Rapid stimulation of human renal ENaC by cAMP in Xenopus laevis oocytes

Among the compensatory mechanisms restoring circulating blood volume after severe haemorrhage, increased vasopressin secretion enhances water permeability of distal nephron segments and stimulates Na+ reabsorption in cortical collecting tubules via epithelial sodium channels (ENaC). The ability of vasopressin to upregulate ENaC via a cAMP-dependent mechanism in the medium to long term is well established. This study addressed the acute regulatory effect of cAMP on human ENaC (hENaC) and thus the potential role of vasopressin in the initial compensatory responses to haemorrhagic shock. The effects of raising intracellular cAMP (using 5 mmol/L isobutylmethylxanthine (IBMX) and 50 ìmol/L forskolin) on wild-type and Liddle-mutated hENaC activity expressed in Xenopus oocytes and hENaC localisation in oocyte membranes were evaluated by dual-electrode voltage clamping and immunohistochemistry, respectively. After 30 min, IBMX + forskolin had stimulated amiloride-sensitive Na+ current by 52 % and increased the membrane density of Na+ channels in oocytes expressing wild-type hENaC. These responses were prevented by 5 ìmol/L brefeldin A, which blocks antegrade vesicular transport. By contrast, IBMX + forskolin had no effects in oocytes expressing Liddle-mutated hENaC. cAMP stimulated rapid, exocytotic recruitment of wild-type hENaC into Xenopus oocyte membranes, but had no effect on constitutively over-expressed Liddle-mutated hENaC. Extrapolating these findings to the early cAMP-mediated effect of vasopressin on cortical collecting tubule cells, they suggest that vasopressin rapidly mobilises ENaC to the apical membrane of cortical collecting tubule cells, but does not enhance ENaC activity once inserted into the membrane. We speculate that this stimulatory effect on Na+ reabsorption (and hence water absorption) may contribute to the early restoration of extracellular fluid volume following severe haemorrhage (AU)

Bioactivatable, membrane-permeant analogs of cyclic nucleotides as biological tools for growth control of C6 glioma cells.

In the present study, the cAMP analogs 8-bromo-cAMP (8-Br-cAMP), N6-2'O-dibutyryl-cAMP (DBcAMP) and 8-para-chlorophenylthio-cAMP (8-CPT-cAMP), as well as the corresponding cAMP-acetoxymethyl (AM)-ester-prodrugs were tested in a HPLC study for their membrane permeability, intracellular accumulation and biotransformation. Antiproliferative activities of these compounds were studied in the rat C6 glioma cell line. Chromatographic analysis revealed that the AM-ester analogs of the cyclic nucleotides penetrate quantitatively into rat C6 glioma cells and generate high amounts of their parent cyclic nucleotides intracellularly within 60 min; however, long-term growth inhibition tested in C6 cells is only slightly enhanced with the AM-ester prodrugs of 8-Br-cAMP or DBcAMP.

Vasodilative effects of urocortin II via protein kinase A and a mitogen-activated protein kinase in rat thoracic aorta.

Four corticotropin-releasing factor (CRF)-related peptides have been found in mammals and are known as CRF, urocortin, urocortin II, and urocortin III (also known as stresscopin). The three urocortins have considerably higher affinities for CRF receptor type 2 (CRF R2) than CRF, and urocortin II and urocortin III are highly selective for CRF R2. In the present study, the authors examined the hypothesis that urocortin II or urocortin III, in addition to urocortin, produces vasodilation as a candidate for natural ligands of CRF R2beta in rat thoracic aorta. Involvement of protein kinases on urocortin-induced vasodilation was also explored. The vasodilative effects of urocortin II and urocortin III were more potent than that of CRF, but less potent than that of urocortin. Urocortin II-induced vasodilation was significantly attenuated by a CRF R2-selective antagonist, antisauvagine-30. Both SQ22536, an adenylate cyclase inhibitor, and Rp-8-Br-cAMPS, a protein kinase A (PKA) inhibitor, were found to attenuate the urocortin II-induced vasodilation. SB203580, a p38 mitogen-activated protein (MAP) kinase inhibitor, also inhibited the effects of urocortin and urocortin II on vasodilation. Thus, urocortins contribute to vasodilation via p38 MAP kinase as well as PKA pathways.

The expression of hCG receptor mRNA in four human ovarian cancer cell lines varies considerably under different experimental conditions.

