Effects of N6, 2‘-0-dibutyryladenosine 3’,5‘-cyclic monophosphate,adenosine, and of oxotremorine and 3-isobutyl-1-methylxanthine on the electrically evoked [3H] acetylcholine secretion in the guinea-pig ileum myenteric plexus

The guinea-pig ileum longitudinal muscle-myenteric plexus preparation, pre-incubated with [³H]choline, was mounted in an organ bath and superfused with Tyrode's solution. [³H]Acetylcholine secretion was evoked by 150 electrical shocks at 0.5 Hz. >N⁶,2′-0-Dibutyryladenosine 3′,5′-cyclic monophosphate (dibutyryl cyclic AMP) enhanced the [³H]acetylcholine secretion in the presence of eserine and the adenosine receptor antagonist 8-phenyltheophylline (10 μmol 1^-1). Conversely, in the absence of 8-phenyltheophylline the [³H]acetylcholine secretion was reduced by dibutyryl cyclic AMP. In the absence and presence of 8-phenyltheophylline (apparent K_D = 12 μ.mol 1^-1), adenosine reduced the [³H]acetylcholine secretion to 33% of control (IC₅₀ = 8, μmol l^-1) and to 48% of control (IC₅₀ = 14 μmol l^-1) respectively. Neither butyrate, dibutyryl cyclic GMP nor guanosine altered the [³H]acetylcholine secretion. Interaction experiments with 3-isobutyl-1-methylxanthine and oxotremorine were done in the absence of eserine, i.e. when oxotremorine is effective. Oxotremorine depressed the fractional secretion of [³H]acetylcholine with a ‘maximal inhibition’ of 13% of control (IC₅₀ = 10 nmol l^-1). In the presence of 3-isobutyl-1-methylxanthine (5 mmol l^-1) oxotremorine depressed the secretion to 2% of control with an apparent IC₅₀) value of 0.9 μmol l^-1 3-Isobutyl-I-methylxanthine (0.01–4 mmol l^-1) enhanced the fractional secretion of [³H]acetylcholine with a ‘maximal enhancement’ value of 232% of control (EC₅₀ = 0.19 mmol l^-1). The presence of oxotremorine (30 nmol l^-1) counteracted, and higher concentrations reversed, the enhancement caused by 3–isobutyl-I-methylxanthine. The results are consistent with the suggestion that the presynaptic muscarinic acetylcholine receptor-mediated control of the secretion of [³H]acetylcholine in the guinea-pig ileum myenteric plexus may be mediated both by cyclic AMP and calcium ions.     

Secretion of 3H-acetylcholine from guinea-pig ileum myenteric plexus is enhanced by 8-Br adenosine 3‘,5’-cyclic monophosphate but not changed by 8-Br guanosine 3‘,5’-cyclic monophosphate

The acetylcholine (ACh) stores of cholinergic neurons of the myenteric plexus of guinea-pig ileum were labelled by preincubation with ³H-(methyl)-choline. The secretion of labelled transmitter evoked by electrical field stimulation at 1 Hz in the presence of eserine increased by 55% after addition of 0.5 mM 8-Br adenosine 3‘,5’-cyclic monophosphate (8-Br cAMP). Atropine further enhanced the secretory response, but not more than in the absence of 8-Br cAMP. 8-Br guanosine 3‘,5’-cyclic monophosphate (8-Br cGMP, 0.5 mM) did not change the secretory response to 0.5 or 1 Hz stimulation, either at 1.8 mM or at 0.6 mM calcium, in the absence of eserine. Nor did 1 mM 8-Br cGMP alter the effects of atropine or of oxotremorine. Activation of guanylate cyclase by 0.1 mM N-methyl-N‘-nit-ro-N-nitroso guanidine failed to alter the secretory response to 0.5 Hz stimulation in the absence of eserine, or to influence the depression of the secretion caused by oxotremorine. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (1 mM) neither altered the secretory response in the presence of eserine, nor the enhancing effect of atropine. The results suggest that cyclic nucleotides are probably not critically involved as “second messengers” in the muscarinic “autoinhibition” of ACh secretion from cholinergic myenteric neurons of guinea-pig ileum.     

