perfused rat hindquarters.

To determine the structural requirements for parathyroid hormone (PTH) activity in mature bone, we perfused the surgically isolated hindquarters of adult male rats with either native bovine PTH-(1-84) [bPTH-(1-84)] or the synthetic amino-terminal fragment, bovine PTH-(1-34) [bPTH-(1-34)]. Changes in the release of cyclic AMP (cAMP) and bone Gla protein (BGP) were monitored as evidence of bone-specific response to PTH; tissue specificity of the cAMP response was confirmed through in vitro examination on nonskeletal tissue response to PTH. Biologically active, monoiodinated 125I-bPTH-(1-84) was administered to determine if mature murine bone cleaves native hormone. We found that perfused rat bone continuously releases BGP, and that both bPTH-(1-84) and bPTH-(1-34) acutely suppress this release. In addition, both hormones stimulate cAMP release from perfused rat hindquarters. When examined on a molar basis, the magnitude of the cAMP response was dose-dependent and similar for both hormones, with doses yielding half-maximal cAMP responses. The response for bPTH-(1-34) was 0.5 nmol and for bPTH-(1-84) was 0.7 nmol. Moreover, biologically active 125I-bPTH-(1-84) was not metabolized in our hindquarter perfusion system. These findings indicate that PTH-(1-84) does not require extraskeletal or skeletal cleavage to an amino-terminal fragment in order to stimulate cAMP generation in, or suppress BGP release from, mature rat bone.

Increased release of cyclic adenosine monophosphate into jugular vein in response to isoproterenol administration

Catecholamine administration elevates plasma cyclic AMP (cAMP) levels but the source of the cAMP is unknown. To determine possible sources, plasma cAMP levels were determined in blood vessels across the head, liver, kidney and lung in anesthetized dogs infused with the β-adrenergic agonist, isoproterenol. Only the head showed an increased release of cAMP into the blood. The kidneys removed cAMP from the blood while liver and lung showed no change. This in vivo demonstration of release of cAMP from the head represents contributions from brain and facial muscles and may be a useful approach to study brain involvement in the action of various hormones and drugs.

Dose-related effects of luteinizing hormone on the pattern of steroidogenesis and cyclic adenosine monophosphate release in superfused preovulatory rat follicles.

The secretion of steroids and the release of cAMP in response to repeated luteinizing hormone (LH) stimulation were examined during superfusion of isolated preovulatory rat follicles. A high dose of ovine LH (1 microgram/ml for 20 min) caused a prolonged increase in the secretion of progesterone (P) and 20 alpha-dihydroprogesterone (20 alpha-OHP) and a transient increase in the secretion of testosterone (T) and estradiol-17 beta (E2), and was accompanied by a peak of cAMP release. A single pulse of LH at a low dose level (10 mg/ml for 20 min) gave a limited increase in T secretion, but no clear change in P, 20 alpha-OHP and E2 secretion or cAMP release. When the follicles were challenged with a second pulse of LH (at 1 microgram/ml), the response varied according to the dose of LH delivered in the preceding pulse. Following exposure to the high dose of LH, the follicles were partially refractory to the second LH challenge in terms of cAMP and P and the secretion of T and E2 remained low. The low dose of LH, however, had a conditioning effect on the follicles since the response to the second LH challenge was amplified in terms of P, 20 alpha-OHP and cAMP. In this case a secondary increase in T and E2 secretion was found. The differential response to varying doses of LH are likely to reflect the physiological control of steroidogenesis during final follicular maturation.

Iontophoretic release of cyclic AMP and dispersion of melanosomes within a single melanophore.

Selective dispersion of melanosomes was often observed after iontophoretic injection of cyclic adenosine monophosphate (AMP) from a glass microelectrode positioned in a target melanophore in frog skin (as viewed from above through a microscope), with other melanophores in the field serving as controls. Because the skin has orderly arrays of several types of closely spaced cells, it is probable that at times the microelectrode also impales cells other than melanophores. When cyclic AMP injection inside a cell resulted in dispersion of melanosomes from a perinuclear position into dendritic processes, the onset of dispersion was relatively rapid, in many cases less than 4 min (mean time of onset, 5.3 +/- 2.9 [SD] min). A much slower dispersion (mean time of onset, 19.0 +/- 5.0 min) of melanosomes was observed when the microelectrode was positioned adjacent to a melanophore, and much larger quantities of cyclic AMP were released. In addition, no changes were observed for injections of 5'-AMP or cyclic guanosine monophosphate (GMP) through electrodes positioned inside or adjacent to melanophores. Potential measurements showed that after impaling a clell, a constant transmembrane potential could often be recorded over many minutes, indicating that the membrane tends to seal around the microelectrode. The results indicate that cyclic AMP acts more rapidly on the inside of a cell than when applied outside a cell and allowed to diffuse through the plasma membrane. This study introduces a model system whereby the properties of the plasma membrane and melanocyte-stimulating hormone (MSH) receptors can be studies within a single target cell.