Possible Involvement Of cAMP Research Articles

Sustained antagonism of acute ethanol-induced ataxia following microinfusion of cyclic AMP and cpt-cAMP in the mouse cerebellum

Ataxia is a conspicuous physical manifestation of alcohol consumption in humans and laboratory animals. Previously we reported possible involvement of cAMP in ethanol-induced ataxia. We now report a sustained antagonism of ataxia due to multiple ethanol injections following intracerebellar (ICB) cAMP or cpt-cAMP microinfusion. Adenylyl cyclase drugs cAMP, cpt-cAMP, Sp-cAMP, Rp-cAMP, adenosine A1 agonist, N6-cyclohexyladenosine (CHA) and GABAA agonist muscimol were directly microinfused into the cerebellum of CD-1 male mice to evaluate their effect on ethanol (2g/kg; i.p.) ataxia. Drug microinfusions were made via stereotaxically implanted stainless steel guide cannulas. Rotorod was used to evaluate the ethanol's ataxic response. Intracerebellar cAMP (0.1, 1, 10fmol) or cpt-cAMP (0.5, 1, 2fmol) 60min before ethanol treatment, dose-dependently attenuated ethanol-induced ataxia in general agreement with previous observations. Intracerebellar microinfusion of cAMP (100fmol) or cpt-cAMP (2fmol) produced a sustained attenuation of ataxia following ethanol administration at 1, 4, 7 and 25h or 31h post-cAMP/cpt-cAMP microinfusion. At 31h post-cAMP, the ataxic response of ethanol reappeared. Additionally, marked antagonism to the accentuation of ethanol-induced ataxia by adenosine A1 and GABAA agonists, CHA (34pmol) and muscimol (88pmol), respectively, was noted 24h after cAMP and cpt-cAMP treatment. This indicated possible participation of AC/cAMP/PKA signaling in the co-modulation of ethanol-induced ataxia by A1 adenosinergic and GABAergic systems. No change in normal motor coordination was noted when cAMP or cpt-cAMP microinfusion was followed by saline. Finally, Rp-cAMP (PKA inhibitor, 22pmol) accentuated ethanol-induced ataxia and antagonized its attenuation by cAMP whereas Sp-cAMP (PKA activator, 22pmol) produced just the opposite effects, further indicating participation of cAMP-dependent PKA downstream. Overall, the results support a role of AC/cAMP/PKA signaling in the expression of ethanol-induced ataxia and its co-modulation by adenosine A1 and GABAA receptors.

The Bombyx mori sex pheromone biosynthetic pathway is not mediated by cAMP

In most moths, sex pheromone production is regulated by pheromone biosynthesis-activating neuropeptide (PBAN). How the extracellular PBAN signal is turned into a biological response has been the focus of numerous studies. In the classical scheme of signal transduction, activated G proteins relay the extracellular signal to downstream effector molecules such as calcium channels and adenylyl cyclase. The role of calcium in PBAN signaling has been clearly demonstrated, but the possible involvement of cAMP is not as straightforward. While cAMP has been shown to be necessary for PBAN signaling in most heliothine species, there has been no definitive demonstration of its role in Bombyx mori. To address this question, we used degenerate RT-PCR to clone two Gs subunits, designated P50Gs1 and P50Gs2, from B. mori pheromone gland (PG) cDNAs. The two Gs proteins were expressed in all tissues examined and were not up-regulated in accordance with adult eclosion. Even though two bands corresponding to the approximate molecular weights of P50Gs1 and P50Gs2 were detected in PG homogenates, the Gs antagonist, NF449, had no effect on sex pheromone production. Furthermore, no changes in the intracellular cAMP levels were detected following PBAN stimulation.

