Increase In Intracellular cAMP Levels Research Articles

Rutaecarpine Induces Chloride Secretion across Rat Isolated Distal Colon

The present study evaluated the effect of rutaecarpine (Rut) on Cl(-) secretion across rat distal colonic mucosa. Basolateral application of Rut elicited an increase in short-circuit current (I(SC)) response in a concentration-dependent manner. Evidence that Rut-stimulated I(SC) was due to Cl(-) secretion is based on 1) inhibition of current by bumetanide; 2) Cl(-) channel blockers diphenylamine-2-carboxylate, 5-nitro-2-(3-phenylpropylamino)-benzoic acid, and glibenclamide; and 3) removal of Cl(-) ions in bath solution. Determination of neurogenic blockers on Rut-induced I(SC) indicated that pretreatment of tissues with tetrodotoxin or indomethacin, but not atropine or hexamethonium, inhibited Rut-induced response. Treatment with Rut led to release and synthesis of prostaglandin E(2) in rat colonic mucosa. Rut-stimulated I(SC) was markedly reduced by pretreatment with MDL-12,330A [cis-N-[2-phenylcyclopentyl]-azacyclotridec-1-en-2-amine] and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), but not with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester, bisindolylmaleimide, and thapsigargin. Elimination of the extracellular Ca(2+) also did not alter Rut response. Rut treatment resulted in the increase in intracellular cAMP levels and the activation of protein kinase A. Depolarizing the basolateral membrane with high K(+) showed that Rut-stimulated apical Cl(-) current was largely prevented by cystic fibrosis transmembrane conductance regulator (CFTR) inhibitors. Permeabilizing apical membrane with nystatin revealed that Rut-stimulated basolateral K(+) current was specifically inhibited by Ba(2+) ions and chromanol 293B. The evidence derived from present study suggests that Rut-stimulated Cl(-) secretion is mediated by generation of endogenous prostaglandin E(2) and that it also involves the stimulation of cAMP and protein kinase A pathways, which subsequently lead to the activation of apical Cl(-) channels, mostly the CFTR and basolateral cAMP-dependent K(+) channels.

Changes in intracellular cyclic adenosine monophosphate levels in peripheral blood leukocytes during immunotherapy with vespid venom

The allergen-induced release of mediators from basophils is known to be decreased after rush venom immunotherapy (VIT) compared with pretreatment values. A rise in the intracellular cyclic adenosine monophosphate (cAMP) level is known to inhibit mediator release. To determine changes in cAMP levels in peripheral blood leukocytes (PBLs) during rush VIT and to evaluate their relation to allergen-specific reactivity of basophils. Ten patients allergic to vespid venom (VV) were investigated before rush VIT and after reaching the maintenance dose. Five VV-allergic patients not undergoing VIT served as controls. Patients' PBLs were incubated with VV, and allergen-induced histamine and leukotriene release from basophils was measured. Levels of cAMP were determined in PBLs and in plasma. Immediately after rush VIT, VV-induced histamine release (P = .04) and VV-induced leukotriene release (P = .01) were significantly reduced. Intracellular cAMP levels increased significantly (P = .047). However, 6 months after VIT, mediator responses in basophils were comparable with pre-VIT values. No significant changes were found in the control group. An increase in intracellular cAMP levels might account for the decreased reactivity of basophils to allergen after 1 week of VIT. However, similar to the decreased mediator release after 1 week of VIT, this is not a long-term effect as values returned to baseline after 6 months.

Nitroglycerin on contractile force in canine cardiac Purkinje fibers

The objective of this study was to investigate the effects of nitroglycerin (NG) on the contractile force in order to elucidate regulatory roles of cyclic guanosine monophosphate (cGMP) in the bundle of canine cardiac Purkinje fibers. NG suppressed the developed tension in canine Purkinje fibers immersed in normal Tyrode's solution. The contraction was also elicited in the depolarizing solution containing 30 mM K+ by adding isoproterenol (Iso) or tetraethylammonium (TEA). The suppression caused by NG was more marked in fibers in the depolarizing solution treated with TEA than in the depolarizing solution treated with Iso. 8-Bromo-cGMP and sodium nitroprusside had a slight inhibition on the contraction in the fibers. Methylene blue did not affect the decrease in contractile force induced by NG. The weaker inhibition caused by NG on the contraction in the presence of Iso might result from a possible increase in intracellular cAMP levels in the fibers.

