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PubMed1984

Interactions of vasopressin, prostaglandins, and cAMP in rat renal papillary collecting tubule cells in culture.

To evaluate the hypothesis that prostaglandins (PGs) inhibit vasopressin stimulation of adenylate cyclase in the collecting tubule, we have studied the interactions of vasopressin, PGs, and intracellular cAMP in rat renal papillary collecting tubule (RPCT) cells in cell culture. Inhibition of PGE2 synthesis with acetylsalicylic acid (ASA) did not potentiate arginine vasopressin (AVP)-stimulated intracellular cAMP. Augmentation of prostanoid synthesis with arachidonic acid or exogenous addition of PGE2 did not decrease AVP-stimulated cAMP in the RPCT cells. Arachidonic acid or PGE2, used alone, increased cAMP and ASA reduced cAMP, consistent with the presence of a PG-sensitive adenylate cyclase. Six-hour incubations of RPCT cells produced clear evidence of homologous desensitization of the PGE2 receptor, after exposure to either PGE2 or arachidonic acid, but not heterologous desensitization of the AVP receptor linked to cAMP synthesis. Preincubation of the RPCT cells with AVP or 1-desamino-8-D-arginine vasopressin (dDAVP) induced homologous desensitization of AVP- or dDAVP-stimulated cAMP. Three-day incubations with dDAVP or AVP apparently induced cyclooxygenase activity since PGE2 synthesis increased in response to arachidonic acid and recovery of cyclooxygenase activity after ASA was enhanced by dDAVP or AVP. In conclusion, our data on AVP-stimulated cAMP in cultured RPCT cells do not support the hypothesis that PGE2 inhibits AVP-dependent collecting tubule cAMP.

Animals↗
PubMed1987

cAMP-associated inhibition of Na+-H+ exchanger in rabbit kidney brush-border membranes.

Adenosine 3',5'-cyclic monophosphate (cAMP) inhibits the rate of bicarbonate reabsorption and the rate of Na+-H+ exchange transport in the apical membrane of the proximal convoluted tubule. To study the relation between cAMP, cAMP-dependent protein kinase, and Na+-H+ exchange transport, brush-border membrane vesicles from the rabbit kidney were phosphorylated in vitro. The rate of proton gradient-stimulated amiloride-inhibitable 22Na+ uptake was measured as an index of Na+-H+ exchange transport activity. The inclusion of cAMP (10(-6) M) in a phosphorylating solution containing ATP decreased the 10-s uptake of amiloride-sensitive sodium from 2.25 +/- 0.21 nmol/mg protein in controls to 1.94 +/- 0.19 (P less than 0.001). Incubation of vesicles in the presence of purified catalytic subunit of cAMP-dependent protein kinase inhibited the amiloride-sensitive uptake of 22Na+ at 10 s from 2.35 +/- 0.49 nmol/mg protein to 2.05 +/- 0.44 (P less than 0.005). The inhibitory effect of both cAMP and catalytic subunit of cAMP-dependent protein kinase was blocked by the specific thermostable protein inhibitor of the kinase. These studies demonstrate that activation of endogenous membrane-bound cAMP-dependent protein kinase or exposure to exogenous catalytic subunit of cAMP-dependent protein kinase inhibits the rate of Na+-H+ exchange transport in the brush-border membrane of the rabbit kidney.

Adenosine Triphosphate↗
PubMed1990

Alpha 2-adrenergic receptors regulate Na(+)-H+ exchange via a cAMP-dependent mechanism.

