Increase In Cyclic AMP Research Articles

Bradykinin-dependent activation of adenylate cyclase activity and cyclic AMP accumulation in tracheal smooth muscle occurs via protein kinase C-dependent and -independent pathways.

Treatment of cultured tracheal smooth-muscle cells (TSM) with phorbol 12-myristate 13-acetate (PMA) (100 nM) or bradykinin (100 nM) elicited enhanced basal and guanosine 5'-[beta gamma-imido]-triphosphate-stimulated adenylate cyclase activities in subsequently isolated membranes. Combined stimulation of cells was non-additive, indicating that both agents activate adenylate cyclase via similar routes. Both PMA (100 nM) and bradykinin (100 nM) allowed the alpha subunit of Gs to act as a more favourable substrate for its cholera-toxin-catalysed ADP-ribosylation in vitro. PMA was without effect on intracellular cyclic AMP in control cells. However, constitutive activation of Gs by treatment in vivo with cholera toxin (0.5 ng/ml, 18 h) sensitized the cells to PMA stimulation, resulting in a concentration-dependent increase in intracellular cyclic AMP accumulation (EC50 = 7.3 +/- 2.5 nM, n = 5). Bradykinin also elicited a concentration-dependent increase in intracellular cyclic AMP (EC50 = 63.3 +/- 14.5 nM, n = 3). Constitutive activation of Gs resulted in an increased maximal response (10-fold) and potency (EC50 = 6.17 +/- 1.6 nM, n = 3) to bradykinin. This response was not affected by the B2-receptor antagonist, NPC567 [which selectively blocks bradykinin-stimulated phospholipase C (PLC), with minor activity against phospholipase D (PLD) activity]. Des-Arg9-bradykinin (a B1-receptor agonist) was without activity. These results suggest that the receptor sub-type capable of activating PLD may also be stimulatory for cyclic AMP accumulation. Furthermore, pre-treatment of the cells with butan-l-ol (0.3%, v/v), which traps phosphatidate derived from PLD reactions, blocked the bradykinin-stimulated increase in intracellular cyclic AMP. These studies suggest that there may be a causal link between PLD-derived phosphatidate and the positive modulation of adenylate cyclase activity. In support of this, the concentration-dependence for bradykinin-stimulated adenylate cyclase activity was identical with that of bradykinin-stimulated phospholipase D activity (EC50 = 5 nM). Bradykinin, but not PMA, was also capable of eliciting the inhibition of cyclic AMP phosphodiesterase activity in TSM cells (EC50 > 100 nM) via an unidentified mechanism. These studies indicate that cross-regulation between the cyclic AMP pathway and phospholipid-derived second messengers in TSM cells does not occur as a consequence of PLC-catalysed PtdIns(4,5)P2 hydrolysis, but may involve, in part, PLD-catalysed phosphatidylcholine hydrolysis.

Transient cyclic AMP increase induced by glutamate in cultured spinal neurons

Transient cyclic AMP increase induced by glutamate in cultured spinal neurons

NMDA Receptor Activation Increases Cyclic AMP in Area CA1 of the Hippocampus via Calcium/Calmodulin Stimulation of Adenylyl Cyclase

We observed previously that activation of N-methyl-D-aspartate (NMDA) receptors in area CA1 of the hippocampus, through either NMDA application or long-term potentiation (LTP)-inducing high-frequency stimulation (HFS), results in an increase in cyclic AMP. In the present study, we performed experiments to determine the mechanism by which NMDA receptor activation causes this increase in cyclic AMP. As the NMDA receptor-mediated increase in cyclic AMP is dependent upon extracellular calcium, we hypothesized that NMDA receptors are coupled to adenylyl cyclase (AC) via calcium/calmodulin. In membranes prepared from area CA1, AC was stimulated by calcium in the presence of calmodulin, and the effect of calcium/calmodulin on AC in membranes was blocked by the calmodulin antagonists N-(6-aminohexyl)-5-chloro-1- naphthalenesulfonamide (W-7) and trifluoperazine (TFP). In intact hippocampal slices, W-7 and TFP blocked the increase in cyclic AMP levels caused by both NMDA application and HFS of Schaffer collateral fibers. Exposure of hippocampal slices to elevated extracellular potassium to induce calcium influx also caused increased cyclic AMP levels; the increase in cyclic AMP caused by high potassium was also blocked by W-7 and TFP. These data support the hypothesis that NMDA receptor activation is positively coupled to AC via calcium/calmodulin and are consistent with a role for cyclic AMP metabolism in the induction of NMDA receptor-dependent LTP in area CA1 of the hippocampus.