Altered pHi regulation and Na+/HCO3− transporter activity in choroid plexus of cilia-defective Tg737orpk mutant mouse

Tg737(orpk) mice have defects in cilia assembly and develop hydrocephalus in the perinatal period of life. Hydrocephalus is progressive and is thought to be initiated by abnormal ion and water transport across the choroid plexus epithelium. The pathology is further aggravated by the slow and disorganized beating of motile cilia on ependymal cells that contribute to decreased cerebrospinal fluid movement through the ventricles. Previously, we demonstrated that the hydrocephalus phenotype is associated with a marked increase in intracellular cAMP levels in choroid plexus epithelium, which is known to have regulatory effects on ion and fluid movement in many secretory epithelia. To evaluate whether the hydrocephalus in Tg737(orpk) mutants is associated with defects in ion transport, we compared the steady-state pH(i) and Na(+)-dependent transport activities of isolated choroid plexus epithelium tissue from Tg737(orpk) mutant and wild-type mice. The data indicate that Tg737(orpk) mutant choroid plexus epithelium have lower pH(i) and higher Na(+)-dependent HCO(3)(-) transport activity compared with wild-type choroid plexus epithelium. In addition, wild-type choroid plexus epithelium could be converted to a mutant phenotype with regard to the activity of Na(+)-dependent HCO(3)(-) transport by addition of dibutyryl-cAMP and mutant choroid plexus epithelium toward the wild-type phenotype by inhibiting PKA activity with H-89. Together, these data suggest that cilia have an important role in regulating normal physiology of choroid plexus epithelium and that ciliary dysfunction in Tg737(orpk) mutants disrupts a signaling pathway leading to elevated intracellular cAMP levels and aberrant regulation of pH(i) and ion transport activity.

CAMP-PKA Signaling Plays an Important Role in Augmented Neutrophil Adhesion and Chemotaxis in Sickle Cell Disease.