In the present study, expression and regulation of hCG receptor mRNA were analyzed in four established human ovarian cancer cell lines using different concentrations of hCG, EGF, and 8-bromo-cAMP for different periods between 6 and 72 h. The cells were examined for the hCG receptor using the reverse-transcriptase polymerase chain reaction with specific primers amplifying the hCG receptor gene. Receptor mRNA was found in all cell lines. In the line OVCAR-3, it was expressed in all samples independent of kind and concentration of the receptor agonist and incubation period. In the line COLO-704, the hCG receptor gene was expressed only in unstimulated samples, but not in the samples incubated with a receptor agonist. The cell line EFO-21 showed a downregulation of receptor mRNA after 24 h of treatment with 25 IU/ml hCG and after 6 h of treatment with 250 IU/ml hCG or 100 ng/ml EGF. The mRNA reappeared within 24-48 h. The cell line EFO-27 showed a downregulation of receptor mRNA after 6 h of incubation with 250 IU/ml hCG. Agarose gel electrophoresis and sequencing of the polymerase chain reaction products revealed four cDNA fragments resulting from an alternative splicing of the primary transcript. The results of the study demonstrate that the expression of hCG receptor mRNA in ovarian cancer cell lines varies considerably under different experimental conditions. We showed that ovarian cancer cells can produce hCG receptors when needed or wanted. The inherent mechanisms which rule this phenomenon need further evaluation.

8-Cl-adenosine mediated cytotoxicity and sensitization of T-lymphoblastic leukemia cells to TNFalpha-induced apoptosis is via inactivation of NF-kappaB.

These data show that 8-Cl-cAMP is cytotoxic to the lymphoblastic leukemia cell line CEM and its vinblastine selected multidrug resistant derivative, CEM/VLB100 although PKA was not involved in these effects. The cytotoxic effects of 8-Cl-cAMP was abrogated by cotreatment with either ADA or IBMX which indicated a degradation form of 8-Cl-cAMP was needed for this cytotoxicity. CEM and CEM/VLB100 cells displayed a notable sensitivity to 8-Cl-adenosine-induced growth inhibition and apoptosis. 8-Cl-adenosine increased the cytosolic levels of IkappaBalpha which prevented NF-kappaB nuclear translocation. 8-Cl-adenosine also prevented TNFalpha-induced IkB decay and NF-kappaB activation in CEM and CEM/VLB100 cells.

Inhibition of basolateral cAMP permeability in the toad urinary bladder.

1. The effect of sulphonylurea drugs on hydrosmotic flow across toad urinary bladder epithelium was re-evaluated in the present study. Glibenclamide, added to the basolateral medium, significantly enhanced the osmotic flow induced by low doses of antidiuretic hormone (ADH) or forskolin (FK), while it inhibited the effect of exogenous cyclic adenosine monophosphate (cAMP) or its non-hydrolysable bromo derivative, 8-Br-cAMP, added to the basolateral medium. These opposite effects of glibenclamide on the transepithelial osmotic flow can be explained by a reduction of cAMP permeability across the basolateral membrane of the epithelium. The decrease in cAMP permeability leads, according to the direction of the cAMP gradient, to firstly an enhanced osmotic flow when cAMP is generated intracellularly by addition of ADH and FK, glibenclamide reducing cAMP exit from the cell, and secondly a decreased osmotic flow in response to cAMP (and 8-Br-cAMP) added to the basolateral medium, glibenclamide inhibiting, in this case, their entry into the cell. 2. The demonstration that glibenclamide actually inhibits the basolateral cAMP permeability rests on the fact that firstly it decreases the release of cAMP into the basolateral medium by about 40 %, at each concentration of ADH or forskolin tested, secondly it increases the cAMP content of paired hemibladders incubated in the presence of ADH or FK, when intracellular degradation was prevented by phosphodiesterase inhibition, and thirdly it decreases also the uptake of basolateral 8-Br-[3H]cAMP into paired toad hemibladders. 3. Taken together, the present data demonstrate that glibenclamide inhibits the toad urinary bladder basolateral membrane permeability to cAMP, most probably by a direct interaction with a membrane protein not yet indentified but distinct from the sulphonylurea receptor.

Phase I study of the novel cyclic AMP (cAMP) analogue 8-chloro-cAMP in patients with cancer: toxicity, hormonal, and immunological effects.

The cyclic AMP (cAMP)-dependent protein kinase regulatory subunit RI is overexpressed in cancer cells. 8-Chloro-cAMP (8-Cl-cAMP) is an RII site-specific analogue that down-regulates RI and inhibits the growth of a wide range of cancer cells in vitro and in vivo. We performed a Phase I trial of 8-Cl-cAMP in 32 patients with malignancies that were refractory to standard treatments. 8-Cl-cAMP was initially given in a 1-month cycle by constant infusion at 0.005 mg/kg/h for 21 days, followed by 1 week of rest. The dose was escalated to 0.045 mg/kg/h, but hypercalcemia became the dose-limiting toxicity. The length of drug administration was, therefore, reduced to 5 days per week for the first 3 weeks of the cycle, but it was not possible to increase the drug dose without producing hypercalcemia. Hence, the length of drug administration was reduced to 3 days per week for the first 3 weeks of the cycle. The maximum tolerated dose for this regimen was 0.15 mg/kg/h, and the dose-limiting toxicities were reversible hypercalcemia and hepatotoxicity. Stable disease for > or =4 months was observed in two patients treated at > or =0.045 mg/kg. cAMP-dependent protein kinase is involved in hormone- and cytokine-mediated signaling, and so representative hormone, cytokine, and peripheral lymphocyte subsets were measured. The drug had a parathyroid hormone-like effect on calcium homeostasis and significantly increased circulating luteinizing hormone and 17-hydoxyprogesterone levels (P < 0.02 and P < 0.0006, respectively). We conclude that 8-Cl-cAMP is well tolerated without attendant myelotoxicity, and in this study, it was associated with biological effects. In Phase II studies, a dose of 0.11 mg/kg/h for 3 days per week would be appropriate.