Adenosine receptor-induced second messenger production in adult guinea-pig cerebellum.

1. The effects of adenosine receptor agonists on cyclic nucleotides accumulation were investigated in adult guinea-pig cerebellar slices by use of radioactive precursors. 2. Adenosine elicited a rapid and maintained increase in cyclic AMP, that was fully reversed upon addition of adenosine deaminase. Adenosine analogues stimulated cyclic AMP generation up to 40 fold with the rank order of potency: 5'-N-ethylcarboxamidoadenosine (0.6 microM) > 2-chloroadenosine (6 microM) > adenosine (13 microM). CGS 21680 (10 microM) elicited only a small stimulation (1.2 fold). 3. The cyclic AMP response to NECA was reversed by the 1,3-dipropylxanthine-based adenosine receptor antagonists 8-[4-[[[[(2-aminoethyl)amino]amino]carbonyl]methyl]oxy]- phenyl]-1,3-dipropylxanthine (XAC), 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and N-[2-(dimethylamino)ethyl]N-methyl-4-(1,3-dipropylxanthine)benzene sulphonamide (PD 115,199) with estimated apparent inhibition constants of 15, 81 and 117 nM, respectively. 4. Pretreatment with adenosine also potentiated the cyclic GMP response to sodium nitroprusside, abolishing the decline in [3H]-cyclic GMP observed with sodium nitroprusside alone, and allowing [3H]-cyclic GMP levels to be maintained for at least an additional 10 min. This potentiation was fully reversed by adenosine deaminase. 5. Adenosine analogues potentiated the sodium nitroprusside-elicited cyclic GMP generation with the rank order of potency: 5'-N-ethylcarboxamidoadenosine (0.7 microM) > 2-chloroadenosine (6 microM) > adenosine (42 microM). 6. NECA potentiation of cyclic GMP formation was reversed by the antagonists XAC, DPCPX and PD 115,199 with apparent inhibition constants of 17, 102 and 242 nM, respectively. 7. The similar potencies of adenosine analogues and xanthine antagonists for stimulation of cyclic AMP and potentiation of cyclic GMP lead to the suggestion that these phenomena are mediated through the same adenosine receptor, the A2b receptor. Furthermore, we suggest that potentiation of the sodium nitroprusside-induced cyclic GMP response may be mediated at the level of phosphodiesterase hydrolysis of the cyclic nucleotides.

Cloning and Functional Expression of a Human Glucagon-like Peptide-1 Receptor

A human glucagon-like 1 peptide receptor has been cloned from the gastric tumor cell line HGT-1. The cDNA clone encodes a protein of 463 amino acids and is a member of the superfamily of seven transmemhrane domain G protein coupled receptors. Transfection of the human GLP-1 receptor into COS-7 cells confers upon them high affinity binding for [ 125I] GLP-1(7-36) amide. In membranes prepared from COS-7 cells transfected with the human GLP-1 receptor, the binding of [ 125I] GLP-1 (7-36) amide is inhibited with the rank order of potency GLP-1 (7-36) amide > glucagon > secretin, characteristic of a GLP-1 receptor. The human GLP-1 receptor is functionally coupled to increases in intracellular cAMP in these cells: incubation of COS-7 cells expressing the human GLP-1 receptor with GLP-1 (7-36) amide gives rise to a 4-fold increase in cyclic AMP over basal levels, with an EC 50 of 25pM. Glucagon is also a full agonist but is 200-fold less potent than GLP-1 (7-36) amide in stimulating the human GLP-1 receptor.

The kidney and essential hypertension: a link to osteoporosis?