A chronic inflammatory state is now thought to underlie much of the pathophysiology of sickle cell disease (SCD). Increased leukocyte numbers are associated with increased morbidity and mortality in SCD and the adhesion of leukocytes to the vascular endothelium and sickle red cells may play an initiating role in the vaso-occlusive process. We sought to better understand the signaling events that lead to alterations in SCD neutrophil adhesive and chemotactic abilities. Neutrophils were isolated from the peripheral blood of healthy controls and SCD individuals in steady state by separation over a ficoll-paque gradient. Following washing and lysis of contaminating red cells, adhesion to fibronectin (FN) and chemotaxis of control and SCD neutrophils (4×106 cells/ml in RMPI medium) were assessed using static adhesion assays and a 96-well chemotaxis chamber assay (ChemoTX, Neuroprobe). Cell adhesion to FN (20mg/ml; 30 min, 37°C, 5% CO2) or IL-8-induced migration (2h, 37°C, 5% CO2) was calculated using a standard curve of the original cell suspension and expressed as the percentage of cells adhered or the number of cells migrated. As previously shown, SCD neutrophils demonstrate a greater ability to adhere to FN-coated plates than control individual leukocytes (13.1±1.48 % neutrophils adhered compared to 5.0±0.6 %; n=11; P<0.001 Mann-Whitney test). Interestingly, SCD neutrophils demonstrate significantly higher levels of the secondary messenger, cAMP, than control neutrophils (4.76±0.38 pMol/106 cells compared to 1.91±0.34 pMol/106 cells; n≥12; P<0.001). When neutrophils were incubated on FN-coated plates in the presence of the cAMP-dependent kinase (PKA) inhibitor, KT5720 (3μM), increased SCD neutrophil adhesion was significantly reversed to levels approaching those of control neutrophils (7.73±1.3 %; n=11; P=0.001). In contrast, control neutrophil adhesion to FN was not significantly affected by the PKA inhibitor (P>0.05). Furthermore, activation of primed SCD neutrophils by the chemokine, IL-8 (500ng/ml), resulted in a significant increase in SCD neutrophil adhesion to FN (19.3±2.2 %; P=0.02; n=7) with a concomitant increase in intracellular cAMP levels (335.8±17.1 % increase, P<0.05, n=3) that could also be reverted by co-incubation with the PKA inhibitor, KT5720 (11.8 ± 0.8 %; P=0.02; n=7). Interestingly, in vitro chemotaxis of SCD neutrophils in response to a stimulus of IL-8 (100 ng/ml) was found to be significantly increased when compared to control neutrophil IL-8-stimulated chemotaxis (14.3±1.1 x105 cells/ml migrated compared to 8.8±0.8x105 cells/ml; P=0.005; n=8). Once again, KT5720 (3 μM, pre-incubation for 15 min, 37°C), was able to significantly reverse (8.5±1.1 x105/ml; P=0.03; n=6) this enhanced IL-8-dependent SCD neutrophil chemotaxis. Results suggest that an up-regulated PKA-dependent pathway may play an important role in the altered adhesive and chemotactic properties of SCD neutrophils and their further activation by cytokines/chemokines such as IL-8, found in increased levels in the serum of SCD patients. Thus, upregulation of the cAMP-PKA pathway in SCD neutrophils may have important consequences for the chronic inflammatory state in SCD patients and may characterize a novel target for the control of altered neutrophil function in SCD.

Anthrax Edema Factor, Voltage-dependent Binding to the Protective Antigen Ion Channel and Comparison to LF Binding

Anthrax toxin complex consists of three different molecules, the binding component protective antigen (PA, 83 kDa), and the enzymatic components lethal factor (LF, 90 kDa) and edema factor (EF, 89 kDa). The 63-kDa N-terminal part of PA, PA(63), forms a heptameric channel that inserts at low pH in endosomal membranes and that is necessary to translocate EF and LF in the cytosol of the target cells. EF is an intracellular active enzyme, which is a calmodulin-dependent adenylate cyclase (89 kDa) that causes a dramatic increase of intracellular cAMP level. Here, the binding of full-length EF on heptameric PA(63) channels was studied in experiments with artificial lipid bilayer membranes. Full-length EF blocks the PA(63) channels in a dose, temperature, voltage, and ionic strength-dependent way with half-saturation constants in the nanomolar concentration range. EF only blocked the PA(63) channels when PA(63) and EF were added to the same side of the membrane, the cis side. Decreasing ionic strength and increasing transmembrane voltage at the cis side of the membranes resulted in a strong decrease of the half-saturation constant for EF binding. This result suggests that ion-ion interactions are involved in EF binding to the PA heptamer. Increasing temperature resulted in increasing half-saturation constants for EF binding to the PA(63) channels. The binding characteristics of EF to the PA(63) channels are compared with those of LF binding. The comparison exhibits similarities but also remarkable differences between the bindings of both toxins to the PA(63) channel.

Extracellular NAD+ Is an Agonist of the Human P2Y11 Purinergic Receptor in Human Granulocytes

Extracellular NAD+ Is an Agonist of the Human P2Y11 Purinergic Receptor in Human Granulocytes

Differential Regulation of Two Distinct Voltage-Dependent Sodium Currents by Group III Metabotropic Glutamate Receptor Activation in Insect Pacemaker Neurons