Pharmacokinetics, metabolism and tumour disposition of 8-chloroadenosine 3',5'-monophosphate in breast cancer patients and xenograft bearing mice.

BACKGROUND: 8-Chloroadenosine 3',5'-monophosphate (8-Cl-cAMP) is undergoing phase I clinical trials as an anticancer drug. However, there is debate as to whether it is a prodrug for its 8-Cl-adenosine metabolite. DESIGN: Pharmacokinetics, metabolism and tumour disposition studies have been performed in 7 breast cancer patients receiving continuous infusion (28 day) 8-Cl-cAMP (0.54 or 1.08 mg/kg/day) and tumour biopsies were obtained before and on the last day of infusion. Parallel studies were performed in nude mice bearing the HT29 human colon cancer xenograft after continuous infusion (7 day) of active drug doses (50 or 100 mg/kg/day). RESULTS: Steady state plasma levels (Css) of 8-Cl-cAMP in patients ranged from 0.15-0.72 microM but 8-Cl-adenosine was not detected in plasma. In contrast, 8-Cl-cAMP was not detectable in 3 tumour biopsies but 8-Cl-adenosine was present in 2 samples at high concentrations (1.33 and 2.02 microM). In mice, Css of 8-Cl-cAMP ranged from 3.2-4.6 microM and 8-Cl adenosine was present in plasma only at the higher dose (100 mg/kg/day, peak concentration of 2.3 microM). In the HT29 xenograft, 8-Cl-cAMP levels were considerably lower than in plasma (0.37-1.22 microM) while 8-Cl-adenosine was present at 5.3-21.0 microM and 8-Cl-AMP was found at 11.3-35.7 microM. CONCLUSIONS: The fate of 8-Cl-cAMP in human tumours is characterised by extensive metabolism to products which are not generally observed in plasma. These data raise the possibility that 8-Cl-cAMP is a prodrug for a product of its metabolism in human tumours.

Inhibition of colon tumor cell growth by 8-chloro-cAMP is dependent upon its conversion to 8-chloro-adenosine.

Recent interest in site-selective cAMP analogs has focused on the role of 8-chloro-adenosine (8-Cl-adenosine) in the inhibition of tumor cell growth by 8-chloro-cAMP (8-Cl-cAMP) (Van Lookeren Campagne, et al. Cancer Res 1991; 51: 1600-5). We have evaluated 8-Cl-cAMP and 8-Cl-adenosine for their growth inhibitory activity against two human colon adenocarcinoma cell lines, HCT116 and FET. Because these cell lines have been adapted to grow in chemically defined medium we were able to evaluate the effect of serum on 8-Cl-cAMP's growth inhibitory activity. In addition, cells grown in serum-free medium were tested for their sensitivity to 8-Cl-cAMP, serum-activated 8-Cl-cAMP and 8-Cl-adenosine. IC50 values, determined by measuring cell growth using a MTT colorimetric assay, showed that 'serum activation' of 8-Cl-cAMP was required to achieve inhibition of HCT116 (IC50 = 1.3 +/- 0.1 microM) and FET (IC50 = 2.0 +/- 0.1 microM) cell growth. IC50 values were not reached at the highest concentrations tested (IC50 > 500 microM) in the absence of serum, permitting us to conclude that 8-Cl-cAMP does not have growth inhibitory activity between 1.0 and 500 microM doses. HCT116 and FET cells grown in media containing serum and in the presence of 8-Cl-adenosine had IC50 values of 0.6 +/- 0.1 and 0.9 +/- 0.2 microM, respectively. HCT116 and FET cells grown in chemically defined medium containing 8-Cl-adenosine exhibited IC50 values of 1.0 +/- 0.1 and 3.1 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of cyclic nucleotide analogs on intraocular pressure and trauma-induced inflammation in the rabbit eye.

In this study the effects of cell-permeable 8-bromo-cAMP and 8-bromo-cGMP on intraocular pressure (IOP) and puncture-induced inflammatory response were investigated. Both 8-bromo-cAMP and 8-Bromo-cGMP reduced IOP when given subconjunctivally, but not topically. Subconjunctival administration of 8-bromo-cAMP induced a moderate disruption of the blood-aqueous barrier (BAB); in addition, subconjunctival 8-bromo-cAMP, but not topical 8-bromo-cAMP or subconjunctival 8-bromo-cGMP, reduced the disruption of the BAB and elevation of the aqueous PGE2 level after puncture trauma. It is concluded that the effects of 8-bromo-cAMP depend on the mode of administration, since this determines the concentration of 8-bromo-cAMP reached in the aqueous humor. It is suggested that 8-bromo-cAMP can partially suppress a slight inflammatory response by interference with the release of arachidonic acid from the tissues surrounding the aqueous humor.