Abnormalities of calcium metabolism independent of changes in intracellular calcium have been described in patients with essential hypertension. These include increased urinary calcium excretion for a given salt intake, a raised parathyroid hormone level, an increase in urinary cyclic AMP, a tendency for a low serum ionized calcium level, a raised 1,25-dihydroxyvitamin D level and an increased intestinal calcium reabsorption. These changes have been seen as a consequence of a primary renal calcium leak. We propose that these changes are secondary to a genetic defect in the ability of the kidney to excrete sodium. On the high salt intake in most Western countries (i.e. approximately 170 mmol/day sodium) compensatory mechanisms occur to try to overcome this defect. These compensatory mechanisms are responsible for the rise in blood pressure, but also cause an increase in central blood volume which is the direct cause of the increase in urinary calcium excretion. This causes a slightly negative calcium balance, and the other abnormalities of calcium metabolism can then be seen as a compensatory response to try to restore calcium balance to normal. This hypothesis explains the increase in kidney stones in essential hypertension and predicts that hypertensives, in the long term, will be more likely to develop bone demineralization (osteoporosis), as has been demonstrated in some animal models of hypertension. Increases in salt intake will not only cause a further rise in blood pressure, but will also increase urinary calcium excretion and aggravate the other abnormalities. A moderate reduction in salt intake from 170 to 70 mmol/day will lower the blood pressure and will tend to correct the abnormalities of calcium metabolism. It should simultaneously reduce the incidence of renal stones and the long-term risk of bone demineralization.

Single-cell [Ca 2+] i analysis and biochemical characterization of pinealocytes immobilized with novel attachment peptide preparation

Single-cell image analysis of rat pinealocytes has been difficult because they do not attach readily to coated or uncoated surfaces and typically adhere in clusters to fibroblast-like cells. In the present report, a new method for the rapid attachment of rat pinealocytes is described. Cells were prepared using papain digestion and density centrifugation and then were placed on coverslips or slides coated with PepTite-2000 TM, a preparation containing the attachment peptide sequence Arg-Gly-Asp. Cells immobilized with this preparation responded to norepinephrine treatment with an increase in cyclic AMP and melatonin production. Single-cell analysis of Fura-2-loaded cells revealed that norepinephrine increase [Ca 2+] i. This development makes it possible to conduct routine single-cell image analysis and other studies of freshly isolated rat pinealocytes.

The bis(adenosin-N6-yl)alkanes, a family of potential dinucleoside-polyphosphate analogue precursors. Cytotoxicity, adenosine-receptor binding and metabolism.

A series of bis(adenosin-N6-yl)alkanes, in which two adenosine residues are linked via their N6 positions by alkyl bridges comprising between 2 and 14 methylene units, were synthesized as potential precursors to dinucleoside-polyphosphate analogues. These compounds were moderately cytotoxic to mammalian cells, the toxicity increasing with the length of the alkyl chain. For example, the dose of bis(adenosin-N6-yl)dodecane, A[CH2]12A, leading to 50% inhibition of cell growth (ID50) for BHK fibroblasts, Walker 256 carcinoma cells and S-49 T-lymphoma cells were 90 +/- 8, 100 +/- 5 and 23 +/- 4 microM respectively. A significant amount of A[CH2]12A bound to serum albumin in the growth media; thus the ID50 for S-49 cells grown in serum-free medium was 9 +/- 2 microM. The corresponding bis-cytidine analogues were much less toxic; however the presence of a second adenosine moeity/molecule had little significant effect on cell growth when compared to N6-alkyladenosines. Toxicity to S-49 cells was unaffected by the nucleoside-transporter inhibitor nitrobenzylthioinosine and was even higher (ID50 = 5 +/- 0.5 microM) towards nucleoside-transport-deficient AE-1 cells, showing that the analogues could pass freely through the plasma membrane. Interaction with A1 adenosine receptors was shown by displacement of [3H]N6-R-phenylisopropyladenosine (Kd = 6 nM) from rat adipocyte membranes, with Ki values of 45, 65, 85 and 390 nM for the compounds containing 12, 8, 6 and 4 methylene units, respectively. Affinity for human platelet membrane A2 adenosine receptors was about 100-fold less, however the compounds were weak A2 agonists, producing up to a threefold increase in intracellular cyclic AMP in WI-38/VA-13 cells. Thus, these compounds behave, not surprisingly, as adenosine analogues. In addition, A[CH2]12A was metabolized in vitro and intracellularly by adenosine kinase (Ki = 70 nM) and adenylate kinase to yield a number of phosphorylated derivatives with the potential to act as diadenosine polyphosphate analogues. One of these, the bismonophosphate, was recognized by and inhibited adenylate kinase more effectively than adenosine(5')tetraphospho(5')adenosine (Ap4A, Ki = 3 microM).