Using whole cell patch-clamp technique and immunocytochemistry on adult dorsal unpaired median (DUM) neurons isolated from the cockroach Periplaneta americana CNS, we reported the characterization of a native mGluR, sharing pharmacological properties with vertebrate metabotropic glutamate receptor III (mGluRIII) that regulated voltage-dependent sodium current (I(Na)). The global I(Na) was dissociated by means of l-glutamate sensitivity, deactivation time constant, voltage dependence of activation and inactivation, recovery from inactivation, and intracellular regulation process. These two currents were respectively designated I(Na1) and I(Na2) for l-glutamate-sensitive and -insensitive sodium currents. l-glutamate selectively reduced I(Na1) by an increase of intracellular cAMP level. Using different activators and/or inhibitors of G proteins and cAMP/PKA cascade, together with St-Ht31 (an inhibitor of PKA binding to AKAP) and AKAP-79 antibodies, we established that mGluRIII was linked to I(Na1) by a Gi/o and a suspected Gs protein. According to the activated signaling pathway, l-glutamate elevated the cAMP level, which thereby activated cytosolic PKA and released PKA bound to AKAP. As expected from both biophysical and pharmacological studies, we showed that, through an inhibition of I(Na1), l-glutamate increased DUM neuron spontaneous electrical activity. These results indicated that such mGluRIII-activated dual processes provided a new physiological control of pacemaker neuronal firing.

Prostaglandin E2 activates EP2 receptors to inhibit human lung mast cell degranulation

The prostanoid, PGE2, is known to inhibit human lung mast cell activity. The aim of the present study was to characterize the EP receptor that mediates this effect. PGE2 (pEC(50), 5.8+/-0.1) inhibited the IgE-mediated release of histamine from mast cells in a concentration-dependent manner. Alternative EP receptor agonists were studied. The EP2-selective agonist, butaprost (pEC50, 5.2+/-0.2), was an effective inhibitor of mediator release whereas the EP1/EP3 receptor agonist, sulprostone, and the EP1-selective agonist, 17-phenyl-trinor-PGE2, were ineffective. The DP agonist PGD2, the FP agonist PGF(2alpha), the IP agonist iloprost and the TP agonist U-46619 were ineffective inhibitors of IgE-mediated histamine release from mast cells. PGE2 induced a concentration-dependent increase in intracellular cAMP levels in mast cells. The effects of the EP1/EP2 receptor antagonist, AH6809, and the EP4 receptor antagonist, AH23848, on the PGE2-mediated inhibition of histamine release were determined. AH6809 (pK(B), 5.6+/-0.1) caused a modest rightward shift in the PGE2 concentration-response curve, whereas AH23848 was ineffective. Long-term (24 h) incubation of mast cells with either PGE2 or butaprost (EP2 agonist), but not sulprostone (EP1/EP3 agonist), caused a significant reduction in the subsequent ability of PGE2 to inhibit histamine release. Collectively, these data suggest that PGE2 mediates effects on human lung mast cells by interacting with EP2 receptors.

Effect of Endothelin‐1 on Lipolysis in Rat Adipocytes

To explore the role of endothelin-1 (ET-1) on lipid metabolism, we examined the effect of ET-1 on lipolysis in rat adipocytes. Adipocytes isolated from male Sprague-Dawley rats, weighing 400 to 450 grams, were incubated in Krebs-Ringer buffer with or without 10(-7) M ET-1 for various times or with various concentrations of ET-1 for 4 hours; then glycerol release into the incubation medium was measured. In addition, selective ET(A)R and ET(B)R blockers were used to identify the ET receptor subtype involved. We also explored the involvement of cyclic adenosine monophosphate (cAMP) in ET-1-stimulated lipolysis using an adenylyl cyclase inhibitor and by measuring changes in intracellular cAMP levels in response to ET-1 treatment. To further explore the underlying mechanism of ET-1 action, we examined the involvement of the extracellular signal-regulated kinase (ERK)-mediated pathways. Our results showed that ET-1 caused lipolysis in rat adipocytes in a time- and dose-dependent manner. BQ610, a selective ET(A)R blocker, blocked this effect. The adenylyl cyclase inhibitor, 2',5'-dideoxyadenosine, had no effect on ET-1-stimulated lipolysis. ET-1 did not induce an increase in intracellular cAMP levels. In addition, ET-1-induced lipolysis was blocked by inhibition of ERK activation using PD98059. Coincubation of cells with ET-1 and insulin suppressed ET-1-stimulated lipolysis. These findings show that ET-1 stimulates lipolysis in rat adipocytes through the ET(A)R and activation of the ERK pathway. The underlying mechanism is cAMP-independent. However, this non-conventional lipolytic effect of ET-1 is inhibited by the anti-lipolytic effect of insulin.