Effects of estrogenic xenobiotics on human and mouse spermatozoa

To investigate human sperm responsiveness to the estrogenic xenobiotic genistein and seek further information regarding the mechanism of action of estrogenic xenobiotics using mouse spermatozoa. Uncapacitated human spermatozoa were incubated with genistein and assessed using chlortetracycline (CTC) fluorescence. CTC was also used to evaluate mouse sperm responses to daidzein and combinations of genistein, 8-prenylnaringenin and nonylphenol. Several steroids were tested to determine structure-function relationships, and possible involvement of cAMP and G proteins in responses was also investigated. Genistein significantly accelerated capacitation and acrosome loss in human spermatozoa, with 1, 10 and 100 nmol/l being equally effective. In mouse spermatozoa, daidzein produced significant responses, and combinations of xenobiotics at low concentrations were more effective than used singly. The compounds appear to act at the cell surface, and responses to three different steroids were nonidentical. A protein kinase-A inhibitor blocked responses to xenobiotics, while genistein and nonylphenol significantly stimulated cAMP production. Pertussis toxin and dideoxyadenosine blocked responses, suggesting involvement of inhibitory G proteins and membrane-associated adenylyl cyclases. Human and mouse sperm responses to genistein are very similar, but human gametes appear to be even more sensitive. The mechanism of action may involve unregulated stimulation of cAMP production, leading to significant acrosome loss, undesirable because already acrosome-reacted cells are nonfertilizing. Xenobiotics were even more effective in combination. Since simultaneous exposure to low concentrations of multiple xenobiotics is likely to occur in animals and humans, further investigation is needed to determine whether this could impair fertility.

Silibinin prevents cholestasis-associated retrieval of the bile salt export pump, Bsep, in isolated rat hepatocyte couplets: Possible involvement of cAMP

Estradiol-17ß- d-glucuronide (E 217G) and taurolithocholate (TLC) induce acute cholestasis-associated with retrieval of the bile salt export pump (Bsep), which parallels with alteration in transport activity. cAMP stimulates the apically directed vesicular trafficking of transporters, partially preventing these alterations. The hepatoprotector, silymarin, which inhibits cAMP-phosphodiesterase, prevents the cholestasis induced in vivo by both estrogens and TLC. We aimed to assess the ability of silibinin (Sil), the silymarin active component, to prevent the retrieval of Bsep induced by TLC and E 217G, and the associated alteration in its transport function. The possible involvement of cAMP as a second messenger and the intracellular signalling pathways implicated were also evaluated. Functional studies were performed analysing the proportion of isolated rat hepatocyte couplets (IRHC) accumulating the fluorescent bile salt analogue, cholyl-lysylfluorescein (CLF), into their sealed canalicular vacuoles. Cellular localisation of Bsep was assessed by immunofluorescent staining. Intracellular levels of cAMP were measured by ELISA. Sil (2.5 μM) elevated by 40 ± 3% intracellular cAMP, and mimicked the ability of dibutyryl-cAMP (10 μM) to prevent internalisation of Bsep and the TLC (2.5 μM)- and E 217G (50 μM)-induced impairment in the capacity of IRHC to accumulate CLF apically. Preventive effects of Sil and dibutyryl-cAMP were not abolished by the specific protein kinase A inhibitors, KT5720 and H89. Contrarily, the intracellular Ca 2+ chelator, BAPTA/AM, significantly blocked the protective effect of both compounds. We conclude that Sil prevented TLC- and E 217G-induced bile salt secretory failure, at least in part, by avoiding redistribution of Bsep, by a mechanism probably involving cAMP-induced cytosolic Ca 2+ elevations.

Changes in cyclic nucleotides during nodule formation

The changes in the cAMP and cGMP contents in root nodules were investigated using pea (Pisum sativum L. cv. Izusen) and soybeans (Glycine max L. (Merr.) cv. Enrei and its supernodulating mutant En6500). The concentration of cAMP increased in the nodules from pea and soybean cv. Enrei with nodule development, but not in the nodules from soybean En6500. The concentrations of cGMP remained low in pea and soybean cv. Enrei, while the levels of cGMP in the roots and nodules from soybean En6500 showed a temporal increase after rhizobium infection. The external application of dibutyryl-cAMP (db-cAMP) significantly decreased nodule production in these leguminous plants, indicating the possible involvement of cAMP in the regulation of nodule formation. On the other hand, dibutyryl-cGMP (db-cGMP) did not significantly affect the nodule fresh weight of these legumes. The data further suggest the existence of a difference of the functional role of cGMP from that of cAMP in the regulation of root nodule formation.

Possible involvement of cAMP in aerial mycelium formation and secondary metabolism in Streptomyces griseus.