The effect of methyldopa and procainamide on suppressor cell activity in relation to red cell autoantibody production

Kirtland et al (1980) suggested that methyldopa caused the production of red cell (RBC) autoantibodies by causing a persistent increase in lymphocyte cyclic AMP, which inhibited suppressor T cell function, leading to unregulated autoantibody production in some patients. They showed that significantly higher lymphocyte cyclic AMP concentrations were generated by lymphocytes from healthy donors after adding methyldopa, and by lymphocytes from patients who were receiving methyldopa compared to lymphocytes from healthy donors without methyldopa present. They also showed that methyldopa affected suppressor cell activity. We measured the effect of methyldopa and procainamide on suppressor cell activity, using a similar approach to Kirtland et al (1980). Suppressor cell activity was measured by measuring the amount of IgG, produced in vitro, by B cells following mitogen stimulation preceded by a 24 h incubation period. We found no significant increase in the amount of IgG generated by normal donor lymphocytes, when methyldopa or procainamide was present during the preincubation period. This is in contrast to the findings of Kirtland et al (1980). We also measured the amount of IgG generated in vitro by mitogen-stimulated lymphocytes from patients (with and without positive direct antiglobulin tests) taking methyldopa and compared this to the amount of IgG generated by lymphocytes from normal donors and patients (with and without positive direct antiglobulin tests). The results were similar for each group. This does not agree with the findings of Kirtland et al (1980) who found that lymphocytes from patients taking methyldopa produced more IgG in vitro than lymphocytes from normal donors. Our results do not support the hypothesis that methyldopa and procainamide induce autoantibodies by affecting suppressor cell function.

Platelet activation in mental stress.

Platelets are known to become activated in vivo by different stressful stimuli such as surgery and dynamic exercise. Mental stress has been shown to increase platelet aggregability. Platelet activation is thought to be of major importance in atherogenesis and cardiac fatalities. In order to clarify further stress-induced platelet activation with special reference to the period after the stress, we studied eight young, healthy volunteers during and for 1 h after a mental stress test (Stroops Colour Word Conflict Test). Using highly standardized techniques, we have measured platelet aggregability ex vivo and the platelet release products beta-thromboglobulin, platelet factor 4 and serotonin in plasma. As markers of the stress response we measured cyclic-AMP in plasma, heart rate, cardiac output and blood pressure. The stress test induced a significant cardiovascular response with increases in heart rate, blood pressure, and cardiac output and as a measure of adrenergic activity an increase in cyclic AMP in plasma during the test. Platelet aggregability was unaffected during the test but decreased following the stress. During the first hour following the test and release products beta-thromboglobulin and serotonin increased significantly in plasma. We conclude that platelets are activated during mental stress and that this activation involves a post-stress release of vasoactive compounds from platelets.

Biochemical and physiological effects of octopamine and selected octopamine agonists on the oviducts of Locusta migratoria

The effects of octopamine and some selected octopamine agonists on neurally-evoked contrations and cyclic AMP levels of the lateral oviducts of the locust, Locusta migratoria, have been examined. Octopamine caused reversible, dose-dependent decreases in both the basal tonus and amplitude of neurally-evoked contractions of the lateral oviducts, and inhibited myogenic contractions. The 2-aminooxazoline, Ac6 [2-(4-chloro- o-toluidino)-2-oxazoline], and the substituted phenyliminoimidazolidines (PIIs), NC5 (2,6-diethyl-PII) and NC7 (2-methyl-4-chloro-PII), were each capable of eliciting similar responses to octopamine on neurally-evoked contractions. The vertebrate α-adrenergic receptor antagonist phentolamine blocked the physiological effects of all agonists tested. The effect of these agonists on cyclic AMP levels was also examined. Octopamine and the three agonists were able to increase the cyclic AMP content of the lateral oviducts in a dose-dependent manner. The increases in cyclic AMP were inhibited in the presence of various vertebrate receptor antagonists. The results of this study indicate that AC6, NC5, and NC7 all act as agonists to the octopamine 2-like receptors present on locust oviduct and confirm previous studies for the agonistic properties of these agents.

Ca(2+)-dependent inactivation of Ca2+ current in Aplysia neurons: kinetic studies using photolabile Ca2+ chelators.