The mechanism by which alpha 2-adrenergic receptors regulate Na(+)-H+ exchange activity in opossum kidney (OK) cells was studied. Because receptors linked to inhibition of adenylate cyclase, like alpha 2-receptors, also can interact with additional signaling mechanisms, we examined specifically the role of adenosine 3',5'-cyclic monophosphate (cAMP) in the signaling pathway controlling Na(+)-H+ activity in OK cells. Parathyroid hormone (PTH), prostaglandin (PGE1), and forskolin, agents that stimulate cAMP production in these cells, inhibited the rate of amiloride-sensitive 22Na+ uptake by up to 40%. Epinephrine and UK 14304, acting through alpha 2-receptors, were able to reverse this inhibition of 22Na+ uptake to near-control levels and also attenuate PTH-, PGE1-, and forskolin-stimulated cAMP accumulation. Likewise, serotonin (5-HT) and SDZ21-009, acting through 5-HT1b receptors, could reverse inhibition of 22Na+ uptake and also attenuate stimulated cAMP accumulation. Neither epinephrine nor serotonin affected the rate of uninhibited 22Na+ uptake. Pertussis toxin pretreatment abolished the effects of alpha 2- and 5-HT1b receptors on both cAMP accumulation and 22Na+ uptake, suggesting that receptor-mediated inhibition of cAMP accumulation is involved in receptor modulation of Na(+)-H+ exchange activity. In contrast, epinephrine was not able to alter the inhibition of 22Na+ uptake mediated by the membrane-permeant cAMP analogues 8-bromo-cAMP and dibutyryl cAMP at any concentration of analogue that significantly inhibited 22Na+ uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Protein kinase C modulates cAMP content in proximal tubular cells: role of phosphodiesterase inhibition.

The present study was designed to evaluate whether protein kinase C (PKC) activation affects hormone-modulated adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in rabbit renal proximal tubular cells in primary culture. When intracellular cAMP content was measured in the presence of Ro 20-1724, a selective inhibitor of type III phosphodiesterase (PDE), activation of PKC by the phorbol ester phorbol 12-myristate 13-acetate (PMA) or by diacylglycerol kinase inhibitor R 59022 reinforced parathyroid hormone (PTH)- and forskolin-stimulated cAMP accumulation. During PKC activation, the inhibitory effect of norepinephrine on cAMP content persisted, whereas that of angiotensin II (ANG II) was blunted. In contrast, PKC activators had no effect on cAMP content during PDE blockade by the nonspecific inhibitor 3-isobutyl-1-methylxanthine (IBMX). These data suggested that PKC might affect cAMP degradation through inactivation of a Ro 20-1724-insensitive PDE. The possibility that the involved PDE was calcium sensitive was assessed; during PDE inhibition by Ro 20-1724, but not by IBMX, calcium ionophore A23187 inhibited PTH-stimulated cAMP accumulation and PMA abolished the effect of A23187. Finally, neither PKC inhibition by staurosporine nor its downregulation modified the magnitude of PTH-induced cAMP accumulation. In conclusion, 1) in proximal tubular cells PKC affects cAMP degradation rather than synthesis, possibly via inactivation of a calcium-sensitive PDE; 2) PKC modulates PTH-ANG II interaction; and 3) this pathway is likely to play a role in the fine tuning of the effect of PTH and ANG II in the proximal tubule.

Angiotensin II↗
PubMed1994

cAMP stimulates proximal convoluted tubule Na(+)-K(+)-ATPase activity.