Regulation of chondrosarcoma metabolism by cyclic adenosine 3'5'-monophosphate

The in vitro effects of cyclic AMP on amino acid transport and synthesis of macromolecules in the Swarm rat chondrosarcoma were investigated using the cyclic AMP analogue, N6-monobutyryl cyclic AMP (MBcAMP), and the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (MIX). Amino acid transport was assessed by measuring alpha-amino-[1-14C]isobutyrate (AIB) uptake. The synthesis of macromolecules was estimated by measuring radiolabeled precursor incorporation into total proteins, proteoglycan, and RNA. MBcAMP stimulated [14C]AIB uptake, [3H]uridine transport, and UTP formation. MBcAMP inhibited 35SO4 and [3H]leucine incorporation into proteoglycan and stimulated [3H]uridine incorporation into RNA. MIX elevated endogenous cyclic AMP levels in the Swarm rat chondrosarcoma and mimicked the effects of MBcAMP on AIB transport and radiolabeled precursor incorporation into macromolecules. For comparative purposes, the effects of MBcAMP on AIB uptake and macromolecule synthesis in rat costal cartilage segments were investigated. MBcAMP and MIX stimulated AIB uptake by costal cartilage segments, inhibited [3H]leucine incorporation into total protein and 35SO4 incorporation into proteoglycan, and had no effect on [3H]uridine incorporation into RNA.     

Histamine, cAMP, and activation of piglet gastric mucosa

The involvement of cAMP as a second messenger for histamine-induced H+ secretion was studied in a physiologically active, in vitro preparation of piglet gastric mucosa. During the first 5--10 min of stimulation with either histamine or the cAMP phosphodiesterase inhibitor 3-isobutyl-1,4-methylxanthine (IBMX), increases (greater than or equal to 5-fold) in tissue cAMP content [(c-AMP]) were well correlated with the characteristic decrease in transepithelial resistance (R); these changes precede H+ secretion by several minutes. Control experiments indicate that, during these treatments, tissue [cAMP] is dominated by the [cAMP] of oxyntic cells alone; change in R and H+ are also related to activity of these cells alone. At the steady state (45 min), histamine and IBMX caused equivalent increases in H+ and decreases in R, but [cAMP] was markedly different in the two cases. With IBMX [cAMP] was elevated at least fivefold, whereas with histamine [cAMP] was less than or equal to 50% above resting levels. The tissue is also stimulated by exogenous additions of dibutyryl cAMP. A histamine-sensitive adenylate cyclase was present in isolated, purified oxyntic cells. The histamine sensitivity of the cyclase was very similar to that which the intact tissue exhibits for histamine-induced changes in H+ and R. The cyclase activity was blocked by cimetidine but not by promethazine. We conclude that during stimulation histamine activates a histamine (H2)-sensitive adenylate cyclase of oxyntic cells, and there is a rapid increase in cellular [cAMP] that is involved in activation of H+ transport and other associated changes of oxyntic cells. An active phosphodiesterase is responsible for reducing [cAMP] to a level much below the "peak" value. Other cellular factors (e.g. protein kinases and Ca2+-calmodulin) must also be involved in the maintenance of the stimulated state of oxyntic cells.     

Metiamide and stimulated acid secretion from the isolated non-distended and distended mouse stomach.