1. The kinetics and sensitivity of the Ca(2+)-dependent inactivation of calcium current (ICa) were examined in intact cell bodies from the abdominal ganglion of Aplysia californica under two-electrode voltage clamp. 2. Rapid changes in the level of intracellular free calcium ([Ca2+]i) were generated at the cell surface by photolytic release of Ca2+ (nitr-5 and dimethoxy nitrophen) or Ca2+ buffer (diazo-4). 3. Diazo-4 increased ICa by 10-15% and slowed the rate of ICa decay when photolysed before a test pulse or between a prepulse and a test pulse. The predominant effect of further light flashes was to increase the amount of non-inactivating current (I infinity) remaining at the end of long (> 1 s) depolarizing pulses. 4. A rapid increase in [Ca2+]i buffering during ICa inactivation did not cause a rapid recovery of current but merely reduced the rate and extent of subsequent inactivation. This effect was not seen when Ba2+ was the charge carrier. 5. Photolytic release of Ca2+ from nitr-5 produced estimated Ca2+ jumps of 3-4 microM at the front surface of the cell but failed to augment inactivation either before or during ICa. In contrast, photolysis of DM-nitrophen 10-90 ms before the test pulse decreased peak ICa by about 30%. A flash given during ICa rapidly blocked 41 +/- 3% of peak current with a time constant of 3-4 ms at 17 degrees C. Similar results were seen with the barium current (IBa). 6. Microinjection of the potent phosphatase inhibitor microcystin-LR (5 microM) had variable effects on ICa inactivation and augmented the cyclic AMP-induced depression of the delayed rectifier (IK(V) by forskolin (100 microM) and 3-isobutyl-1-methylxanthine (IBMX; 200 microM). 7. Full recovery from inactivation measured in two-pulse experiments took at least 20 s. This slow recovery process was unaffected by increases in intracellular cyclic AMP elicited by direct injection or by bath application of forskolin and IBMX. It was also unaffected by decreases in cyclic AMP induced by injecting 2',5'-dideoxyadenosine (1 mM) or bath application of the Rp isomer of cyclic adenosine 3',5'-monophosphothioate (Rp-cAMPS; 200 microM). 8. A 'shell' model relating submembrane Ca2+ to inactivation was inconsistent with the experimental results since it greatly overestimated the effects of diazo-4 and predicted significant inactivation by nitr-5 photolysis. 9. A model linearly relating [Ca2+]i in a single Ca2+ channel 'domain' to inactivation more closely matched the experimental results with diazo-4 and DM-(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of flosequinan on ischaemia-induced arrhythmias and on ventricular cyclic nucleotide content in the anaesthetized rat.

1. Flosequinan, milrinone, isoprenaline and forskolin given intravenously at similarly hypotensive doses have been evaluated in separate studies for their effect on ischaemia-induced arrhythmias and on ventricular cyclic nucleotide content following coronary artery ligation in the pentobarbitone anaesthetized rat. 2. Flosequinan did not affect mortality or arrhythmias following coronary artery ligation in either study and no change in ventricular cyclic nucleotide content was observed. 3. Isoprenaline caused a significant increase in mortality (P < 0.05) in both studies whereas milrinone and forskolin caused a significant increase in mortality in only one of the two studies conducted. All three agents caused significant increases in cyclic AMP which were associated with increased incidence of arrhythmias. 4. When compared at similarly hypotensive doses, flosequinan, in contrast to milrinone, isoprenaline and forskolin, did not influence ischaemia-induced arrhythmias or raise ventricular cyclic nucleotide levels in the anesthetized rat.

Cyclic AMP-induced inhibition of collagen lattice contraction by fibroblasts may be attenuated by both cyclic AMP dependent and independent mechanisms.

The contraction of collagen lattices made with foreskin fibroblasts in medium containing 1% fetal bovine serum was inhibited by intracellular cyclic AMP-raising drugs including cholera toxin (CT), forskolin, and dibutyryl-cAMP. The inhibition by CT was attenuated by insulin, acidic fibroblast growth factor (aFGF), and transforming growth factor-beta (TGF-beta). All three peptide factors have previously been reported to promote collagen lattice contraction by arterial smooth muscle cells and/or fibroblasts. Incubation of cells suspended in collagen gels with CT and forskolin resulted in a transient rise of the intracellular cyclic AMP levels, which peaked at 2 hr and 30 min, respectively, after drug exposure. Cholera toxin-induced intracellular cyclic AMP increase was attenuated by TGF-beta, but not by aFGF and insulin, when added simultaneously. Thus, TGF-beta may attenuate CT's inhibition on collagen lattice contraction by attenuating CT-induced intracellular cyclic AMP increase, whereas the attenuation by insulin and aFGF on the inhibition of lattice contraction may be mediated by a cyclic AMP-independent mechanism.