In Streptomyces griseus, A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) triggers secondary metabolism and morphogenesis by binding a repressor protein (ArpA) and dissociating it from DNA. UV-mutagenesis of the A-factor-deficient mutant HH1 generated strain HO2, defective in the synthesis of ArpA and therefore able to form aerial mycelium, spores and streptomycin. Shotgun cloning of chromosomal DNA from wild-type S. griseus in strain HO2 yielded a gene that suppressed aerial mycelium formation and streptomycin production. Nucleotide sequencing and subcloning revealed that the gene encoded a eukaryotic-type adenylate cyclase (CyaA). In mutant HO2 production of cAMP was growth-dependent until the middle of the exponential growth stage; the production profile was the same as in the wild-type strain. However, the amount of cAMP produced was five times larger when mutant HO2 harboured cyaA on the high-copy-number plasmid pIJ486. Consistent with this, supplying cAMP exogenously at a high concentration to mutant HO2 suppressed formation of both aerial mycelium and streptomycin. On the other hand, some lower concentrations of cAMP stimulated or accelerated aerial mycelium formation. No effects of exogenous cAMP on morphogenesis and secondary metabolism were apparent in the wild-type strain. In addition, disruption of the chromosomal cyaA gene in the wild-type strain had almost no effect. Introducing cyaA cloned in either a low- or a high-copy-number plasmid suppressed morphogenesis and secondary metabolism not only in mutant HO2 but also in other arpA mutants, implying that the effects of cAMP became apparent in the arpA-defective background. When mutant HO2 carried cyaA on a plasmid, synthesis of the stringent response factor ppGpp was greatly reduced; this may account for the observed suppression by cAMP of morphogenesis and secondary metabolism. cAMP also affected protein tyrosine phosphorylation, as determined with antiphosphotyrosine antibody.

Leukotriene D 4 induces a rapid increase in cAMP in the human epithelial cell line, Int 407: a potential role for this signal in the regulation of calcium influx through the plasma membrane

Although the LTD 4-induced Ca 2+ influx in human epithelial cells has been shown to be regulated by a pertussis toxin-sensitive heterotrimeric G-protein, most likely a Gα i3 protein [Adolfsson J.L.P., Öhd J.F., Sjölander A. Leukotriene D 4-induced activation and translocation of the G-protein α 13-subunit in human epithelial cells. Biochem Biophys Res Commun 1996; 226: 413–419], the signalling pathway further downstream is still unclear. In the present study, we investigated the possible involvement of CAMP and protein kinase A activity in the LTD 4-induced Ca +2, influx in the epithelial cell line Int 407. Stimulation with LTD 4, but not with the calcium ionophore ionomycin, triggered a rapid increase (peak at 7 s) in the cellular CAMP level, an effect that was totally abolished by pertussis toxin. Furthermore, the LTD 4-induced Ca 2+ signal was reduced by 60% when cells that had been pre-incubated with the protein kinase A inhibitor Rp-cAMPS (50 μM for 30 min) were stimulated in a calcium containing medium. In contrast, Rp-cAMPS had no apparent effect on the LTD 4-induced Ca 2+ signal when the cells were stimulated in a calcium-depleted medium. The immediate LTD 4-induced protein tyrosine phosphorylation (15 s), previously shown to be necessary for the subsequent Ca 2+ influx, was abolished not only by pretreatment with pertussis toxin but also by exposure to Rp-cAMPS. Furthermore, direct activation of the cellular adenylyl cyclase activity by treatment with forskolin alone induced a prompt Ca 2+-signal in the presence, but not in the absence, of extracellular Ca 2+ identical results were obtained with the cell permeable cAMP analogue 8-bromo-cAMP. In addition, forskolin induced protein tyrosine phosphorylation similar to that seen with LTD 4. These results suggest that protein kinase A activity participates in the regulation of the LTD 4-induced Ca 2+ influx at a site that is downstream of the activation of the pertussis toxin-sensitive G-protein but upstream of a LTD 4-stimulated tyrosine kinase(s).

Down-regulation by prostaglandins of type-II phospholipase A2 expression in guinea-pig alveolar macrophages: a possible involvement of cAMP.