1. The action of metiamide, a specific histamine H2-receptor antagonist, on the acid secretory response to various gastric stimuli in the perfused isolated whole mouse stomach is described. 2. Two kinds of stomach preparations, the non-distended stomach and distended stomach, were used. The distended stomach gave a marked and dose-related acid secretory response to histamine (10(-6) to 10(-3) M), pentagastrin (10(-8) to 10(-5) M), acetylcholine (5 X 10(-5) to 10(-5) M), eserine (10(-5) to 10(-3) M) to dibutyryl cyclic AMP (5 X 10(-5) to 10(-3) M). In the nondistended stomach, dibutyryl cyclic AMP regularly stimulated acid secretion in a dose-dependent manner; in contrast to dibutyryl cyclic AMP, histamine, pentagastrin or acetylcholine did not always stimulate acid secretion. 3. Histamine or pentagastrin but not acetylcholine always caused significant stimulation of acid secretion from the non-distended stomach in the presence of a phosphodiesterase inhibitor such as caffeine, theophylline or the I.C.I. compound, 63197. At the concentration of 10(-4) M, these phosphodiesterase inhibitors markedly potentiated the stimulatory effect of histamine or pentagastrin on acid secretion and the order of effectiveness was 63197 greater than theophylline greater than caffeine. 63197 also produced profound potentiation of histamine- or pentagastrin-stimulated acid secretion in the distended stomach. 4. Metiamide (5 X 10(-5) to 10(-4) M) did not antagonize stimulation of acid secretion by dibutyryl cyclic AMP in the non-distended or distended stomach. 5. In the distended stomach, metiamide (5 X 10(-4) M) produced significant inhibition of histamine-stimulated acid secretion with a linear and parallel displacement of the histamine dose--response curve to the right. Although at this concentration metiamide did not depress maximal acid secretory response to histamine, it caused marked reduction of the maximal acid secretory response attainable with pentagastrin. 6. In the distended stomach, metiamide (5 X 10(-5) M) did not cause significant inhibition of acetylcholine-induced acid secretion. Atropine (5 X 10(-6) M) abolished the stimulatory effect of acetylcholine; it also produced marked inhibition of pentagastrin-stimulated acid secretion but it had little effect on acid secretion induced by histamine. 7. The present results indicate that metiamide inhibited histamine-induced acid secretion by competitive antagonism of the histamine H2-receptor, but its inhibitory effect on pentagastrin-induced acid secretion seemed to be of non-competitive nature. The failure of metiamide to inhibit acid secretion induced by dibutyryl cyclic AMP suggests that cyclic AMP might regulate gastric acid secretion at a site beyond the histamine H2-receptor activation. It is also considered that the present results support the hypothesis that cyclic AMP may be involved in histamine- or pentagastrin-induced acid secretion in the isolated mouse stomach. 8...     

Adenosine-induced depression of synaptic transmission in the isolated olfactory cortex: receptor identification

We have investigated the type of purine receptor in the guinea-pig olfactory cortex, using pial surfaces slices maintained in vitro. Adenosine (0.1 to 100 mumol/l) bath applied in the presence of the uptake inhibitor nitrobenzylthioinosine, depressed the evoked potentials in a dose related fashion. Synthetic and uptake resistant adenosine analogues had the same effect as adenosine and the order of potency of these was: 5'-N-ethylcarboxamide adenosine greater than L-N6-phenylisopropyl adenosine (L-PIA) = N6-cyclohexyladenosine = 2-chloroadenosine greater than adenosine greater than D-N6-phenylisopropyladenosine (D-PIA). The D-stereoisomer of PIA was 45 times less potent than L-PIA. The methylxanthine compounds 8-phenyltheophylline (3 mumol/l) and 3-isobutyl-1-methylxanthine (50 mumol/l) antagonised the depression produced by L-PIA. Rolipram, a phosphodiesterase inhibitor, in concentrations up to 100 mumol/l had no effect on the evoked potentials or on adenosine action. Forskolin, a cAMP stimulant, slightly increased the amplitude of the evoked potential, and partly reversed the depressant effect of adenosine. Noradrenaline had no effect either alone or in the presence of adenosine. The results of these experiments indicate the existence of A1 subtype adenosine receptors in the guinea pig olfactory cortex probably linked to a depression of intracellular cAMP.     