Hypoxia-induced remodelling of PDE4 isoform expression and cAMP handling in human pulmonary artery smooth muscle cells

Human pulmonary artery smooth muscle cells (hPASM cells) express PDE4A10, PDE4A11, PDE4B2, PDE4C and PDE4D5 isoforms. Hypoxia causes a transient up-regulation of PDE4B2 that reaches a maximum after 7 days and sustained up-regulation of PDE4A10/11 and PDE4D5 over 14 days in hypoxia. Seven days in hypoxia increases both intracellular cAMP levels, protein kinase A (PKA) activity and activated, phosphorylated extracellular signal regulated kinase (pERK) but does not alter either PKA isoform expression or total cAMP phosphodiesterase-4 (PDE4) activity or cAMP phosphodiesterase-3 (PDE3) activity. Both the cyclooxygenase inhibitor, indomethacin and the ERK inhibitors, UO126 and PD980589 reverse the hypoxia-induced increase in intracellular cAMP levels back to those seen in normoxic hPASM cells. Challenge of normoxic hPASM cells with prostaglandin E 2 (PGE 2) elevates cAMP to levels comparable to those seen in hypoxic cells but fails to increase intracellular cAMP levels in hypoxic hPASM cells. The adenylyl cyclase activator, forskolin increases cAMP levels in both normoxic and hypoxic hPASM cells to comparable elevated levels. Challenge of hypoxic hPASM cells with indomethacin attenuates total PDE4 activity whilst challenge with UO126 increases total PDE4 activity. We propose that the hypoxia-induced activation of ERK initiates a phospholipase A 2/COX-driven autocrine effect whereupon PGE 2 is generated, causing the activation of adenylyl cyclase and increase in intracellular cAMP. Despite the hypoxia-induced increases in the expression of PDE4A10/11, PDE4B2 and PDE4D5 and activation of certain of these long PDE4 isoforms through PKA phosphorylation, we suggest that the failure to see any overall increase in PDE4 activity is due to ERK-mediated phosphorylation and inhibition of particular PDE4 long isoforms. Such hypoxia-induced increase in expression of PDE4 isoforms known to interact with certain signalling scaffold proteins may result in alterations in compartmentalised cAMP signalling. The hypoxia-induced increase in cAMP may represent a compensatory protective mechanism against hypoxia-induced mitogens such as endothelin-1 and serotonin.

Regulation of sodium-proton exchanger isoform 3 (NHE3) by PKA and exchange protein directly activated by cAMP (EPAC)

The Na(+)/H(+) exchanger 3 (NHE3) is expressed in the brush border membrane (BBM) of proximal tubules (PT). Its activity is down-regulated on increases in intracellular cAMP levels. The aim of this study was to investigate the contribution of the protein kinase A (PKA) and the exchange protein directly activated by cAMP (EPAC) dependent pathways in the regulation of NHE3 by adenosine 3',5'-cyclic monophosphate (cAMP). Opossum kidney cells and murine kidney slices were treated with cAMP analogs, which selectively activate either PKA or EPAC. Activation of either pathway resulted in an inhibition of NHE3 activity. The EPAC-induced effect was independent of PKA as indicated by the lack of activation of the kinase and the insensitivity to the PKA inhibitor H89. Both PKA and EPAC inhibited NHE3 activity without inducing changes in the expression of the transporter in BBM. Activation of PKA, but not of EPAC, led to an increase of NHE3 phosphorylation. In contrast, activation of PKA, but not of EPAC, inhibited renal type IIa Na(+)-coupled inorganic phosphate cotransporter (NaPi-IIa), another Na-dependent transporter expressed in proximal BBM. PKA, but not EPAC, induced the retrieval of NaPi-IIa from BBM. Our results suggest that EPAC activation may represent a previously unrecognized mechanism involved in the cAMP regulation of NHE3, whereas regulation of NaPi-IIa is mediated by PKA but not by EPAC.

