Inhibition by acidosis of adenosine 3',5'-cyclic monophosphate accumulation and lipolysis in isolated rat fat cells.

Lipolysis and cyclic AMP accumulation were studied in isolated rat fat cells at normal (7.4) and decreased (7.0, 6.6) pH. Acidosis inhibited lipolysis and cyclic AMP accumulation due to NA non-competetively. Maximal lipolysis (3 muM NA) was inhibited by 25% at pH 7.0 and by 61% at pH 6.6 Cyclic AMP accumulation 5 min after 3 muM NA was inhibited by 57% at pH 7.0 and by 83% at pH 6.6. Between 10 and 60 minutes of incubation NA-stimulated lipolysis was linear at pH 7.4, whereas a progressively increasing inhibition was seen at lower pH. The FFA production was inhibited to the same degree as glycerol production by acidosis. The fraction of FFA associated with the cells was the same at all pHs. Thus, we have no evidence that acidosis inhibits lipolysis via accumulation of FFA intracellularly. NA-induced accumulation of 3H-cAMP from 3H-ATP, endogenously formed by prelabelling the cells with 3H-adenine, was inhibited by acidosis both in the presence and absence of theophylline in the incubation medium (by 48 and 44% respectively at pH 7.0 and by 74 and 68% at pH 6.6). Cyclic nucleotide phosphodiesterase in homogenates of fat cells was inhibited by decreasing the pH, whether measured at high or low substrate concentrations. Basal adenylyl cyclase activity in a cell membrane fraction from fat cells was affected to a minor degree, while NA-stimulated activity was inhibited by decreased pH. The response to 3 muM NA at pH 6.6 was inhibited by 43% relative to control. The results show that acidosis inhibits NA-induced cyclic AMP accumulation by interfering with the formation, rather than the inactivation of the nucleotide. Since NA-induced lipolysis is a cyclic AMP-mediated process it is suggested that at least part of the antilipolytic effect of acidosis is due to inhibition of cyclic AMP formation.     

Dopamine- and adenosine-3':5'-monophosphate (cAMP)-regulated phosphoprotein of Mr 32,000 (DARPP-32) in the retina of cat, monkey and human.

The cellular localization of a dopamine- and cAMP-regulated phosphoprotein of Mr 32,000 (DARPP-32) was investigated in cat, monkey and human retina by immunohistochemistry. In cat, DARPP-32-immunoreactive cell bodies identified as Müller cells were demonstrated in the inner nuclear layer (INL) with processes closely surrounding the cell soma of photoreceptors in the outer nuclear layer. Some DARPP-32-IR cells were also seen in the nerve fiber layer (NFL) sending processes to the inner plexiform layer. In monkey and human retina, DARPP-32-IR cell bodies were also demonstrated in the INL, with few cells located in the NFL.     

Regulation of 3', 5'-cyclic guanosine monophosphate content in deep cerebellar nuclei.

The 3',5'-cyclic guanosine monophosphate (cGMP) content of deep cerebellar nuclei is decreased following activation of γ-aminobutyrate (GABA) receptors by muscimol and diazepam and is increased following a reduction of GABA content caused by isoniazid. Since previous work has shown that, in deep cerebellar nuclei, GABA receptor stimulation decreases the firing rate of neurones (Ito, Yoshida, Obata, Kawai &;Udo, 1970), it appears that the cGMP content of deep cerebellar nuclei may be an index of the activity of these neurones. Neither the destruction nor the specific activation of climbing fibers changes the cGMP content of deep cerebellar nuclei. Morphine and haloperidol decrease the cGMP content of deep cerebellar nuclei without changing the activity of enzymes that metabolize cGMP in cell free preparations or by activating GABA receptors or changing GABA content. Thus, it is tempting to conclude that a decrease in mossy fiber activity could mediate the decrease in the cGMP content of deep cerebellar nuclei induced by morphine and haloperidol injected parenterally.     

Mode of stimulation by adenosine 3':5'-cyclic monophosphate of the sodium efflux in barnacle muscle fibres.