Characterization and quantitative autoradiographic distribution of [3H]acetylcholine muscarinic receptors in mammalian brain. Apparent labelling of an M2-like receptor sub-type

[3H]Acetylcholine receptor binding characteristics (under muscarinic conditions) have been investigated using membrane binding assays and in vitro receptor autoradiography. In rat, guinea-pig and monkey brain membrane preparations, [3H]acetylcholine binds with high affinity (25-50 nM) to an apparently single class of sites which is differentially distributed across brain regions. The ligand selectivity pattern reveals that the potency of (-)quinuclidinyl benzylate is greater than (greater than) atropine greater than scopolamine greater than oxotremorine greater than carbamylcholine greater than pirenzepine greater than methylcarbamyl-choline = nicotine in competing for [3H]acetylcholine binding sites, indicating that [3H]acetylcholine selectively binds to muscarinic sites under these incubation conditions. Moreover, the low potency of pirenzepine suggests that [3H]acetylcholine does not label a significant proportion of the M1 receptor sub-type but most likely binds to putative M2-like receptor sites. This hypothesis is also supported by the autoradiographic distribution of [3H]acetylcholine binding sites in all species studied here. High densities of [3H]acetylcholine binding sites are seen in various nuclei of the medulla and pons, certain thalamic nuclei, medial septum, laminae III, V and VI of the cortex and just above the pyramidal cell layer of the hippocampus. Such localization is much different from that seen with the non-selective antagonist [3H]quinuclidinyl benzylate and the selective M1 receptor ligand [3H]pirenzepine, although it resembles that of the selective M2 receptor antagonist [3H]AF-DX 116. Thus, [3H]acetylcholine apparently mostly binds with high affinity mainly to non-M1 muscarinic receptor types in mammalian brain tissues. Moreover, the ligand selectivity pattern and in vitro receptor autoradiographic data suggest that at low concentrations (10-20 nM) most of [3H]actylcholine labelled sites are of the M2-like receptor class.     

Effect of adenosine analogues and cAMP-raising agents on TNF-, GM-CSF-, and chemotactic peptide-induced degranulation in single adherent neutrophils.

Adenosine and adenosine analogues are potent inhibitors of the respiratory burst in neutrophils. Most investigators, however, have found little or no effect of these compounds on neutrophil degranulation from cytochalasin B-treated neutrophils in suspension. We have instead investigated the effect of adenosine and 2-chloroadenosine on degranulation in adherent neutrophils in the absence of cytochalasin B. Both adenosine and 2-chloroadenosine were effective inhibitors of lactoferrin secretion induced by the chemotactic peptide N-formyl-methionine-leucyl-phenylalanine (fMLP) [50% inhibitory concentration (IC50) of less than 10(-6) M]. Secretion induced by tumor necrosis factor (TNF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) was inhibited only at high concentrations (IC50 of approximately 10(-4) M). In the presence of cytochalasin B no inhibitory effect of 2-chloroadenosine was seen. The effect of cAMP-raising agents on secretion from adherent neutrophils was also investigated. Dibutyryl cAMP at 0.2 mM reduced secretion in response to fMLP by 50% but did not inhibit TNF- and GM-CSF-induced degranulation. At a concentration of 2.0 mM dibutyryl cAMP also inhibited exocytosis in response to the two cytokines. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) at 300 microM reduced fMLP-induced degranulation, whereas a concentration of 1 mM was required to inhibit TNF- and GM-CSF-mediated secretion. The adenylate cyclase activator forskolin (50 microM) alone did not inhibit secretion in response to TNF or fMLP. However, in combination with IBMX (300 microM), forskolin (50 microM) reduced both TNF- and fMLP-induced secretion to less than 10%. PMA-induced exocytosis was unaffected by all these agents. In conclusion, adenosine appears to be an effective inhibitor of neutrophil granule protein secretion induced by fMLP but only a weak inhibitor of exocytosis in response to TNF or GM-CSF. Secretion in response to fMLP was also found to be more susceptible to a rise in cAMP than degranulation induced by TNF and GM-CSF.     

