Inhibition of forskolin-stimulated cAMP formation in vitro by paraoxon and chlorpyrifos oxon in cortical slices from neonatal, juvenile, and adult rats

Parathion (PS) and chlorpyrifos (CPF) are organophosphorus insecticides, which elicit toxicity following biotransformation to the potent acetylcholinesterase inhibitors, paraoxon (PO) and chlorpyrifos oxon (CPO). Both oxons have also been shown to interact directly with muscarinic receptors coupled to inhibition of adenylyl cyclase. Immature animals are more sensitive than adults to the acute toxicity of PS and CPF but little is known regarding possible age-related differences in interactions between these toxicants and muscarinic receptors. We compared the inhibition of forskolin-stimulated cAMP formation by PO and CPO (1 nM-1 mM) in vitro in brain slices from 7-, 21-, and 90-day-old rats to the effects of well-known muscarinic agonists, carbachol and oxotremorine (100 microM). Both agonists inhibited cAMP formation in tissues from all age groups and both were more effective in adult and juvenile (20-26% inhibition) than in neonatal (12-13% inhibition) tissues. Atropine (10 microM) completely blocked agonist-induced inhibition in all cases. PO maximally inhibited (37-46%) cAMP formation similarly in tissues from all age groups, but atropine blocked those effects only partially and only in tissues from 7-day-old rats. CPO similarly inhibited cAMP formation across age groups (27-38%), but ATR was partially effective in tissues from all three age groups. Both oxons were markedly more potent in tissues from younger animals. We conclude that PO and CPO can directly inhibit cAMP formation through muscarinic receptor-dependent and independent mechanisms and that the developing nervous system may be more sensitive to these noncholinesterase actions.     

Developmental Changes in the Modulation of Cyclic AMP Formation by the Metabotropic Glutamate Receptor Agonist 1S,3R-Aminocyclopentane-1,3-Dicarboxylic Acid in Brain Slices

Metabotropic glutamate receptors (mGluRs) have been recently described as a family of guanine nucleotide-binding regulatory protein-coupled receptors with multiple signal transduction pathways. At least one of these receptors appears to be negatively coupled to adenylyl cyclase when stably expressed in transfected cells. We have studied how activation of native mGluRs modulates cyclic AMP (cAMP) formation in brain slices prepared from rats at different ages. 1S,3R-1-Aminocyclopentane-1,3-dicarboxylic acid (1S,1R-ACPD), a selective agonist of mGluRs, slightly increased basal cAMP formation but reduced forskolin-stimulated cAMP formation in adult hippocampal slices, in agreement with previous results. The action of 1S,3R-ACPD on basal cAMP formation was not reproduced by the ionotropic receptor agonists N-methyl-D-aspartate, kainate, and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate and was antagonised by L-2-amino-3-phosphonopropionate (L-AP-3). L-AP-3, however, did not prevent but rather mimicked the inhibitory action of 1S,3R-ACPD on forskolin-stimulated cAMP formation. In hippocampal slices from 1-, 8-, or 15-day-old rats, 1S,3R-ACPD increased basal cAMP formation but failed to reduce the action of forskolin. A similar development pattern of modulation was observed in hypothalamic slices with the difference that 1S,3R-ACPD did not stimulate basal cAMP formation in the hypothalamus of adult animals. These results suggest that inhibition of forskolin-stimulated cAMP formation by 1S,3R-ACPD is mediated by a specific mGluR subtype that is preferentially expressed in the adult.     

Adenosine A 2B receptors modulate cAMP levels and induce CREB but not ERK1/2 and p38 phosphorylation in rat skeletal muscle cells

The present study examined the existence of the adenosine A(1),A(2A), and A(2B) receptors and the effect of receptor activation on cAMP accumulation and protein phosphorylation in primary rat skeletal muscle cells. Presence of mRNA and protein for all three receptors was demonstrated in both cultured and adult rat skeletal muscle. NECA (10(-9)-10(-4)M) increased the cAMP concentration in cultured muscle cells with an EC(50) of (95% confidence interval)=15 (5.9-25.1) micro M, whereas CGS 21680 (10(-9)-10(-4)M) had no effect on cAMP accumulation. Concentrations of [R]-PIA below 10(-6)M had no effect on cAMP accumulation induced by either isoproterenol or forskolin. NECA resulted in phosphorylation of CREB with an EC(50) of (95% confidence interval)=1.7 (0.40-7.02) micro M, whereas ERK1/2 and p38 phosphorylation was unchanged. The results show that, although the A(1),A(2A), and A(2B) receptors are all present in skeletal muscle cells, the effect of adenosine on adenylyl cyclase activation and phosphorylation of CREB is mainly mediated via the adenosine A(2B) receptor.     