We have demonstrated previously that isolated guinea-pig alveolar macrophages (AM) synthesize type-II phospholipase A2 (PLA2-II) through a tumour necrosis factor-alpha (TNF-alpha)-dependent process. This synthesis is enhanced by lipopolysaccharide (LPS) and accompanied by a release of prostaglandin E2 (PGE2) into the medium. Because agents elevating intracellular cAMP, such as PGE2, have been shown to stimulate PLA2-II expression in various cell types, we investigated the modulation of PLA2-II synthesis by cAMP in AM. Surprisingly, incubation of AM with PGE2, dibutyryl-cAMP, cholera toxin or rolipram (an inhibitor of specific cAMP-phosphodiesterase) inhibited both basal and LPS-stimulated PLA2-II expression. The inhibitory effect of PGE2 was observed at concentrations similar to those released by AM. Moreover, treatment of AM with either aspirin or neutralizing PGE2 monoclonal antibody stimulated PLA2-II synthesis. These effects were closely correlated with the ability of these agents to modulate TNF-alpha release, which was decreased by dibutyryl-cAMP and exogenous PGE2, whereas neutralizing PGE2 antibody markedly increased this release. Hence, in contrast to other cell systems, we report that: (i) agents elevating intracellular cAMP levels down-regulate both basal and LPS-induced PLA2-II synthesis, (ii) prostaglandins exert a negative feedback effect on this synthesis, probably through an elevation of intracellular cAMP levels, and (iii) inhibition of TNF-alpha release may account, at least in part, for the down-regulation of PLA2-II expression by endogenously produced prostaglandins and cAMP-elevating agents.

Mouse cerebellar adenosinergic modulation of ethanol-induced motor incoordination: possible involvement of cAMP

As an extension of our previous work pertaining to brain adenosinergic modulation of ethanol-induced motor incoordination, the effect of direct intracerebellar administration of the A 1-selective adenosine agonist, N 6-cyclohexyladenosine (CHA) on ethanol-induced motor incoordination was evaluated. Marked accentuation of ethanol-induced motor impairment by CHA was observed. No change in the normal motor coordination was noted when CHA administration was followed by saline instead of ethanol. Intracerebellar cAMP or its analog, 8-(4-chlorophenylthio)-cAMP, significantly inhibited ethanol's motor impairment in a dose-related manner as well as abolished CHA's accentuating effect on ethanol-induced motor incoordination. These observations suggested a possible involvement of cAMP in the adenosinergic modulation and in the expression of ethanol-induced motor incoordination. Further support was provided by the observation of a marked accentuation and attenuation in a dose-related manner of ethanol-induced motor impairment as well as CHA's accentuation of ethanol's motor impairment by intracerebellar miconazole and forskolin, respectively. However, equimolar intracerebellar doses of miconazole and forskolin (inhibitor and stimulator of adenylyl cyclase, respectively) failed to significantly alter ethanol-induced motor incoordination probably due to their mutual functional antagonism. The expression of adenosinergic modulation and that of ethanol-induced motor impairment most likely involved G i protein-coupled receptor(s) (such as adenosine receptors). The involvement of receptors linked to pertussis toxin-sensitive G-proteins was suggested because intracerebellar pertussis toxin pretreatment markedly inhibited ethanol-induced motor incoordination as well as CHA's accentuation of ethanol's motor impairment. Finally, cAMP, unlike its antagonism to CHA's accentuation, failed to antagonize the accentuation of ethanol-induced motor impairment by intracerebellar GABA A agonist (+)-muscimol. This indicated selectivity of cAMP participation in G protein coupled receptor (such as adenosine)-mediated response and not in ionic channel coupled receptor (such as GABA A)-mediated mechanism. Overall, the data suggested a possible involvement of cerebellar adenylyl cyclase-cAMP signalling pathway in the adenosinergic modulation of ethanol's ataxia.

Transcription of the rat liver uricase-encoding gene is regulated via a cis-acting element responsive to cAMP

Glucagon was found to increase the mRNA level of the uricase-encoding gene ( UOX), but not that of genes encoding other peroxisomal enzymes, such as catalase, acyl-CoA oxidase and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase. The possible involvement of cAMP in the glucagon-induced transcription of rat UOX was studied by measuring the enhancer activity of the isolated 5′-untranslated region of the gene. An 84-bp sequence spanning positions −169 to −86 was found to be essential for cAMP-mediated expression of rat UOX, on deletional analysis of the upstream 1.4-kb portion by means of a transient transfection assay (CAT assay). The 30-mer oligodeoxyribonucleotide (positions from −169 to −140) was found to form a DNA-protein complex by an electrophoretic mobility shift assay. The core sequence for the DNA-protein complex formation, 5′-CAAAAATGTC-3′, was found to be located in positions from −164 to −155. In addition, the binding assays suggested that the DNA-binding protein(s) was different from cAMP-response element binding protein (CREB). Thus, this report shows that a novel cis-acting element of rat UOX and the binding protein(s) possibly play an essential role in the glucagon-induced transcription via cAMP.