Alpha-adrenoceptor stimulation, but not muscarinic stimulation, increases cyclic AMP accumulation in brain slices due to protein kinase C mediated enhancement of adenosine receptor effects

In the present study, using rat hippocampal slices, we have further examined the stimulatory effect of alpha 1-adrenoceptors on the accumulation of cyclic AMP, which is known to depend on calcium and adenosine. The addition of noradrenaline (NA) stimulated the accumulation of [3H]inositol phosphates in [3H]inositol-treated slices. This effect was shared by carbachol (10-100 mumol l-1) but not by the adenosine receptor agonist 2-chloroadenosine (100 mumol l-1). The stimulatory effect of the alpha-agonists (phenylephrine or NA + propranolol) on cyclic AMP was shared by a diacylglycerol derivative, sn-1-oleyl-2-acetyl glycerol (OAG), and by the tumour-promoting phorbol esters phorboldibutyrate (PDiBu) and tetradecanoyl phorbol acetate (TPA). PDiBu caused a translocation of protein kinase C from soluble to particulate fractions. The effects of PDiBu and alpha-adrenoceptor stimulation on cyclic AMP were not additive. Surprisingly, carbachol (1-1000 mumol l-1) did not stimulate cyclic AMP accumulation in rat hippocampal slices either in the presence or in the absence of an adenosine receptor agonist. The results are compatible with the opinion that alpha-adrenoceptor stimulating drugs enhance the formation of inositol phosphates and diacylglycerol, which synergistically activate protein kinase C, which in turn augments the stimulation of cyclic AMP formation. Thus, a neurotransmitter whose principal biological effect is to stimulate inositol phosphate formation can influence cyclic AMP formation by virtue of an interaction with the actions of the ubiquitous neuromodulator adenosine. The fact that the effect of the alpha-receptor stimulation was not mimicked by a muscarinic agonist could indicate that other factors besides activation of inositol phospholipid hydrolys are important for this receptor-receptor interaction.     

Cholinergic agonists and dibutyryl cyclic guanosine monophosphate inhibit the norepinephrine-induced accumulation of cyclic adenosine monophosphate in the rat cerebral cortex

Addition of cholinergic agonists, namely carbamylcholine (carbachol), acetylcholine, eserine, eserine plus acetylcholine and eserine plus choline chloride, and dibutyryl cyclic guanosine moue-phosphate inhibited the norepinephrine-induced accumulation of cyclic adenosine monophosphate in incubated slices of rat cerebral cortex. Methacholine was ineffective and atropine did not overcome the action of carbachol on the stimulation of cyclic adenosine monophosphate synthesis by norepinephrine. Carbachol stimulated the production of cyclic guanosine monophosphate in the tissue slices white norepinephrine did not influence cyclic guanosine monophosphate levels to a significant degree.The data are in keeping with the ‘Yin-Yang’ hypothesis in which under particular situations the intracellular levels of cyclic guanosine monophosphate may modulate cyclic adenosine monophosphate concentrations.     

In vivo release of [3H]gamma-aminobutyric acid in the rat neostriatum--I. Characterization and topographical heterogeneity of the effects of dopaminergic and cholinergic agents