The role of inner membrane in the realization of cAMP-dependent activation of mitochondrial enzymes

It was shown that the increase in the activities of transhydrogenase and NAD(+)-dependent isocitrate dehydrogenase after incubation of mitochondria with cAMP is due to the stimulating effect of cAMP on mitochondria, but not to the increased stability of mitochondria to the incubation procedure. Treatment of mitochondria with trypsin prevents the action of cAMP on the both enzymes. The integrity of the inner mitochondrial membrane is necessary for the manifestation of cAMP effect. Pretreatment of mitochondria with the local anesthetic, lidocaine, prevents the activation of NAD(P)(+)-transhydrogenase and NAD(+)-dependent isocitrate dehydrogenase during subsequent incubation of mitochondria with cAMP. It is concluded that the role of the inner mitochondrial membrane consists in the reception of the cAMP signal for the internal compartment of mitochondria, i.e. for mitoplasts. Peripheral protein(s) on the external side of the inner mitochondrial membrane seems to play a role in cAMP reception.     

cAMP induces a rapid and reversible modification of the chemotactic receptor in Dictyostelium discoideum

Stimulation, within 1 min after cAMP stimulation, of aggregation-competent Dictyostelium discoideum amebae was found to cause a rapid (within 1 min) modification of the cell's surface cAMP receptor. The modified receptor migrated on SDS PAGE as a 47,000-mol-wt protein, as opposed to a 45,000-mol-wt protein labeled on unstimulated cells. The length of time this modified receptor could be detected depended upon the strength of the cAMP stimulus: 3-4 min after treatment with 10(-7) M cAMP, cells no longer possessed the 47,000-mol-wt form of the cAMP receptor. Instead, the 45,000-mol-wt form was present. Stimulation of cells with 10(-5) M cAMP, however, resulted in the persistent (over 15 min) expression of the modified receptor. The time course, concentration dependence, and specificity of stimulus for this cAMP-induced shift in the cAMP receptor were found to parallel the cAMP-stimulated phosphorylation of a 47,000-mol-wt protein. In addition, both phenomena were shown to occur in the absence of endogenous cAMP synthesis. The possibility that the cAMP receptor is phosphorylated in response to cAMP stimulation, and the role of this event in cell desensitization, are discussed.     

Ovarian dysfunction in streptozotocin-induced diabetic rats

The effect of streptozotocin diabetes on some ovarian functions in adult rats was examined. Diabetic diestrus animals showed reduced ovary weight and lower circulating levels of progesterone. Scatchard plots of binding data derived from ovarian particulate fractions of normal and streptozotocin diabetic rats revealed the presence of one class of binding sites with high affinity for 125I-hCG. The apparent association constant of the hCG receptors of diabetic ovaries was comparable to that of normal gonads. However, a marked decrease (42%) in the number of hCG binding sites was found in diabetic animals. With isolated luteal cells similar results were obtained, and the administration of insulin to streptozotocin diabetic rats restored to normality the number of hCG binding sites. The maximal response of progesterone production by luteal cells from control ovaries was obtained with 10(-10) M hCG. A 100-fold higher concentration of hCG was required for the maximum stimulation of cAMP synthesis. The cAMP response of cells from diabetic rats was significantly higher than that of control cells. However, luteal cells from diabetic rats showed some loss of sensitivity in the synthesis of progesterone during incubation with hCG. Most of the alterations seen in diabetic female rats could be restored with insulin therapy, indicating that insulin plays an important role in the regulation and maintenance of normal reproductive functions. It is suggested that the diminution of the LH receptor population causes the disruption of normal luteal cell function. This fact could be responsible for some of the reproductive alterations in the diabetic female rat.     

The in vitro effects of histamine and metiamide on neutrophil motility and their relationship to intracellular cyclic nucleotide levels.

Histamine at concentrations of 1 x 10(-5) M to 5 x 10(-5) M consistently increased neutrophil movement as measured in Boyden chambers. This effect was entirely caused by stimulation of chemokinesis (stimulated random migration) and true chemotaxis was inhibited by these concentrations. This inhibition of chemotaxis could be abolished by pretreatment with metiamide, an H-2 receptor antagonist, and levamisole, but not by diphenylhydramine, an H-1 receptor antagonist. Metiamide at similar concentrations produced a mild stimulation of chemokinesis but has no effect on true chemotaxis. The histamine effects on neutrophil motility were associated with increased levels of intracellular cAMP wehreas cAMP levels were unaffected. Agents known to elevate intracellular cAMP levels produced effects on neutrophil motility similar to those of histamine. It is suggested that histamine exerts a 2-fold effect on neutrophil motility mediated via an H-2 receptor site and associated with elevated levels of cAMP.     