The effect of adenosine 3',5'-cyclic monophosphate (cAMP) was examined on the electrophysiological properties of nonperfused proximal convoluted tubule in vitro. In 5 mM bath K+, the basolateral membrane potential (Vbl) was -66 +/- 1 mV (n = 26). Low bath K+ (0.1 mM) led to a transient hyperpolarization of Vbl followed by a sustained decrease to reach -48.6 +/- 5.0 mV. Return to 5 mM bath K+ produced a rapid and transient Vbl hyperpolarization of 24.6 +/- 1.4 mV (n = 5). This hyperpolarization was completely blocked by 100 microM strophanthidin (n = 4), demonstrating that the hyperpolarization was caused by reactivation of the Na(+)-K(+)-adenosinetriphosphatase (ATPase). Addition of 1 microM forskolin (forsk) + 100 microM 8-(4-chlorophenylthio)-cAMP (cp-cAMP) significantly increased this hyperpolarization to 30.8 +/- 10 mV (P < 0.005, n = 5). In a separate series of experiments, addition of 1 microM forsk + 100 microM 3-isobutyl-1-methylxanthine increased this hyperpolarization from 21.7 +/- 2.8 to 27.1 +/- 1.6 mV (P < 0.05, n = 5), which excludes any nonspecific effect of cp-cAMP. Forsk + cp-cAMP decreased the apparent partial conductance to Cl- (tCl) from 0.049 +/- 0.003 to 0.031 +/- 0.007 (P < 0.06, n = 6), decreased that to K+ (tK) from 0.56 +/- 0.05 to 0.43 +/- 0.03 (P < 0.05, n = 6), slightly decreased that mediated by the Na-HCO3 cotransporter (tNaHCO3) from 0.26 +/- 0.03 to 0.21 +/- 0.05, and had no effect on the absolute conductance mediated by the Na-HCO3 cotransporter. Forsk + cp-cAMP had no effect on tK when determined using bath K+ steps from 15 to 45 mM (tK = 0.84 +/- 0.02, n = 5) instead of K+ steps from 5 to 15 mM as previously done, and did not affect the value of tK measured in the presence of strophanthidin (tK = 0.41 +/- 0.03, n = 5). These results demonstrate that the decrease of tK by forsk + cp-cAMP observed using K+ steps from 5 to 15 mM is due to modulation by these agents of the stimulated hyperpolarizing Na(+)-K(+)-ATPase current produced by the bath K+ steps. Consequently, the increased Vbl initial recovery from low bath potassium observed when intracellular cAMP is increased could not be the result of modulation of passive basolateral membrane properties and represents a stimulation of the pump current. The present work thus demonstrates that the Na(+)-K(+)-ATPase is stimulated by cAMP.

Animals↗
PubMed1995

Feedback regulation of Na channels in rat CCT. III. Response to cAMP.

The effects of exogenous adenosine 3',5'-cyclic monophosphate (cAMP) on apical Na channels in the rat cortical collecting tubule were studied using the patch-clamp technique and fura 2 fluorescence measurements of intracellular Ca2+ (Ca2+i). When the permeant analogue, 8-(4-chlorophenylthio)-cAMP (CPT-cAMP, 200 microM), was added to the superfusate during recording from cell-attached patches, both the mean number of open channels (NPo) and the single-channel current (i) decreased within 3 min. When the superfusate also contained amiloride (10 microM), there was no effect of CPT-cAMP on either NPo or i. When CPT-cAMP was added to the bath before formation of the patch, the density of conducting channels was increased from 10 +/- 2 to 37 +/- 6 per patch, as estimated by analysis of channel-induced noise. This suggests that cAMP increases open-channel density in the regions of the apical membrane outside the patch but not within the patch. Channels already active in the patch before stimulation with the nucleotide are subject to feedback inhibition secondary to increased Na entry into the cell. CPT-cAMP increased Ca2+i from 104 to 198 nM. This increase in Ca2+i was abolished by benzamil (0.5 microM) or by low extracellular Ca2+. The cAMP-dependent reduction in NPo was still observed in Ca(2+)-free medium, indicating that a rise in Ca2+i was not essential for the feedback response. The decrease in NPo was attenuated, however, when cAMP was added in the absence of Ca2+ and in the presence of ouabain (1 mM) in the superfusate.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗
PubMed1994

Reversal of pulmonary capillary ischemia-reperfusion injury by rolipram, a cAMP phosphodiesterase inhibitor.