The release of [3H]gamma-aminobutyric acid continuously synthesized from [3H]glutamine was studied in the striatum of halothane-anaesthetized rats superfused with a push-pull cannula. The levels of spontaneously released [3H]GABA were identical in all striatal regions examined, but were found to be higher at the junction between the striatum and the globus pallidus. Superfusion with a medium enriched in K+ ions induced a concentration-dependent increase in [3H]GABA release. Superfusion with a Ca2+-free medium did not affect the spontaneous outflow of [3H]GABA but sharply reduced the release of [3H]GABA evoked by 30 mM K+. Locally applied tetrodotoxin (50 microM) decreased slightly the spontaneous release of [3H]GABA (-22%). When acetylcholine (50 or 500 microM) was added to a superfusion medium containing eserine (50 microM), the spontaneous release of [3H]GABA was enhanced in the ventral but not in the dorsal region of the striatum. The local application of 2,3,4,5-tetrahydro, 7,8,-dihydroxy, 1-phenyl, 1-H, 3-benzazepine (10 microM), a dopaminergic agonist acting preferentially on D1 receptors increased the release of [3H]GABA in the dorsal striatum (+32%) but decreased it slightly (-19%) in the ventral striatum. 3-(2-(N-3 hydroxyphenylethyl)N-propylamino)ethyl-phenol (50 microM), a preferential D2 receptor agonist, decreased [3H]GABA release when it was applied dorsally (-23%) but not ventrally in the striatum. It is concluded that the regulation of the release of [3H]GABA by acetylcholine and dopaminergic drugs is different in the dorsal and ventral regions of the striatum. These differences may be related to the existence of subpopulations of GABA neurons and may well have functional implications as suggested by behavioural studies.     

Somatostatin inhibition of phospholipase C activity in isolated rat pancreatic islets.

We have assessed the effect of somatostatin on the phospholipase C activity in isolated rat pancreatic islets. The phospholipase C activity was measured as the generation of inositol 1,4,5-trisphosphate and its metabolite inositol 1,3,4-trisphosphate from the hydrolysis of polyphosphoinositides. Inositol phosphates were measured using anion-exchange fast protein liquid chromatography analysis of extracts from islets prelabelled with myo-[3H]inositol. Somatostatin (1-1000 nmol l-1) significantly inhibited the glucose-induced (12 mmol l-1) phospholipase C activity in a concentration-dependent manner. The Ca2+ channel blocker verapamil (25 mumol l-1) also inhibited the glucose-induced (12 mmol l-1) phospholipase C, whereas the combination of somatostatin and verapamil did not induce any additional inhibition. At 3.3 mmol l-1 glucose, the hypoglycaemic sulphonylurea, tolbutamide (1 mmol l-1), increased the phospholipase C activity. This effect was reversed by somatostatin (100 nmol l-1). Tolbutamide did not further increase the glucose-induced (12 mmol l-1) phospholipase C activity. However, the somatostatin inhibition of glucose-induced (12 mmol l-1) phospholipase C was reversed by tolbutamide. The activator of adenylyl cyclase, forskolin (20 mumol l-1), did not exert any effect on the PLC-inhibition of somatostatin, whereas forskolin alone inhibited the phospholipase C activation at 12 mmol l-1 glucose. Our study demonstrates that somatostatin inhibits the hydrolysis of polyphosphoinositides in pancreatic islets, apparently via a mechanism dependent on Ca2+ and not on cAMP.     

The interaction between cAMP-dependent and cAMP-independent mechanisms in mediating the somatostatin inhibition of insulin secretion in isolated rat pancreatic islets.

To characterize the intracellular mechanisms by which somatostatin modulates the insulin secretion, studies were performed with isolated rat pancreatic islets at 12 mmol l-1 glucose. Somatostatin (0.1-1000 nmol l-1) inhibited the glucose-induced insulin secretion concentration-dependently. Increasing intracellular cAMP concentration either with dibutyryl-cAMP (1 mmol l-1) or by the adenylate cyclase activator forskolin (20 mumol l-1) partly reversed the inhibition by somatostatin (100 nmol l-1). Neither somatostatin (100 nmol l-1) nor dibutyryl-cAMP (1 mmol l-1 were able to affect the low insulin secretion observed in the absence of extracellular Ca2+. To study cAMP-independent mechanisms of somatostatin, the experiments were performed with and without dibutyryl-cAMP (1 mmol l-1) present. Both somatostatin (100 nmol l-1) and the Ca(2+)-channel blocker verapamil (25 mumol l-1) inhibited the insulin secretion both with and without dibutyryl-cAMP present. An additional inhibition of the insulin secretion was observed when somatostatin was combined with verapamil in the absence, but not in the presence of dibutyryl-cAMP. We conclude that somatostatin inhibits the glucose-induced insulin secretion both by cAMP-dependent mechanism which requires extracellular Ca2+, and by cAMP-independent/verapamil-sensitive Ca(2+)-channel-dependent mechanism.     