Adaptation in the motility response to cAMP in Dictyostelium discoideum

When developing amebae of Dictyostelium discoideum are treated with constant concentrations of cAMP above 10(-8)M, the average rate of motility is depressed, with maximum inhibition at roughly 10(-6)M. It is demonstrated that shifting the concentration of cAMP from 0 M to concentrations ranging from 10(-8) to 10(-6)M in a perfusion chamber results in the immediate inhibition of motility. After shifting from 0 M to 10(-8) or 10(-7)M, the rate of cell motility remains low, then rebounds to a higher level, exhibiting a standard adaptation response. No adaptation is exhibited after a shift from 0 M to 10(-6)M, a concentration resulting in maximum inhibition. It is demonstrated that the level of inhibition and the extent of the adaptation period are dependent upon the concentration of cAMP after the shift, and that submaximal inhibition is additive. The characteristics of adaptation in this motility response are very similar to the characteristics of adaptation for the relay system and phosphorylation of the putative cAMP receptor.     

Agonist but not phorbol ester induced desensitization of human lymphocyte prostaglandin receptor is dependent on tubulin polymerization

The regulation of prostaglandin stimulated cAMP accumulation in cells of the human T-cell leukemia line Jurkat was examined. Pretreatment with PGE2 (0.1-10 nM) for 2 hour caused a concentration dependent desensitization of the prostaglandin receptor. Tumor promoting phorbol esters (1-1000 nM) could also inhibit PGE2 stimulated cAMP production dose dependently. Inhibition of tubulin polymerization with colchicine or nocodazole (1 microM) eliminated prostaglandin but not phorbol ester induced desensitization of the receptor. It is concluded that agonist and phorbol ester induced desensitization are mediated by two distinct mechanisms and that tubulin polymerization appear to be required only for agonist induced desensitization of the prostaglandin receptor.     

Deregulated hepatic metabolism exacerbates impaired testosterone production in Mrp4-deficient mice

The physiological role of multidrug resistance protein 4 (Mrp4, Abcc4) in the testes is unknown. We found that Mrp4 is expressed primarily in mouse and human Leydig cells; however, there is no current evidence that Mrp4 regulates testosterone production. We investigated its role in Leydig cells, where testosterone production is regulated by cAMP, an intracellular messenger formed when the luteinizing hormone (LH) receptor is activated. Because Mrp4 regulates cAMP, we compared testosterone levels in Mrp4(-/-) and Mrp4(+/+) mice. Young Mrp4(-/-) mice had significantly impaired gametogenesis, reduced testicular testosterone, and disruption of Leydig cell cAMP homeostasis. Both young and adult mice had impaired testosterone production. In Mrp4(-/-) primary Leydig cells treated with LH, intracellular cAMP production was impaired and cAMP-response element-binding protein (CREB) phosphorylation was strongly attenuated. Notably, expression of CREB target genes that regulate testosterone biosynthesis was reduced in Mrp4(-/-) Leydig cells in vivo. Therefore, Mrp4 is required for normal Leydig cell testosterone production. However, adult Mrp4(-/-) mice are fertile, with a normal circulating testosterone concentration. The difference is that in 3-week-old Mrp4(-/-) mice, disruption of gonadal testosterone production up-regulates hepatic Cyp2b10, a known testosterone-metabolizing enzyme. Therefore, defective testicular testosterone production de-regulates hepatic Cyp-mediated testosterone metabolism to disrupt gametogenesis. These findings have important implications for understanding the side effects of therapeutics that disrupt Mrp4 function and are reported to alter androgen production.     

Induction of post-aggregative differentiation in Dictyostelium discoideum by cAMP: Evidence of involvement of the cell surface cAMP receptor

Exogenous cAMP is known to induce post-aggregative differentiation in Dictyostelium discoideum under conditions that normal development is blocked. We have analysed the cyclic nucleotide specificity, the effect of modulation of the cAMP signal and the dose-response relationship of the induction of two independent markers of post-aggregative differentiation, i.e., a prespore cell-specific antigen detected by a monoclonal antibody, and the activity of glycogen phosphorylase. Our results confirm that high concentrations of cAMP (10(-6)-10(-3)M) are required for the induction of these markers. The cells are shown not to adapt to the cAMP signal. The cyclic nucleotide specificity of induction agrees with the specificity of the cell surface cAMP receptor, but is very dissimilar to the specificity of the intracellular cAMP-dependent protein kinase. It is thus unlikely that cAMP leaks into the cell and activates the cAMP-dependent protein kinase directly. Instead, the induction of post-aggregative differentiation by cAMP seems to be mediated by cell surface cAMP receptors.     