Isoproterenol (ISO) and forskolin, agents that increase adenosine 3',5'-cyclic monophosphate (cAMP) via adenylyl cyclase activation, reverse lung injury associated with increased microvascular permeability. We studied the role of rolipram, a relatively isozyme-selective cAMP phosphodiesterase (PDE) inhibitor, in reversing increased capillary permeability due to ischemia-reperfusion (I/R), a form of oxidant injury in the lung, by using the isolated perfused rat lung model. Rolipram (2 microM) administered after 45 min of ischemia and 45 min of reperfusion reduced I/R-increased permeability as measured by the capillary filtration coefficient to control lung values. Computer image analysis of air space edema and perivascular cuffing, as well as wet-to-dry weight ratios, confirms the permeability reversal by rolipram administration. Rolipram inhibition of cAMP PDE in the lung was assessed by using [3H]adenine prelabeling adapted for the whole lung and perfusate [3H]cAMP accumulation. Rolipram failed to increase perfusate cAMP alone but dramatically increased perfusate cAMP above ISO alone. Dose-response relationships of ISO or rolipram show a close correlation of the half-maximal effective dose (ED50) for injury reversal and perfusate cAMP production. The combination of rolipram and ISO produced synergistic reversal of I/R injury. We conclude that reversal of I/R-induced increased microvascular permeability can be achieved with rolipram and that the mechanism of action of rolipram is probably through PDE isozyme-selective inhibition. The similarity of the ED50 values for cAMP efflux and reversal of permeability increases also supports a close coupling between cAMP accumulation and endothelial cell permeability.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1980

Effects of gastrointestinal hormones and carbamylcholine on cAMP accumulation in isolated pancreatic duct fragments from the rat.

A modification of a technique to isolate kidney tubule fragments and pancreatic acinar cells has been used to prepare a suspension of pancreatic duct fragments from rats with pancreatic lipomatosis due to pretreatment with penicillamine and a copper-free diet. This suspension is 90-95% pure almost without any acinar cell contamination. The accumulation of 3',5'-cyclic adenosine monophosphate (cAMP) in response to various gastrointestinal hormones, hormone-like substances and theophylline was studied in these isolated pancreatic duct fragments. In the absence of theophylline, secretin increased the level of cAMP in a dose-dependent manner with a maximum at 10(-6) M. With supramaximal doses the concentration of cAMP decreased. During maximal stimulation with secretin the level of cAMP was dependent on the concentration of fragments in the incubation mixture. Vasoactive intestinal peptide (VIP) also increased the formation of cAMP. However, VIP was 10 times less effective than secretin on a molar basis. The addition of various concentrations of VIP to a submaximal dose of secretin did not alter cAMP levels as compared to the levels observed with the same concentration of secretin alone. Theophylline (5 x 10(-3) and 10(-2) M) stimulated cAMP accumulation and 5 x 10(-3) M theophylline potentiated the response to secretin and VIP. Pancreozymin (20 and 99% pure), glucagon bovine pancreatic polypeptide and carbamylcholine did not effect the level of cAMP when given alone or in combination with secretin. These data lend support to the hypothesis that cAMP is the intracellular mediator of the action of secretin and VIP on the pancreatic duct cells.

Animals↗
PubMed1987

Mechanism of action of an antiallergic agent, amlexanox (AA-673), in inhibiting histamine release from mast cells. Acceleration of cAMP generation and inhibition of phosphodiesterase.

Amlexanox markedly inhibits histamine release from rat mast cells. To clarify the mechanism of this inhibition, we investigated the effect of amlexanox on cAMP content, which, when increased, inhibits histamine release in rat peritoneal mast cells. At concentrations of 10(-8)-10(-6)M, amlexanox or isoproterenol increased the cAMP content of mast cells over that of control cells about 2-fold. When the mast cells were incubated with 10(-8), 10(-7) and 10(-6) M of amlexanox combined with 10(-7) M isoproterenol, the cAMP contents were synergistically increased 15-, 60- and 88-fold, respectively. 3-Isobutyl-1-methylxanthine (IBMX) at 10(-6)-10(-4) M increased the cAMP content 1.7-3.8-fold, and a combination of 10(-4) M IBMX and 10(-7) M isoproterenol synergistically increased the cAMP content 41-fold. A combination of amlexanox and IBMX synergistically increased the cAMP content 19-fold. The increase in cAMP content, when amlexanox and isoproterenol were combined, was transient; it peaked at 0.5 min after the drugs were administered, then decreased to 20-30% of the peak value about 2 min later. Pretreatment of mast cells with amlexanox reduced the effect of the combination of amlexanox and isoproterenol, indicating tachyphylaxis; pretreatment with IBMX had no such effect. The cAMP content of macrophages was also increased by amlexanox, but when combined with isoproterenol or PGE2, the effect was additive. Amlexanox inhibited cAMP phosphodiesterase in rat mast cells; its IC50 value was 1.4 X 10(-5) M, and its inhibitory activity was half that of IBMX.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminopyridines↗
PubMed1988

In vivo evidence that an adenylate cyclase-cAMP system dilates cerebral arterioles in mice.