Changes in total cAMP levels during oocyte maturation in the catfish, Clarias batrachus

Oocyte maturation (meiosis reinitiation) in the catfish ( Clarias batrachus) is induced by maturation-inducing steroid (MIS), 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-DP). The results of the present study demonstrate the possible involvement of cAMP in oocyte maturation in vivo or induced by 17α,20β-DP in vitro. Measurement of total cAMP levels at different morphological stages of oocytes obtained under in vivo conditions showed a significant reduction between the values at the centrally located germinal vesicle (C-GV), subperipheral (SP-GV), peripheral (P-GV), and germinal vesicle breakdown (GVBD) stage. In the time course study, MIS-treated oocytes showed a significant reduction in the total cAMP level at 2 hr and maintained it up to the duration of the study (24 hr). These results suggest that a decrease in the cAMP level within the follicle triggers oocyte maturation in this species. The in vitro effects of the adenylate cyclase activator, forskolin, and the phosphodiesterase inhibitor, theophylline, on 17α,20β-DP-induced GVBD was also investigated. Both substances inhibited 17α,20β-DP-induced GVBD, suggesting indirectly the inhibitory role of cAMP in catfish oocyte maturation.

Age-related decrease in the effect of parathyroid hormone-related protein on cytosolic free calcium level and tension in rat aortic smooth muscle.

Age-related changes in the effect of parathyroid hormone-related protein (PTHrP) on the cytosolic free calcium level ([Ca2+]i) and on the tension in rat aortic smooth muscle were investigated. The possible involvement of cAMP, a second messenger of PTHrP, in such changes was also investigated. Spiral aortic strip preparations without endothelium from 8-weeks, 6-months, and 24-months old rats were treated with fura 2/AM, and the fluorescence ratio R340/380, an index of [Ca2+]i was measured. Simultaneously, the tension of the preparations was measured. PTHrP-(1-34) and dibutyryl cAMP produced concentration-dependent decreases in the tension and in R340/380 in aortas precontracted with phenylephrine (10(-7) M). These effects were significantly lower in the aortas of 24-months old rats than in the vessels of 8-weeks and 6-months old rats. The effects were similar in the aortas of 8-weeks and 6-months old rats. PTHrP-(1-34) concentration-dependently increased cAMP production in vascular smooth muscle cells (VSMCs) from 8-weeks old rats. However, the increase in cAMP production was significantly lower in cultured VSMCs from 6-months and 24-months old rats than in cells from 8-weeks old rats. These results suggest that the reduced cAMP production stimulated by PTHrP and the reduced effects of cAMP with aging might contribute to the age-related changes in the decreases in tension and [Ca2+]i in response to PTHrP in the rat aorta.

DBI mRNA is expressed in endocrine pancreas and its post-translational product DBI33–50 inhibits insulin release

It has been previously demonstrated that DBI is present in endocrine pancreas and it is able to inhibit insulin release in isolated rat islets. Its mechanism of action has been investigated, demonstrating the possible involvement of cAMP and ATP-dependent K(+) channels. DB1(33-50), a post-translational product of DBI, is also able to inhibit insulin release, but its action has not been characterized. In the present study, we have investigated the presence of DBI mRNA in pancreas, islets and cultured ß cells. The possible mechanism of action of DBI(33-50) and the involvement of BZ/GABA(A) receptors has been studied.

Potentiation of acetylcholine responses in Xenopus embryonic muscle cells by dibutyryl cAMP.