Presence of VIP receptors in a human pancreatic adenocarcinoma cell line. Modulation of the cAMP response during cell proliferation

It is known that the human exocrine pancreas responds to secretin stimulation more than does VIP, a structurally related peptide. We looked for the receptors for those polypeptides in a human pancreatic cancer cell line grown in culture and in nude mice. By analysing the cAMP responses and the 125I-VIP binding we found VIP receptors with a KD of 1.5 10(-9) M. Secretin stimulates the adenylate cyclase through the VIP receptor sites with a KD of 1.7. 10(-6) M. We noted also that during cell proliferation in culture there was about a 5 fold increase of the cAMP response to VIP.     

The stress induced increase of liver phosphorylase A and cyclic AMP in adrenomedullectomized and adrenalectomized rats.

In adult male rats injected with Pentobarbital, 50 mg.kg-1 i.p. and subjected immediately after administration of the anaesthetic to 400 revolutions (lasting of 6 min and 40 s) in rotating Noble-Collip drums the activity of the active form of hepatic phosphorylase was increased at time 0 after injury without respect to previous adrenomedullectomy or adrenalectomy (7 weeks or 10 days before, respectively). Completeness of surgery was checked by plasma catecholamines which were essentially near zero. The level of liver cAMP was increased in intact and adrenomedullectomized animals at time zero. 90 min after injury a recovery of enzyme activity towards basal levels was observed in contrast to cAMP which was increased in all the three groups. A net glycogenolytic response was found in the injured animals irrespective of previous surgery. It is concluded that for the stress induced activation of hepatic phosphorylase the presence of adrenal cortical and medullary tissue is not always indispensable.     

D1 Dopamine Receptors of NS20Y Neuroblastoma Cells Are Functionally Similar to Rat Striatal D1 Receptors

Dopamine or agonists with D1 receptor potency stimulated cyclic AMP (cAMP) accumulation in whole cell preparations of NS20Y neuroblastoma cells. The accumulation of cAMP after D1 stimulation was rapid and linear for 3 min. Both dopamine and the novel D1 receptor agonist dihydrexidine stimulated cAMP accumulation two- to three-fold over baseline. The pseudo-Km for dopamine was approximately 2 microM, whereas for dihydrexidine it was approximately 30 nM. The effects of both drugs were blocked by either the D1-selective antagonist SCH23390 (Ki, 0.3 nM) or the nonselective antagonist (+)-butaclamol (Ki, 5 nM). Both (-)-butaclamol and the D2-selective antagonist (-)-sulpiride were ineffective (Ki greater than 3 microM). Forskolin (10 microM), prostaglandin E1 (1 microM), and adenosine (10 microM) also stimulated cAMP accumulation, but none were antagonized by SCH23390 (1 microM). Finally, muscarinic receptor stimulation (100 microM carbachol) inhibited both D1- and forskolin-stimulated increases in cAMP accumulation by 80%. The present results indicate that NS20Y neuroblastoma cells have D1 receptors that are coupled to adenylate cyclase, and that these receptors have a pharmacological profile similar to that of the D1 receptor(s) found in rat striatum.     

Expression, binding, and signaling properties of CRF2(a) receptors endogenously expressed in human retinoblastoma Y79 cells: passage-dependent regulation of functional receptors

Endogenous expression of the corticotropin-releasing factor type 2a receptor [CRF2(a)] but not CRF2(b) and CRF2(c) was observed in higher passage cultures of human Y79 retinoblastoma cells. Functional studies further demonstrated an increase in CRF2(a) mRNA and protein levels with higher passage numbers (> 20 passages). Although the CRF1 receptor was expressed at higher levels than the CRF2(a) receptor, both receptors were easily distinguishable from one another by selective receptor ligands. CRF(1)-preferring or non-selective agonists such as CRF, urocortin 1 (UCN1), and sauvagine stimulated cAMP production in Y79 to maximal responses of approximately 100 pmoles/10(5) cells, whereas the exclusive CRF2 receptor-selective agonists UCN2 and 3 stimulated cAMP production to maximal responses of approximately 25-30 pmoles/10(5) cells. UCN2 and 3-mediated cAMP stimulation was potently blocked by the approximately 300-fold selective CRF2 antagonist antisauvagine (IC50 = 6.5 +/- 1.6 nmol/L), whereas the CRF(1)-selective antagonist NBI27914 only blocked cAMP responses at concentrations > 10 microL. When the CRF(1)-preferring agonist ovine CRF was used to activate cAMP signaling, NBI27914 (IC50 = 38.4 +/- 3.6 nmol/L) was a more potent inhibitor than antisauvagine (IC50 = 2.04 +/- 0.2 microL). Finally, UCN2 and 3 treatment potently and rapidly desensitized the CRF2 receptor responses in Y79 cells. These data demonstrate that Y79 cells express functional CRF1 and CRF2a receptors and that the CRF2(a) receptor protein is up-regulated during prolonged culture.     