Pial arterioles of living mice anesthetized with urethane were monitored by television microscopy. I tested the existence of an adenylate cyclase-cyclic adenosine monophosphate (cAMP) system for dilating the arterioles by topically applying the following drugs: cAMP (10(-3) M), its more potent analogue dibutyryl cAMP (10(-3) and 10(-4) M), and forskolin (10(-6) M). Forskolin activates endogenous adenylate cyclase, which leads to increases in endogenous cAMP. Each drug was applied for 30 seconds; all three produced dilation. I then applied either cAMP or forskolin in the presence or absence of 10(-4) M isobutylmethylxanthine (IMX), an inhibitor of endogenous phosphodiesterase, which destroys cAMP. The presence of IMX significantly potentiated the dilation produced by exogenous cAMP and forskolin. These data indicate that cerebral surface arterioles of mice respond to cAMP with dilation and contain the enzymes for producing and inactivating this dilator. The existence of an adenylate cyclase-cAMP dilating mechanism in pial arterioles does not rule out the simultaneous existence of other dilating mechanisms.

1-Methyl-3-isobutylxanthine↗
PubMed1992

Differential expression of guanine nucleotide-binding proteins enhances cAMP synthesis in regenerating rat liver.

Events leading to cAMP accumulation after partial hepatectomy (PH) and effects of cAMP on hormonal induction of DNA synthesis in hepatocytes were characterized. Hepatic cAMP peaked biphasically post-PH and paralleled changes in adenylyl cyclase activity. Fluctuations in cyclase activity were not explained by variations in glucagon receptor kinetics, but reflected altered G-protein expression. Membrane levels of the stimulatory G-protein, Gs alpha, increased early after PH and were sustained. Levels of the inhibitory G-protein, Gi2 alpha, increased more slowly, peaked later, and quickly fell. Levels of both G-proteins correlated poorly with levels of their mRNAs, suggesting posttranscriptional factors modify their membrane concentrations. When growth factor-induced DNA synthesis was compared in hepatocyte cultures grown with or without agents that increase intracellular cAMP, DNA synthesis was inhibited by sustained high levels of cAMP but was enhanced when high cAMP levels fell. In both regenerating liver and hepatocyte cultures, the expression of a "differentiated" hepatocyte gene, phosphoenolpyruvate carboxykinase, correlated with elevated cAMP levels. These data suggest that the differential expression of G-proteins integrates signals initiated by several growth factors so that the accumulation of cAMP is tightly regulated post-PH. The ensuing variations in cAMP levels modulate both growth and differentiated functions during liver regeneration.

Animals↗
PubMed2002

cAMP inhibits both Ras and Rap1 activation in primary human T lymphocytes, but only Ras inhibition correlates with blockade of cell cycle progression.

Cyclic adenosine monophosphate (cAMP) is a negative regulator of T-cell activation. However, the effects of cAMP on signaling pathways that regulate cytokine production and cell cycle progression remain unclear. Here, using primary human T lymphocytes in which endogenous cAMP was increased by the use of forskolin and 3-isobutyl-1-methylxanthine (IBMX), we show that increase of cAMP resulted in inhibition of T-cell receptor (TCR)/CD3 plus CD28-mediated T-cell activation and cytokine production and blockade of cell cycle progression at the G(1) phase. Increase of cAMP inhibited Ras activation and phosphorylation of mitogen-induced extracellular kinase (MEK) downstream targets extracellular signal-related kinase 1/2 (ERK1/2) and phosphatidylinositol-3-kinase (PI3K) downstream target protein kinase B (PKB; c-Akt). These functional and biochemical events were secondary to the impaired activation of ZAP-70 and phosphorylation of LAT and did not occur when cells were stimulated with phorbol ester, which bypasses the TCR proximal signaling events and activates Ras. Increase of cAMP also inhibited activation of Rap1 mediated by TCR/CD3 plus CD28. Importantly, inhibition of Rap1 activation by cAMP was also observed when cells were stimulated with phorbol ester, although under these conditions Ras was activated and cells progressed into the cell cycle. Thus, TCR plus CD28-mediated activation of ERK1/2 and PKB, cytokine production, and cell cycle progression, all of which are inhibited by cAMP, require activation of Ras but not Rap1. These results indicate that signals that regulate cAMP levels after encounter of T cells by antigen will likely determine the functional fate toward clonal expansion or repression of primary T-cell responses.