1. Giant fibres of the barnacle Balanus nubilus have been used as a preparation for studying the mode of action of cAMP on sodium transport. 2. It is shown that a concentration of cAMP as low as 10(-6)M, when micro-injected, causes a sharp rise in the radio-Na efflux. Ouabain fails to reverse the cAMP effect. 3. The magnitude of the response of the Na efflux to cAMP is markedly reduced by pre-injecting 100 or 500 mM-EGTA solutions or by omitting Ca2+ from the bathing medium. Both together fail to bring about a greater reduction in the response. 4. The response to cAMP is greatly reduced by pre-injecting the protein inhibitor of Walsh and practically abolished by pre-injecting 500 mM-EGTA and soaking in Ca-free artificial sea water, ASW. 5. The Ca2+-independent component of the Na efflux which is also stimulated by cAMP is shown to involve Na for H exchange. The magnitude of this exchange is governed by external pH. 6. The Na efflux into Ca2+-free, Li+-ASW is shown to be markedly stimulated by injecting cAMP, an effect which is enhanced by reducing external pH. 7. The Na efflux at 0 degrees C is stimulated by injecting cAMP. This is shown to be related to activation of the protein kinase by cAMP and to depend on the presence of external Ca2+. 8 (i) Ethacrynic acid when injected reduces the ouabain-insensitive Na efflux into HEPES-Ca2+-free ASW at pH 6-3. These same fibres show a marked response to cAMP. (II) The ouabain-insensitive Na efflux into HCO3-, Ca2+-free ASW from fibres pre-treated with ethacrynic acid fails to respond to external acidification. This is interpreted as indicating that ethacrynic acid inactivates the CO2-sensitive adenyl cyclase system. These same fibres when injected with cAMP show a marked response. (iii) Stimulation of the ouabain-insensitive Na efflux into HCO-3, Ca2+-free ASW by external acidification is reversed by injecting ethacrynic acid. These fibres when injected with cAMP show a reduced response. 9. It is concluded that: (i) stimulation of the Na efflux by injected cAMP is mainly due to activation of cAMP-dependent protein kinase; (ii) the underlying exchange mechanism consists of Na:Ca and Na:H exchange. Interaction of Ca2+ with a phosphorylated membrane, thereby modifying permeability remains as a real possibility; (iii) the site of action of CO2 and ethacrynic acid is the adenyl cyclase system. 10. The implications of activation of the adenyl cyclase system by CO2 and Na:H exchange are briefly touched upon.     

A new approach to immunocytochemistry of 3',5'-cyclic guanosine monophosphate: preparation, specificity, and initial application of a new antiserum against formaldehyde-fixed 3',5'-cyclic guanosine monophosphate

The development of a new 3',5'-cyclic guanosine monophosphate (cGMP) antiserum was initiated starting from the following considerations: (a) adequate fixation of cGMP is a prerequisite for a reliable demonstration of soluble cGMP, and (b) fixation might influence the specificity of the immunocytochemical demonstration of cGMP. Therefore, cGMP-protein conjugate was prepared in a way which equals tissue fixation. cGMP was coupled to bovine thyroglobulin using formaldehyde. Antibodies against this conjugate were raised in rabbits. The specificity of the antisera was evaluated in a gelatin model system. No immunoreactivity was observed with nucleotides other than cGMP or with rabbit preimmune sera. Immunoinhibition experiments showed that only the cGMP-formaldehyde-thyroglobulin conjugate and, to a lesser extent free cGMP, absorbed onto the antiserum. In rat brain an extensive localization of cGMP-immunostaining was found. Examples are hippocampus CAI and CAII, and cortical layers II and V. No cGMP-immunostaining was found in the cerebellum. In vitro incubated superior cervical ganglia showed cGMP-immunostaining in the large postganglionic neuronal cell bodies; this cGMP-immunostaining increased upon incubation of the ganglia in iso-osmolar 100 mM K+. In conclusion, we prepared a new-type highly specific antiserum against cGMP, suitable to demonstrate cGMP-immunoreactivity in tissue material.     

Hyperpolarization of myenteric neurons by opioids does not involve cyclic adenosine-3',5'-monophosphate.

To investigate the role of cyclic adenosine-3'5'-monophosphate on the inhibitory actions of opioids in guinea-pig ileum, we made intracellular recordings from the two electrophysiologically defined classes of neurons (S and AH) in the myenteric plexus. The selective opioid mu agonist (D-Ala2,N-Me-Phe4,Gly5-ol)-enkephalin caused a membrane hyperpolarization in 34 out of 67 S neurons but did not affect the membrane potential of AH neurons. The mean amplitude (+/- S.E.M.) of the hyperpolarization was 8.2 +/- 0.8 mV. Forskolin, which activates adenylate cyclase and increases intracellular cyclic adenosine-3',5'-monophosphate levels, caused a membrane depolarization in AH neurons (9.4 +/- 1.9 mV) but did not alter the resting membrane potential of S neurons. Similarly, neither the phosphodiesterase inhibitor, isobutylmethylxanthine, nor the membrane permeable analogue of cyclic adenosine-3',5'-monophosphate, dibutyryl cyclic adenosine-3'-5'-monophosphate, altered the resting membrane properties of S neurons. Furthermore, none of these agents affected significantly the amplitude of the hyperpolarization of S neurons by (D-Ala2,N-Me-Phe4,Gly5-ol)-enkephalin. The experiments indicate that changes in intracellular cyclic adenosine-3',5'-monophosphate are not important in the processes that link occupation of mu receptors to the opening of potassium channels on myenteric neurons.     