Rhythmic Expression of Adenylyl Cyclase VI Contributes to the Differential Regulation of Serotonin N-Acetyltransferase by Bradykinin in Rat Pineal Glands

The rhythmic nocturnal production of melatonin in pineal glands is controlled by the periodic release of norepinephrine from the superior cervical ganglion. Norepinephrine binds to the beta-adrenergic receptor and stimulates an increase in intracellular cAMP levels, leading to the transcriptional activation of serotonin N-acetyltransferase, which in turn promotes melatonin production. In the present study, we report that bradykinin inhibits basal- and forskolin-stimulated adenylyl cyclase activity, norepinephrine-induced cAMP generation, and N-acetyltransferase expression in a calcium-dependent manner. These effects were blocked by pretreatment with U73122 (a selective phospholipase C inhibitor), and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (a Ca(2+) chelator), but not pertussis toxin. The calcium ionophore, ionomycin, inhibited isoproterenol-mediated cAMP generation, similar to bradykinin. Interestingly, the inhibitory effect of bradykinin was evident only during the daytime. At midday, bradykinin inhibited the cAMP level by approximately 50% but markedly stimulated cAMP production (by approximately 50%) at night. Northern blotting and immunoblotting data disclosed circadian expression of calcium-inhibitable adenylyl cyclase type 6. Expression of adenylyl cyclase type 6 was maximal at Zeitgeber Time (ZT) 01 and very low at ZT 13. Our results suggest that bradykinin-induced calcium inhibits melatonin synthesis through the mediation of adenylyl cyclase type 6 expression.

Kinetic analysis of the activation of photoactivated adenylyl cyclase (PAC), a blue-light receptor for photomovements of Euglena

Photoactivated adenylyl cyclase (PAC) was first purified from a photosensing organelle (the paraflagellar body) of the unicellular flagellate Euglena gracilis, and is regarded as the photoreceptor for the step-up photophobic response. Here, we report the kinetic properties of photoactivation of PAC and a change in intracellular cAMP levels upon blue light irradiation. Activation of PAC was dependent both on photon fluence rate and duration of irradiation, between which reciprocity held well in the range of 2--50 micromol m(-2) s(-1)(total fluence of 1200 micromol m(-2)). Intermittent irradiation also caused activation of PAC in a photon fluence-dependent manner irrespective of cycle periods. Wavelength dependency of PAC activation showed prominent peaks in the UV-B/C, UV-A and blue regions of the spectrum. The time course of the changes in intracellular cAMP levels corresponded well with that of the step-up photophobic response. From this and the kinetic properties of PAC photoactivation, we concluded that an increase in intracellular cAMP levels evoked by photoactivation of PAC is a key event of the step-up photophobic response.

Characterization of vasopressin V2 receptor mutants in nephrogenic diabetes insipidus in a polarized cell model