1-Methyl-3-isobutylxanthine↗
PubMed2005

cAMP-induced PKCzeta activation increases functional CXCR4 expression on human CD34+ hematopoietic progenitors.

Chemokines are key regulators of hematopoiesis and host defense. We report here that functional expression of the chemokine receptor CXCR4 on human immature CD34+ hematopoietic progenitors was increased as a result of sustained elevation in cellular cAMP by dbcAMP and prostaglandin E2. This effect of cAMP was specifically mediated by PKCzeta activity. CXCR4 expression and PKCzeta activation by cAMP were decreased after the inhibition of cAMP effector-Rap1 by Spa1 overexpression. Interference with the activation of Rac1, a downstream target of Rap1, prevented the cAMP-induced increase in PKCzeta activity and CXCR4 levels. Functional manifestation of the effects of cAMP-elevating agents revealed an increased ability of human CD34+ cells to transmigrate the bone marrow (BM) endothelial layer and adhere to BM stroma in vitro, and it augmented the homing potential to the BM and spleens of immunodeficient mice in a Rac1- and a PKCzeta-dependent manner. cAMP- and TNFalpha-stimulated pathways converged in PKCzeta-activated CXCR4 expression and MMP-2/MMP-9 secretion. cAMP treatment had a beneficial effect on CD34+ cell survival in a PKCzeta-mediated fashion. Taken together, our data reveal major roles for cAMP-induced PKCzeta activation in signaling governing the motility and development of CD34+ cells.

Adult↗
PubMed2002

Potential role of nuclear factor kappaB and reactive oxygen species in cAMP and cytokine regulation of surfactant protein-A gene expression in lung type II cells.

The human surfactant protein-A2 (hSP-A2) gene is developmentally regulated, expressed in type II pneumonocytes, and induced by cAMP. cAMP induction of hSP-A2 expression is O2 dependent and mediated by increased phosphorylation, DNA binding, and transcriptional activation of thyroid transcription factor-1 (TTF-1). The TTF-1-binding element (TBE) at -175 bp contains a reverse-oriented nuclear factor-kappaB (NF-kappaB) binding site. IL-1 increased SP-A expression in lung type II cells and had additive stimulatory effects with cAMP. Nuclear extracts from cAMP- or IL-1-treated type II cells manifested increased binding to NF-kappaB consensus and TBE probes; cAMP and IL-1 had additive effects. Competitive and antibody supershift EMSA revealed that NF-kappaB and TTF-1 interact with TBE. IL-1 treatment of type II cells caused rapid (1 h) increases in nuclear levels of NF-kappaB (p50 and p65) and in binding to NF-kappaB and TBE probes; nuclear levels of TTF-1 were unaffected. Bt2cAMP increased binding to NF-kappaB and TBE probes more slowly; no changes in nuclear levels of p50, p65, or TTF-1 were evident, suggesting that IL-1 and cAMP act by different mechanisms. A role for endogenous NF-kappaB in cAMP and IL-1 regulation of SP-A was suggested by findings that dominant-negative forms of inhibitor of kappaB reduced binding of type II cell nuclear proteins to TBE and inhibited SP-A expression. In cotransfection assays, NF-kappaB and TTF-1 cooperatively interacted at TBE to stimulate SP-A promoter activity; this was further enhanced by IL-1. In coimmunoprecipitation assays using type II cell nuclear extracts, TTF-1 was found to interact with p65 in vivo. Finally, antioxidant inhibitors of NF-kappaB reduced type II cell nuclear protein binding to TBE and blocked stimulatory effects of cAMP on SP-A expression. This provides intriguing evidence that permissive effects of O2/reactive oxygen species on cAMP regulation of SP-A expression may be mediated by cooperative interactions of TTF-1 and NF-kappaB at the TBE.