Increased responsiveness of left ventricular apical myocardium to adrenergic stimuli.

The aim was to determine whether left ventricular apical myocardium has mechanisms to compensate for sparse sympathetic innervation. Contractile and metabolic responses to various adrenergic stimuli and beta adrenergic receptor density were compared between left ventricular basal and apical regions in 26 anaesthetised mongrel dogs, weight 12-28 kg. Regional contractile changes in response to graded cardiac sympathetic nerve stimulation were compared among three basal (anterior, middle, and posterior) regions, and between basal middle and apical regions. There were significant differences in contractile changes among the three basal regions with distinct regions of innervation from right and/or left sided sympathetic ganglia, but not between apical and basal regions. Constant infusion of noradrenaline (0.2-0.4 microgram.kg-1.min-1) produced a greater response in normalised end systolic length in the apical myocardium than in the basal region, at 9.86(SEM 0.06) mm v 10.14(0.04) mm (n = 5, p < 0.025), and a greater increase in tissue cyclic AMP: 1.04(0.20) v 0.60(0.08) pmol.mg-1 (n = 5, p < 0.05). Giving a forskolin derivative (30 micrograms.kg-1, n = 5) produced a greater increase in cyclic AMP in the apical region than in the basal region: 1.26(0.18) v 0.88(0.19) pmol.mg-1 (p < 0.02). beta Adrenergic receptor density in the apical region was greater than in the basal region: 455(45) v 341(35) fmol.mg-1 protein (n = 5, p < 0.05). Greater beta adrenergic receptor density and/or increased myocardial responsiveness to adenylate stimulation in apical myocardium compensates, at least in part, for its sparse sympathetic innervation.

ICI 147,798 causes insurmountable antagonism against T-0509, a selective beta 1-adrenoceptor agonist, but surmountable antagonism against isoproterenol.

In canine right ventricular muscle, the influence of ICI 147,798, a slowly dissociable beta 1-adrenoceptor antagonist, on the positive inotropic effect and cyclic AMP production of T-0509, a selective beta 1-agonist, and isoproterenol, a nonselective beta-agonist, were examined to elucidate the properties of the high- and low-affinity states or subtypes of beta 1-adrenoceptor. ICI 147,798 (1 x 10(-8) - 1 x 10(-5) M) produced biphasic concentration-response curves for the positive inotropic effect of T-0509 that were due to its insurmountable antagonism against the lower concentrations of T-0509. The maximum of the biphasic curves were almost the same as Camax. Analysis of hemiequilibrium antagonism by ICI 147,798 against T-0509 showed the dissociation constant (pKB) of ICI 147,798 to be approximately 8. On the other hand, ICI 147,798 produced a parallel rightward shift of the curves of isoproterenol. Schild analysis showed pA2 to be 7.98 +/- 0.04, suggesting competitive antagonism. The concentration-response curve for the increase in cyclic AMP elicited by T-0509 in the presence of 1 x 10(-6) M ICI 147,798 was also biphasic, but slightly depressed as compared with that of control, whereas that elicited by isoproterenol was shifted to the right. The positive inotropy and increase in cyclic AMP produced by T-0509 and isoproterenol in the presence of ICI 147,798 may be mediated by the low-affinity state of beta 1-adrenoceptor, which mechanism may be different from that producing the positive inotropy through the high-affinity state of beta 1-adrenoceptor.

Prostaglandins Stimulate Serotonin Acetylation in Chick Pineal Cells: Involvement of Cyclic AMP‐Dependent and Calcium/Calmodulin‐Dependent Mechanisms

The effects of prostaglandins (PGs) on the activity of the rate-limiting enzyme of melatonin biosynthesis, arylalkylamine-N-acetyltransferase (NAT) were investigated on primary cultures of dispersed chick pineal cells. In indomethacin-treated cells, PGs caused a four-fold increase in NAT activity. This response was associated with an eight-fold increase in cyclic AMP (cAMP) levels. The potency order of PGs was the same for NAT and for cAMP responses (PGE1 > PGE2 > PGF2 alpha >> cloprostenol). However, each PG tested was 30- to 200-fold more potent to increase NAT activity than to stimulate cAMP accumulation. As a result, half-maximal stimulation of NAT by PGs was not associated with an increase in cAMP levels. Half-maximal stimulation of NAT by PGE1 was highly sensitive to inhibition by a calcium/calmodulin antagonist (W-7). In contrast, maximal stimulation of NAT by PGE1 as well as stimulations evoked by either forskolin or 8-bromo-cAMP were poorly sensitive to inhibition by W-7. These results indicate that an increase in cAMP levels may be responsible for the maximal stimulation of NAT evoked by PGs, whereas half-maximal stimulation of NAT by PGs would rely principally on a calcium/calmodulin-dependent mechanism.