Trans-2-carboxy-3-pyrrolidineacetic acid (CPAA), a novel agonist at NMDA-type receptors

Although 2-carboxy-3-pyrrolidineacetic acid (CPAA) analogs are associated with activity as agonists at kainate-type receptors, here we report that CPAA is an agonist at N-methyl-D-aspartic acid-type receptors. CPAA evoked the release of [3H]acetylcholine (ACh) from striatal slices with the same efficacy as NMDA, and an EC50 of 20.0 microM, compared to an EC50 of 45.8 microM for NMDA. CPAA-evoked [3H]ACh release was inhibited by CPP (IC50 = 5.1 microM), tiletamine (IC50 = 0.53 microM), MK-801 (IC50 = 0.12 microM), and MgCl2 (IC50 = 26 microM). CPAA produced a tachyphylaxis when applied continuously for 18 min or more, and a cross-tachyphylaxis to NMDA. Similarly, NMDA generated a cross-tachyphylaxis to CPAA. All of these data suggest that CPAA is an agonist at NMDA-type receptors.     

The binding of cholinesterase inhibitors tacrine (tetrahydroaminoacridine) and 7-methoxytacrine to muscarinic acetylcholine receptors in rat brain in the presence of eserine

Cholinesterase inhibitor tacrine (1,2,3,4-tetrahydro-9-aminoacridine) is known to interfere with the binding of specific ligands to muscarinic receptors with unusually steep binding inhibition curves. We investigated whether the concentration dependence of the inhibition of binding is associated with the inhibitory effect of tacrine on the activity of cholinesterases, and compared the effect of tacrine with that of 7-methoxytacrine. Tacrine was found to inhibit the specific binding of [3H]quinuclidinyl benzilate (QNB) in rat brain cortex with IC50 values of 11 microM both in the absence and in the presence of 100 microM eserine, which had been added to ensure complete inhibition of cholinesterases at all concentrations of tacrine; in the cerebellum, the IC50 value was 10 microM in the absence and 14 microM in the presence of eserine; Hill slope factors (nH) were in the range of 1.55-1.79 and were not significantly affected by the presence of eserine. 7-Methoxytacrine inhibited the binding of [3H]QNB with an IC50 value of 2.3 microM in the cortex and of 2.6 microM in the cerebellum. The results indicate that the degree and the steep course of the inhibition of [3H]QNB binding to M1 and M2 muscarinic receptors by tacrine do not depend on its inhibitory effect on cholinesterases, and that 7-methoxytacrine is likely to interfere with the function of muscarinic receptors 4-5 times more strongly than tacrine.     

Stimulus-secretion coupling: role of cyclic AMP, cyclic GMP and calcium in mediating enzyme (kallikrein) secretion in the submandibular gland.