Application of membrane-permeable analogues of adenosine 3',5'-(cyclic)monophosphate (cAMP) or forskolin to embryonic muscle cells of 1-day-old Xenopus cultures altered the response of the myocyte to iontophoretically applied acetylcholine (ACh). The initial amplitude and the decay time of the ACh-induced currents were elevated, and the rate of ACh-induced channel desensitization was increased. Single-channel recordings showed that cAMP analogues increased the mean open time of the low-conductance ACh channels, without affecting the single-channel conductance. Interestingly, this effect on ACh channels disappeared in myocytes of 3-day-old cultures, suggesting developmental changes in the susceptibility of the ACh channel to modulation. A possible involvement of cAMP in modulating the synaptic activity of early developing synapses was further indicated by prolonged decay times of spontaneous synaptic currents following treatment with dibutyryl-cAMP (Bt2cAMP). Factors released from the nerve terminals, if they activate the muscle adenylate cyclase system, could thus enhance the synaptic response during synaptogenesis.

Cyclic AMP modulates sensory-neural communication at the vestibular end organ

Adenosine 3′:5′-cyclic phosphate (cAMP) is a second messenger that plays an important role in mediating neuronal interactions in many systems. A possible role for cAMP in sensorineural communication at the vestibular end organ was studied. The putative roles for cAMP action investigated here were: the ability of cAMP to act as the second messenger for the efferent transmitter, acetylcholine, and the possible involvement of cAMP in modulating spontaneous or mechanically-evoked afferent nerve firing. Levels of cAMP were increased pharmacologically with forskolin, 3-isobutyl-1-methyl xanthine (IBMX) and dibutyryl cAMP. Changes in multiunit afferent nerve firing measured from the ampullar nerve of the semicircular canal, and the transepithelial potential measured across the neuroepithelium of the semicircular canal were recorded. At selected doses, all drugs produced a similar increase in spontaneous multiunit afferent nerve firing with a concomitant decrease in the transepithelial potential. Mechanically-evoked hair cell activity and the response to exogenously applied acetylcholine were unaffected by these drugs. We are suggesting that the excitatory aspects of the acetylcholine response are not mediated via a cAMP-dependent mechanism. However, cAMP does play an important role in modulating spontaneous afferent nerve firing in the semicircular canal. The finding that spontaneous afferent nerve firing can be biochemically modulated without altering mechanically-induced afferent firing is novel and deserves further investigation.

Rat heart organ culture for the study of trophic substances involved in the maintenance of normal sensitivity to norepinephrine: possible involvement of cAMP and search for other factors

In organ culture conditions, in the absence of in vivo factors, the newborn rat right atria acquire a high sensitivity to agonists similar to that seen before sympathetic innervation and after denervation. In the present study, we examined the effects of various extracts and substances on the development of supersensitivity to norepinephrine (NE) to obtain information on the in vivo factors that regulate myocardial sensitivity. Addition of rat serum, right atrial extract, superior cervical ganglionic extract, vas deferens extract, carbachol, insulin, cortisone, thyroxin, and neuropeptide Y in the culture medium did not prevent the development of supersensitivity. Addition of NE completely inhibited the development of supersensitivity. This effect of NE was blocked by sotalol but not by phentolamine. Addition of calcitonin gene related peptide, forskolin, and 8-bromo-cAMP partially inhibited the development of supersensitivity. These results are consistent with the view that NE released from sympathetic nerve terminals in the newborn atria maintains myocardial sensitivity at normal level by acting on beta-adrenergic receptors, and that the effect may be partially mediated by a rise in intracellular cAMP concentration.

Early effects of parathyroid hormone on membrane potential of rat osteoblasts in culture: Role of cAMP and Ca2+

Microelectrodes were used to investigate the possible involvement of cAMP and Ca2+ ions in the parathyroid hormone's, bPTH(1-34), effect on the membrane potential of rat osteoblasts in primary culture. Parathyroid hormone (10(-7) M) depolarized cell membrane by 25.0 +/- 6.1 mV (mean +/- standard deviation, SD; n = 17). Blocking Ca2+ influx with the Ca channel blocker cobalt revealed two phases in the hormone effect: a rapid and slight membrane hyperpolarization followed by sustained depolarization. In addition, cobalt significantly (p less than 0.01) decreased the magnitude of the PTH depolarizing action. The addition of dibutyryl-cAMP (10(-3) M) to the perfusion solution also resulted in a biphasic effect. At a lower concentration (10(-4) M), dibutyryl-cAMP produced only membrane hyperpolarization, suggesting a cAMP dose dependence of the opposite membrane potential changes. Forskolin (10(-5) M) and the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) (10(-4) M) mimicked the depolarizing effect of PTH. IBMX at a low concentration (5 x 10(-6) M) potentiated the depolarizing effect of PTH. Increases in [Ca2+]i using Ca2+ ionophore A23187 and intracellular injection of CaCl2 or inositol trisphosphate decreased the PTH depolarizing action, whereas intracellular injection of EGTA enhanced this effect. These results indicate that PTH evokes a biphasic change in rat osteoblast membrane potential that seems to be mediated by an increase in cAMP and modulated by intracellular calcium.