X-linked nephrogenic diabetes insipidus (NDI) is caused by mutations in the gene encoding the vasopressin V2 receptor (V2R). For the development of a tailored therapy for NDI, knowledge of the cellular fate of V2R mutants is needed. It would be useful when this fate could be predicted from the location and type of mutation. To identify similarities and differences in localization, maturation, stability, and degradation of COOH-terminal GFP-tagged V2R mutants, we stably expressed nine mutants in polarized Madin-Darby canine kidney cells. The mutants V2R-L44P, -Delta62-64, -I130F, -S167T, -S167L, and -V206D were mainly expressed in the endoplasmic reticulum (ER) as immature proteins. These mutants had relatively short half-lives due to proteasomal degradation, except for V2R-Delta62-64. In contrast, V2R-R113W, -G201D, and -T204N were expressed in the ER and in the basolateral membrane as immature, high-mannose glycosylated, and mature complex-glycosylated proteins. The immature forms of V2R-R113W and -T204N, but not V2R-G201D, were rapidly degraded. The mature forms varied extensively in their stability and were degraded by only lysosomes (V2R-T204N and wild-type V2R) or lysosomes and proteasomes (V2R-G201D, -R113W). These data reveal that most missense V2R mutations lead to retention in the ER and suggest that mutations that likely distort a transmembrane domain or introduce a charged amino acid close to it make a V2R mutant more prone to ER retention. Because six of the mutants tested showed significant increases in intracellular cAMP levels on transient expression in COS cells, activation of these six receptors following rescue of cell-surface expression might provide a cure for NDI patients.

Receptors for Relaxin Family Peptides

Recent studies have identified four receptors that are the physiological targets for relaxin family peptides. All are class I (rhodopsin like) G-protein-coupled receptors with LGR7 (RXFP1) and LGR8 (RXFP2) being type C leucine-rich repeat-containing receptors, whereas GPCR135 (RXFP3) and GPCR142 (RXFP4) resemble receptors that respond to small peptides such as somatostatin and angiotensin II. The cognate ligands for the receptors have been identified: relaxin for RXFP1; INSL3 for RXFP2; relaxin 3 for RXFP3 and INSL5 for RXFP4. RXFP1 and RXFP2 receptors produce increases in intracellular cAMP levels upon stimulation, although the response is complex and contains a component sensitive to PI-3-kinase inhibitors. There is also evidence that RXFP1 can activate Erk1/2 and nitric oxide synthase, and relaxin has been reported to enter cells and activate glucocorticoid receptors. In contrast, RXFP3 and RXFP4 couple to Gi by a pertussis toxin-sensitive mechanism to cause inhibition of cAMP production. Now that the receptors for relaxin family peptides and their cognate ligands have been identified, we suggest a nomenclature for both the peptides and the receptors that we hope will be helpful to researchers in this rapidly advancing field.

Cyclooxygenase Inhibitors Suppress Aromatase Expression and Activity in Breast Cancer Cells

Estradiol is biosynthesized from androgens by the aromatase enzyme complex. Previous studies suggest a strong association between aromatase (CYP19) gene expression and the expression of cyclooxygenase (COX) genes. Our hypothesis is that higher levels of COX-2 expression result in higher levels of prostaglandin E2, which, in turn, increases CYP19 expression through increases in intracellular cAMP levels. This biochemical mechanism may explain the beneficial effects of nonsteroidal antiinflammatory drugs on breast cancer. The effects of nonsteroidal antiinflammatory drugs, COX-1 and COX-2 selective inhibitors on aromatase activity and expression were studied in human breast cancer cells. The data from these experiments revealed dose-dependent decreases in aromatase activity after treatment with all agents. Real-time PCR analysis of aromatase gene expression showed a significant decrease in mRNA levels when compared with control for all agents. These results were consistent with enzyme activity data, suggesting that the effect of COX inhibitors on aromatase begins at the transcriptional level. Exon-specific real-time PCR studies suggest that promoters I.3, I.4, and II are involved in this process. Thus, COX inhibitors decrease aromatase mRNA expression and enzymatic activity in human breast cancer cells in culture, suggesting that these agents may be useful in suppressing local estrogen biosynthesis in the treatment of hormone-dependent breast cancer.