Tolvaptan inhibits ERK-dependent cell proliferation, Cl⁻ secretion, and in vitro cyst growth of human ADPKD cells stimulated by vasopressin

In autosomal dominant polycystic kidney disease (ADPKD), arginine vasopressin (AVP) accelerates cyst growth by stimulating cAMP-dependent ERK activity and epithelial cell proliferation and by promoting Cl(-)-dependent fluid secretion. Tolvaptan, a V2 receptor antagonist, inhibits the renal effects of AVP and slows cyst growth in PKD animals. Here, we determined the effect of graded concentrations of tolvaptan on intracellular cAMP, ERK activity, cell proliferation, and transcellular Cl(-) secretion using human ADPKD cyst epithelial cells. Incubation of ADPKD cells with 10(-9) M AVP increased intracellular cAMP and stimulated ERK and cell proliferation. Tolvaptan caused a concentration-dependent inhibition of AVP-induced cAMP production with an apparent IC(50) of ～10(-10) M. Correspondingly, tolvaptan inhibited AVP-induced ERK signaling and cell proliferation. Basolateral application of AVP to ADPKD cell monolayers grown on permeable supports caused a sustained increase in short-circuit current that was completely blocked by the Cl(-) channel blocker CFTR(inh-172), consistent with AVP-induced transepithelial Cl(-) secretion. Tolvaptan inhibited AVP-induced Cl(-) secretion and decreased in vitro cyst growth of ADPKD cells cultured within a three-dimensional collagen matrix. These data demonstrate that relatively low concentrations of tolvaptan inhibit AVP-stimulated cell proliferation and Cl(-)-dependent fluid secretion by human ADPKD cystic cells.     

Effect of thyrotropic hormone on the membrane potential of thyroid gland cells and thyroid hormone secretion with aging

Effect of thyrotropic hormone (TTH) on membrane potential (MP) of thyroid cells and thyroid hormone secretion was studied in experiments on male rats of two age groups (7--12- and 27--32-month-old animals). It was found that during the first 3 hours after TTH administration (5 U/100 g i. v.) the depolarization of secretory cell membranes of adult rats was done pronounced and developed more rapidly than in old ones and that an increase in free thyroxin (T4) correlation with MP changes with time. In a dose of 0.5 U/100 g TTH caused a significant rise in T4 secretion only in old rats. The cAMP level in the thyroid gland declined with aging. In a dose of 5 U/100 g TTH provoked a significant increase in the cAMP content in adult rats and had no effect on its content in old ones. A relationship between the MP level of thyroid secretory cells and thyroid hormone secretion is discussed.     

Suppression of insulin-stimulated glucose transport in L6 myocytes by calcitonin gene-related peptide

The binding of calcitonin gene-related peptide (CGRP) to L6 myocytes, the coupling of this receptor to adenylyl cyclase and the resultant effects on insulin-stimulated 2-deoxyglucose uptake were examined. L6 cells express specific binding sites for CGRP. Binding of human [125I]CGRP was inhibited by rat CGRP with an IC50 of approximately 10(-9) M. Synthetic human calcitonin at concentrations up to 10(-6) M had no effect on the binding of CGRP, suggesting that L6 cells express CGRP receptors, rather than calcitonin receptors which are also capable of binding CGRP. The CGRP receptor appeared to be coupled to adenylyl cyclase. Concentrations of CGRP greater than 3 x 10(-9) M increased the cellular content of cAMP. At 3 x 10(-8) M, CGRP increased cAMP 500-fold. CGRP at 10(-10) M and above suppressed the stimulation of 2-deoxyglucose uptake by insulin. Acute incubation of L6 cells with insulin stimulated 2-deoxyglucose uptake 1.6-fold, which was inhibited up to 70% by CGRP. Our results demonstrate that the specific binding of CGRP to L6 cells causes large increase in the cellular content of cAMP - and inhibition of insulin-stimulated 2-deoxyglucose uptake, but the differences in the dose-response curves suggest that the suppression of insulin action by CGRP cannot be solely explained by the increase in cAMP.