Neurite outgrowth in individual neurons of a neuronal population is differentially regulated by calcium and cyclic AMP

In identified Helisoma neurons, intracellular calcium can regulate neurite elongation and growth cone motility. Neurotransmitters such as 5-HT suppress both neurite elongation and the filopodial and lamellipodial movements of growth cones by causing increases in intracellular calcium (Haydon et al., 1984; Cohan et al., 1987; Mattson and Kater, 1987). Since an additional second messenger, cyclic AMP (cAMP), is known to mediate many physiological effects of neurotransmitters, we tested (1) the possible involvement of cAMP in the regulation of neurite outgrowth from Helisoma buccal neurons and (2) calcium-cAMP interrelationships in the regulation of outgrowth. The cAMP-elevating agents forskolin (5 x 10(-6)-10(-4) M) and dibutyryl cAMP (dbcAMP; 5 x 10(-3)-10(-2) M) suppressed neurite elongation and growth cone movements in identified neurons B19 (5-HT sensitive) and B5 (5-HT insensitive); the suppression was reversible. Exposure of these particular identified neurons to the calcium channel blocker La3+ (10(-5) M) or a culture medium with reduced calcium prevented and reversed the suppressive effects of forskolin and dbcAMP. In order to determine if the results on neurons B5 and B19 were representative of all neurons or only a subset, we examined a larger population of neurons. Calcium ionophore A23187 suppressed outgrowth from all neurons in mass dissociate cultures of buccal neurons, while forskolin or dbcAMP plus IBMX suppressed outgrowth from only one-half of buccal neurons. Finally, we found that 2 subpopulations exist among the neurons whose outgrowth is suppressed by cAMP: One subpopulation requires calcium influx for cAMP to act, while the other does not. Thus, even within the relatively small population of neuronal types comprising the buccal ganglion of Helisoma, second messengers within different neurons can act and interact in different ways to regulate outgrowth.

Effects of ATP and cAMP on salt and sugar (responses of chemosensilla in the blowfly)

1. 1. The addition of cAMP to stimulating solutions of NaCl, fructose (furanose sugar), sucrose, or glucose (pyranose sugars) decreases the responsiveness of labellar chemosensilla in Phormia. 2. 2. The addition of ATP, while decreasing the responsiveness to NaCl or fructose enhances the responsiveness to glucose and sucrose. 3. 3. The inhibiting effect of ATP on NaCl or fructose responses is suppressed by GDPßS, an inhibitor of adenylate cyclase (and thus of cAMP synthesis); moreover GDPßS further enhances the increase in response due to ATP when added to the sucrose or glucose solutions. 4. 4. Results suggest a possible involvement of cAMP and ATP in the taste reception mechanism in the blowfly.

Comparison of 3-isobutyl-1-methylxanthine-induced accumulation of putrescine in rat pancreas and liver

3-Isobutylmethylxanthine (IBMX), a potent phosphodiesterase inhibitor, causes accumulation of putrescine of same magnitude in rat pancreas and liver. IBMX produces increases of acetyl CoA : polymine N′-acetyltransferase (PAT) and of ornithine decarboxylase (ODC) activities in both organs. However ODC activity is 300 times higher in liver than in pancreas. In the latter organ, there is a transient increase of N 1-acetylspermidine, followed by a decrease of spermidine. α-Difluoromethylornithine (DFMO), a potent ODC inhibitor, impairs the accumulation of putrescine in liver but not in pancreas. These results suggest that in pancreas the accumulated putrescine is essentially formed from spermidine, via N 1-acetylation and oxidation, while in liver it is formed from decarboxylation of ornithine. A possible involvement of cAMP in the stimulation of the polyamine interconversion pathway is discussed.