Adenosine 3',5'-cyclic monophosphate in perfused rat hearts exposed to isoprenaline and dopamine.

Isoprenaline and dopamine increased cyclic AMP (cAMP) content and contractile activity of isolated perfused rat hearts. The changes of cAMP levels depended on the mode of drug administration. Isoprenaline (4 x 10(-10) mol) administered to the perfused heart as a relatively concentrated bolus, caused a substantial, rapid and transient increase of cAMP. Isoprenaline (2 x 10(-10) mol and 4 x 10(-10) mol) and dopamine (10(-7) mol) diluted in 40 ml of perfusate which was continuously recirculated through the heart, caused a gradual increase of cAMP content which approached an apparent steady state. cAMP accumulation occurred at isoprenaline concentrations above 10(-9) M and at dopamine concentrations above 10(-6) M. Both agents also increased cAMP labelling from 14C-adenine in the perfusate, probably indicating increased cAMP synthesis. Isoprenaline at 2 x 10(-8) M and 10(-7) M increased labelling more than content of cAMP. Isoprenaline and dopamine also increased phosphorylase a activity. An association between increased cAMP contents and increased contractile activity was revealed by both the time-response and the dose-response curves of hearts exposed to isoprenaline and dopamine. Since both agents stimulate adrenergic beta-receptors in cardiac muscle, the results are concordant with the hypothesis that cAMP is involved as a mediator of the inotropic response to adrenergic beta-stimulation.     

Excitatory amino acid receptors and guanosine 3',5'-cyclic monophosphate in incubated slices of immature and adult rat cerebellum

Addition of the excitatory amino acids l-glutamate, l-aspartate and their analogues kainate and N-methyl-d-aspartate to incubated slices of adult rat cerebellum led to large increases in cyclic GMP levels. The order of apparent potencies was kainate greater than N-methyl-d-aspartate greater than glutamate and aspartate. D-alpha-aminoadipate and Mg2-+ inhibited responses to N-methyl-d-aspartate while glutamic acid diethyl ester was most effective against those to glutamate; responses to kainate were least affected by the antagonists. The exicitant amino acids also elicited large elevations of cyclic GMP levels in slices of immature (8 day) cerebellum. Kainate was less effective than in adult but induced two responses distinguishable by their different time courses, concentration dependencies and sensitivity to antagonists. N-methyl-d-aspartate, glutamate and aspartate were 5 to 10-fold more potent than in the adult. Responses to N-methyl-d-aspartate were similarly inhibited by d-alpha-aminoadipate and Mg2+ but those to glutamate were more resistant to glutamic acid diethyl ester than in the adult. It is concluded that the accumulation of cyclic GMP in response to excitant amino acids in the adult cerebellum is mediated via the operation of receptor types showing pharmacological characteristics expected of excitatory amino acid receptors. The actions of kainate in the immature cerebellum appear to be mediated by receptors different from those on which it acts primarily in the adult.     

The effect of presynaptic receptor stimulation on adenosine 3',5'-cyclic monophosphate concentrations in rat cortical synaptosomes

Changes in adenosine 3',5'-cyclic monophosphate (cAMP) concentration were measured in cortical synaptosomes. Preincubation with adenosine deaminase reduced cAMP concentration by 45%. Oxotremorine, clonidine, gamma-aminobutyric acid (GABA) and baclofen produced no change in basal concentration. 2-Chloroadenosine and noradrenaline (NA), acting at beta-adrenoceptors, both caused a dose-dependent increase in cAMP; the NA-stimulated increase was depressed by GABA and by baclofen.     

Effect of extracellular magnesium on secretagogue-evoked amylase secretion in the isolated rat parotid gland segments.