Magnolol stimulates lipolysis in lipid‐laden RAW 264.7 macrophages

This study investigated the effect of magnolol, a compound isolated from Magnolia officinalis, on lipolysis in lipid-laden RAW 264.7 macrophages. Treatment of macrophages with magnolol led to dissolution of lipid droplets. This phenomenon was accompanied by a dose-dependent release of glycerol and cholesterol and a concomitant reduction in intracellular levels of glycerol and cholesterol. Furthermore, adipose differentiation-related protein (ADRP), a lipid droplet-associated protein, was down-regulated by magnolol in a dose- and time-dependent manner by Western blot analysis. Immunofluorescence studies also showed that ADRP became detached from the surface of lipid droplets after magnolol treatment. The lipolytic effect of magnolol was not mediated through the cAMP-protein kinase A (PKA) system, an authentic lipolytic pathway for macrophages, since magnolol did not induce an increase of intracellular cAMP levels, and pretreatment with either of PKA inhibitors, PKI and KT5720, did not abrogate the lipolytic response to magnolol. We conclude that magnolol induce-lipolysis of lipid-laden macrophages by down-regulation of ADRP expression and detachment of ADRP from the lipid droplet surface by a cAMP-independent mechanism. Lipolysis of lipid-laden macrophages may occur when the amount of ADRP on the surface of lipid droplets is not enough to stabilize the lipid droplets.

A study of signal transduction for the two diuretic peptides of Diploptera punctata

We investigated second messengers involved in the action of the CRF-related peptide Dippu-DH 46 and the calcitonin-like peptide Dippu-DH 31 in Diploptera punctata. Dippu-DH 46 causes a dose-dependent increase in intracellular cAMP levels, its diuretic activity is mimicked by cAMP agonists, but is attenuated by Rp-cAMPS. Dippu-DH 46 acts synergistically with kinins and thapsigargin; both mobilize intracellular Ca 2+. Dippu-DH 46 also acts synergistically with cAMP agonists, and its effect is inhibited by a PKC inhibitor, suggesting it also activates intracellular Ca 2+. Dippu-DH 31 has no effect on cAMP levels and its activity is not blocked by cAMP agonists. Neither peptide stimulated cGMP levels in a dose-dependent manner, nor does cGMP have any effect on fluid secretion.

Overexpression of the integrin $beta;1A subunit and the $beta;1A cytoplasmic domain modifies the $beta;-adrenergic regulation of the cardiac L-type Ca2+current

<h2>Abstract</h2> Integrins are a family of cell-surface receptors that link the extracellular matrix (ECM) to the cellular cytoskeleton. The goal of this study was to determine the importance of the integrin β₁ subunit in regulating cardiac L-type Ca²⁺ channel function. Neonatal rat ventricular myocytes were cultured on collagen membranes and infected with adenovirus expressing either the human β_1A integrin (Adβ_1A) or a chimeric protein consisting of the cytoplasmic tail domain of the β_1A integrin and the extracellular/transmembrane domain of the interleukin-2 receptor (AdTAC-β₁). Expression of the free β₁ integrin tail (TAC-β₁), but not the full-length β_1A integrin, altered cell morphology and disrupted normal cell adhesion. When compared with myocytes infected with control virus, neither Adβ_1A nor AdTAC-β₁ infection produced any significant change in the current vs. voltage relationship of the whole-cell Ca²⁺ current (<i>I</i>_Ca) or the kinetics of <i>I</i>_Ca decay. Expression of TAC-β₁, but not β_1A, induced a negative shift in the Ca²⁺ channel steady-state inactivation curve. Application of the β-adrenergic receptor agonist isoproterenol produced over a 90% increase in <i>I</i>_Ca in control cells, but caused only an 18% increase in myocytes overexpressing the full-length β_1A integrin. In addition, β-adrenergic stimulation resulted in a 5–10-fold increase in intracellular cAMP levels in control cells, but produced no significant response in Adβ_1A-infected cells. In contrast, expression of TAC-β₁ was associated with an augmentation in the Ca²⁺ channel response to isoproterenol (160% increase) and the Ca²⁺ channel agonist BayK8644. Thus, integrin/ECM interactions may be critical in the regulation of <i>I</i>_Ca