Cell Line↗
PubMed1998

Sequence-specific protein interaction with a transcriptional enhancer involved in the autoregulated expression of cAMP receptor 1 in Dictyostelium.

Major stages of Dictyostelium development are regulated by secreted, extracellular cAMP through activation of a serpentine receptor family. During early development, oscillations of extracellular cAMP mobilize cells for aggregation; later, continuous exposure to higher extracellular cAMP concentrations downregulates early gene expression and promotes cytodifferentiation and cell-specific gene expression. The cAMP receptor 1 gene CAR1 has two promoters that are differentially responsive to these extracellular cAMP stimuli. The early CAR1 promoter is induced by nM pulses of cAMP, which in turn are generated by CAR1-dependent activation of adenylyl cyclase (AC). Higher, non-fluctuating concentrations of cAMP will adapt this AC stimulus-response, repress the activated early promoter and induce the dormant late promoter. We now identify a critical element of the pulse-induced CAR1 promoter and a nuclear factor with sequence-specific interaction. Mutation of four nucleotides within the element prevents both in vitro protein binding and in vivo expression of an otherwise fully active early CAR1 promoter and multimerization of the wild-type, but not mutant, sequence will confer cAMP regulation to a quiescent heterologous promoter. These cis and trans elements, thus, constitute a part of the molecular response to the cAMP transmembrane signal cascade that regulates early development of Dictyostelium.

Animals↗
PubMed1994

cAMP does not regulate [Ca2+]i in human tracheal epithelial cells in primary culture.

Human tracheal epithelial cells in primary culture respond to different receptor agonists with different peak intracellular calcium concentrations. From resting concentration 138 +/- 13 nM, bradykinin (0.1 microM) produces an increase to a maximum of 835 +/- 195 nM, histamine (10 microM) to 352 +/- 51 nM, and ATP (5-500 microM) to more than 1500 nM. Nine of 14 cultures also responded to isoproterenol (10 microM), though with a smaller increase, to 210 +/- 29 nM. A response was observed with isoproterenol, and epinephrine, but not norepinephrine, phenylephrine or methoxamine, was inhibited by propranolol but not phentolamine, and so this appeared to be a beta-adrenergic response. However, no response could be detected to adenosine, prostaglandin E2 or forskolin, agents that activate adenylate cyclase, or to permeant analogs of cAMP (CPT-cAMP or db-cAMP). The intracellular calcium response to isoproterenol did not follow either the time-course or the desensitization pattern of the cAMP response. Thus, this response to isoproterenol is not mediated by cAMP. No relation was demonstrated between cAMP production by other agonists and the response of intracellular calcium. Pretreatment with agents that increase cAMP did not affect the calcium responses to ATP or bradykinin. Thus, cAMP does not regulate intracellular calcium concentration in human tracheal epithelial cells. The variation in peak intracellular calcium responses to various agonists may be explained by the presence of multiple second messengers (other than cAMP), multiple intracellular pools of calcium, or cell heterogeneity. The agonists tested had the same relative potency in cells from patients with cystic fibrosis as in non-cystic fibrosis cells.

Adenosine Triphosphate↗
PubMed2002

Increase of cAMP upon release from prophase arrest in surf clam oocytes.