This study investigates the effect of perturbation of extracellular magnesium [Mg2+]o on basal and acetylcholine (ACh), noradrenaline (NA) and phenylephrine (PHE)-evoked amylase secretion from isolated rat parotid gland segments. Both zero (0 mM) and elevated (5 mM and 10 mM) [Mg2+]o can significantly (P < 0.05) inhibit basal and secretagogue-evoked amylase secretion compared to the responses obtained in normal (1.1 mM) [Mg2+]o. The inhibitory effect of zero [Mg2+]o was more pronounced compared 10 mM [Mg2+]o. A concentration of 5 mM, [Mg2+]o was less effective at a secretagogue concentration of 10(-5) M but more pronounced in inhibiting amylase secretion when the concentration of the secretagogue was reduced to 10(-6) M. The results indicate that both hypo and hypermagnesaemia are associated with reduced salivary amylase secretion and both conditions may be associated with 'the dry mouth syndrome'.     

An action of disodium cromoglycate: inhibition of cyclic 3',5'-AMP phosphodiesterase.

Disodium cromoglycate (DSCG) prevents allergic asthma by inhibiting the release of chemical mediators of immediate-type allergic reactions. The mechanism of this action is unclear and prompted us to examine the effect of DSCG on cyclic adenosine 3',5'-monophosphate (cAMP), the implicated regulator of IgE-mediated reactions. We used the peripheral blood lymphocyte as a model to mirror the biochemical events occurring in the allergic shock organs. Isolated peripheral blood lymphocytes from perennial allergic asthmatic children receiving only DSCG had significantly (p less than 0.005) lower phosphodiesterase (PDE) activity (mean 1.05 +/- 0.17 SE per 10(6) cells) than normal individuals (2.93 +/- 0.14) and allergic children receiving methylxanthines (4.08 +/- 0.28) or no medications (3.58 +/- 0.2). DSCG (10 mug/ml) significantly lowered PDE activity in normal lymphocytes (p less than 0.005) in a beef heart extract (p less than 0.001), and 100 mug/ml lowered PDE activity in fetal rabbit lung homogenates (p less than 0.001). DSCG (10 mug/ml) significantly elevated (p less than 0.01) cAMP concentration in normal human lymphocytes (118 +/- 38 vs 30 +/- 10 picomoles cAMP/10(6) lymphocytes). Thus, DSCG appears to inhibit chemical mediator release by increasing intracellular cAMP through the inhibition of cAMP PDE.     

Endogenous prostaglandins, adenosine 3':5'-monophosphate and sodium transport across isolated frog skin.

1. Sodium transport across isolated frog skin, as measured by the short-circuit current, was decreased by acetylsalicylic acid, mefenamic acid, paracetamol and phenylbutazone. Indomethacin (6 X 10(-6) M) had a biphasic effect on the short-circuit current: a transient increase followed by a sustained decrease. 2. The release of prostaglandin-like material from the skin was reduced by acetylsalicylic acid and indomethacin. Paracetamol caused a significant reduction in the short-circuit current response of the skin to low doses of arachidonic acid, but the response to the highest dose tested was not significantly altered. 3. Indomethacin (6 X 10(-6) M) increased the sensitivity of the skin to applied prostaglandin E1. The other prostaglandin synthetase inhibitors did not have this effect. Indomethacin (6 X 10(-6) M) also enhanced the effect of antidiuretic hormone on the short-circuit current. 4. Indomethacin (30 X 10(-6) M) increased the short-circuit current and diminished the response to applied prostaglandin E1. 5. In sulphate Ringer, theophylline increased the short-circuit current and diminished the response to prostaglandin E1. 6. Prostaglandin E1 increased the levels of cyclic AMP in frog skin and these increases preceded the increases in short-circuit current. There was a seasonal variation in the level of cyclic AMP in the skin: the levels in winter exceeded those in summer. There was also a seasonal variation in the cyclic AMP response to prostaglandin E1: the winter response was greater than that in summer. 7. Indomethacin (6 X 10(-6) M) had a biphasic effect on cyclic AMP levels in the skin, an initial increase followed by a decrease. Indomethacin also potentiated prostaglandin E1 stimulated cyclic AMP accumulation. 8. Theophylline increased cyclic AMP levels in the skin and potentiated prostaglandin E1 stimulated cyclic AMP accumulation. 9. Pre-treatment of the skin with theophylline reversed the effects of cyclic AMP on the short-circuit current and open-circuit potential. 10. It is concluded that endogenous prostaglandins help to maintain sodium transport across isolated frog skin and that the effects of E-type prostaglandins on the short-circuit current are mediated by increased cyclic AMP levels. The transient increase in short-circuit current and the increased skin sensitivity caused by indomethacin (6 X 10(-6) M) are attributed to inhibition of phosphodiesterase activity. The failure of theophylline to potentiate the short-circuit current response of the skin to prostaglandin E1 is attributed to alteration of cyclic AMP action on the skin by theophylline.