Characterization and Genetic Analysis of Functional Corticotropin‐Releasing Hormone Receptors in Primary Cerebellar Cultures

We demonstrate that granular cerebellar neurons express functional corticotropin-releasing hormone (CRH) receptors. Activation of these receptors with CRH receptor agonists leads to a dose-dependent increase in cyclic AMP (cAMP) levels with an apparent EC50 close to 10(-9) M. Using the c-fos protooncogene as a system to evaluate genomic effects of CRH, we show that activation of CRH receptors regulates gene expression at the transcriptional level. CRH rapidly induced c-fos mRNA accumulation. Genetic studies, using chimera genes containing human c-fos promoter sequences coupled to a chloramphenicol acetyltransferase (CAT) reporter gene, confirmed and extended this observation. When protein kinase A (PKA) was specifically inactivated by gene transfer of a mutated regulatory subunit of PKA lacking cAMP binding sites, CRH-stimulated c-fos transcription was suppressed but the increase in cAMP level was not affected, indicating a key role of PKA in mediating CRH-stimulated transcription. As CRH clearly modulates gene expression via the cAMP pathway, we analyzed the genomic effect of this neurohormone on a deleted c-fos-CAT construct containing only the cAMP-responsive element (CRE) and on a heterologous promoter construct bearing the minimal palindromic consensus CRE (core sequence TGACGTCA). These minimal cAMP-responsive genes are induced by CRH. These inductions are dependent on functional PKA. Taken together, our results demonstrate the presence of functional CRH receptors in primary cerebellar cultures. Activation of these receptors stimulates gene expression via the cAMP/PKA pathway and the transacting factor CREB (cAMP-responsive element binding protein).

The cyclic nucleotide-activated conductance in olfactory cilia: effects of cytoplasmic Mg2+ and Ca2+.

Olfactory receptor neurons depolarize in response to odorants. This depolarization is mediated by an increase in intracellular cyclic AMP, which directly gates channels in the membranes of the neuronal cilia. Previous evidence suggests that a Ca2+ influx during the odorant response may ultimately play a role in terminating the response. One way Ca2+ inside the cell could terminate the odorant response would be to directly inhibit the cAMP-gated channels. In this report the effects of cytoplasmic Ca2+ and Mg2+ on the cAMP-activated current were measured in single olfactory cilia. Near the neuronal resting potential, cytoplasmic Ca2+ and Mg2+ only slightly reduced the cAMP-activated current. Even at high levels (1.0 mM Ca2+ or 5.0 mM Mg2+), the average inhibition was only around 20%. It is therefore unlikely that an influx of divalent cations terminates the odorant response by a direct effect on the cAMP-gated channels.

Single-channel mechanism of beta-adrenergic enhancement of cardiac L-type calcium current.

The cardiac L-type Ca2+ channel current is enhanced by beta-adrenergic stimulants by phosphorylation mediated by an increase in cyclic AMP. Single-channel studies have revealed that the Ca2+ channels are not always allowed to open at test steps from large negative holding potentials and beta-agonists increase the ratio of current-containing sweeps (availability, Ps). The duration of available (Ts) and unavailable states (TF) are measurable by applying a large number of test pulses. beta-Agonists shorten TF and prolong TS, possibly by enhancing channel phosphorylation by activating cyclic AMP-dependent protein kinase and by inhibiting channel dephosphorylation by phosphorylating phosphatase inhibitor 1, respectively, if the available state reflects the phosphorylated state of the channel. The modulation of the slow gating process, the increase in PS, plays a primary role in increasing the L-type Ca2+ current at moderate increase in sympathetic activity. When a larger increase in cyclic AMP is resulted, moduration of burst-kinetics including mode 2 openings also produced by phosphorylation enhances further the Ca2+ current.