Surf clam (Spisula solidissima) oocytes are spawned at the prophase I stage of meiosis, and they remain arrested at this stage until fertilization. Full oocyte meiosis reinitiation, first evidenced by germinal vesicle breakdown (GVBD), may be induced by artificial activators mimicking sperm, such as high K(+) or serotonin. Previous reports indicated that treatments thought to increase the level of oocyte cAMP inhibited sperm- or serotonin-induced, but not KCl-induced, GVBD in clam oocytes. These observations extend the well known requirement for a drop in oocyte cAMP levels in mammalian, amphibian or starfish oocytes and support the view that such a drop is universally important throughout the animal kingdom. We have re-examined the cAMP dependency of GVBD in clam oocytes and found that various treatments that raise oocyte cAMP levels did not, surprisingly, affect either KCl- or serotonin-induced GVBD. Such treatments, however, inhibited GVBD upon insemination of the oocytes, but this was due to the failure of sperm to fuse/penetrate the oocytes; thus, it was not an inhibition of oocyte activation as such. Direct measurements of oocyte cAMP levels after activation by serotonin, KCl or sperm showed that, contrary to expectations, there is a rise in cAMP levels before GVBD. Using SQ22536, an adenylyl cyclase inhibitor, the increase in oocyte cAMP level was partly prevented and GVBD proceeded, but with a significant retardation, indicating that the normal cAMP rise facilitates GVBD. Our work sheds light on the diversity of upstream pathways leading to activation of MPF and provides a unique model whereby the onset of meiosis reinitiation is associated with an increase, not a decrease, in oocyte cAMP levels.

1-Methyl-3-isobutylxanthine↗
PubMed1985

[The role of Ca ions and cAMP in ACTH lipolysis].

Ca ions and cAMP are known to be involved in the action of hormones. In this study, the actions of Ca ions and cAMP in ACTH lipolysis were investigated by comparing ACTH1-24, ACTH1-10 and ACTH11-24 with regard to: lipolytic activity, binding affinity to the fat cell membrane, changes in Ca ion binding to the fat cell membrane and cAMP formation. Rat adipocytes were used. Lipolytic activity was measured in terms of the amount of FFA released from fat cells into the medium after incubation with the ACTH analogs at 37 degrees C for 30 minutes. Binding affinity to the fat cell membrane was expressed as competitive binding affinity, determined by simultaneously incubating fat cell ghosts with 125I-ACTH1-24 and unlabelled ACTH analogs at 4 degrees for 40 minutes. Changes in Ca ion binding to the fat cell membrane was expressed as Ca binding capacity, measured by simultaneously incubating fat cell ghosts with 45Ca and ACTH analogs at 24 degrees C for 20 minutes. CAMP formation was measured by RIA as the amount of cAMP formed in fat cells incubated with 10(-6)M ACTH analogs at 37 degrees C for 0, 3, 5 and 10 minutes. FFA release after incubation with each of the ACTH analogs at 10(-6) M was 1.17 mEq/L/tube for ACTH1-24, 0.26 mEq/L/tube for ACTH1-10 and 0.25 mEq/L/tube for ACTH11-24, showing a high value only for ACTH1-24. The binding affinity to the fat cell membrane (high affinity constant) was 4.4 X 10(-8) M for ACTH1-24, 4.6 X 10(-8) M for ACTH1-10 and 4.6 X 10(-8) M for ACTH11-24. There were no significant differences among the binding affinities of these ACTH analogs. The binding capacity of 45Ca++ to the fat cell membrane (high affinity constant) was 3.64 X 10(-6) M for ACTH1-24, 4.49 X 10(-6) M for ACTH1-10 and 5.46 X 10(-6) M for ACTH11-24. There were no significant differences among these ACTH analogs. The increase in cAMP formation during the first 3 minutes of incubation was 35.5 fmol/mg dry weight of fat cells for ACTH1-24, 2.2 fmol/mg for ACTH1-10 and 1.8 fmol/mg for ACTH11-24. An increase was only observed for ACTH1-24. The fact that FFA release and cAMP formation were high only with ACTH1-24 among the ACTH analogs tested indicates that cAMP formation plays an important role in the lipolytic activity of ACTH in fat cells and that the three-dimensional structure of ACTH is important for cAMP formation.

Adipose Tissue↗