1. The role of adenosine 3':5'-phosphate (cyclic AMP) and guanosine 3':5'-phosphate (cyclic GMP) as second messengers for the enzyme secretory response evoked by the autonomic neurotransmitters, noradrenaline and acetylcholine, is examined in this in vitro study on the guinea-pig submandibular gland. 2. Noradrenaline increased enzyme (kallikrein) secretion. The initial stimulation of enzyme release appeared to be dose-dependent. The time course of cumulative kallikrein secretion revealed a complex pattern. Isoprenaline and phenylephrine were almost as potent as noradrenaline in releasing kallikrein. Both propranolol and phentolamine were required to fully inhibit the noradrenaline-stimulated enzyme secretion. 3. The cumulative secretion of kallikrein evoked by acetylcholine was dose-dependent. The onset of secretion showed a significantly greater time-lag than that observed with noradrenaline. Atropine effectively blocked the release of kallikrein by acetylcholine. 4. Dibutyryl cyclic AMP stimulated enzyme secretion. Dibutyryl cyclic GMP caused an initial increase which was not maintained. 5. The cyclic nucleotide phosphodiesterase inhibitors, theophylline and papaverine, increased basal kallikrein secretion. The action of the cyclic phosphodiesterase inhibitors on the secretory response to noradrenaline, acetylcholine, dibutyryl cyclic AMP and dibutyryl cyclic GMP was complex. In general, the increase in enzyme release produced by the secretagogues was additively enhanced by both inhibitors. 6. Omission of calcium inhibited both acetylcholine and dibutyryl cyclic GMP stimulated kallikrein release, but to a lesser degree than that of noradrenaline and dibutyryl cyclic AMP. High concentrations of extracellular calcium (10 mM) appeared to enhance the action of acetylcholine. 7. Noradrenaline produced a rise in the intracellular level of cyclic AMP. The increase preceded the stimulated secretion of kallikrein. Of the various adrenergic agonists, noradrenaline and isoprenaline were the most potent, whereas phenylephrine was significantly less effective in raising basal cyclic AMP values. Acetylcholine was without effect, even in the presence of a cyclic phosphodiesterase inhibitor. 8. Acetylcholine and noradrenaline raised intracellular levels of cyclic GMP only when the tissue incubations were performed in the presence of a cyclic phosphodiesterase inhibitor. The increase in cyclic GMP produced by acetylcholine preceded enzyme secretion. 9. Morphological data substantiated the finding that the in vitro release of kallikrein evoked by the secretagogues was associated with the depletion of secretory granules and vacuolations in acinar cells of the gland slices. 10. The molecular mechanisms which control enzyme secretion in the exocrine submandibular gland are discussed. Models are presented for the role of transmitter-specific cyclic nucleotides and calcium in stimulus-secretion coupling.     

Autoradiographic localization of particulate cyclic AMP-dependent protein kinase in mammalian brain using [3H]cyclic AMP: implications for organization of second messenger systems

Cyclic AMP's regulatory role as an intracellular second messenger is well established. In brain and other tissues, specific proteins that bind cyclic AMP have been shown to be the regulatory subunits of cystolic and particulate cyclic AMP-dependent protein kinases. This study of the autoradiographic localization of specific [3H]cyclic AMP binding revealed the heterogeneous distribution of particulate cyclic AMP-dependent protein kinase in the mammalian central nervous system. Specific [3H]cyclic AMP binding to tissue sections was of high affinity (KD = 60 nM) and saturable (Bmax = 5 pmol/mg protein). Purine and pyrimidine nucleotide analogues demonstrated inhibition constants against [3H]cyclic AMP binding consistent with the specific labelling of cyclic AMP-dependent protein kinase (e.g. 8'-bromo-cyclic AMP: IC50 = 130 nM; inosine 3',5'-cyclic monophosphate: IC50 = 1 microM; uridine 3',5'-cyclic monophosphate: IC50 = 60 microM). Variations in the levels of [3H]cyclic AMP binding presumably reflect the presence of differing amounts of particulate cyclic AMP-dependent protein kinase in different neuronal populations. Highest densities were associated with neuronal cell layers such as the pyramidal cells of the piriform cortex and hippocampus, and granule cells of the dentate gyrus and cerebellum. High levels of binding were also found in other cortical and limbic structures, while moderate levels were found in hypothalamic, thalamic and midbrain areas. Excitotoxic lesions confirmed the localization of the enzyme in hippocampal pyramidal cells and cerebellar granule cells. Localizations reported in this study are largely consistent with results obtained using immunohistochemical methods to label cyclic AMP-dependent protein kinases. Recently, [3H]forskolin, a potent and selective activator of adenylate cyclase, the enzyme responsible for the formation of cyclic AMP from adenosine 5'-triphosphate, has been used to localize the activated catalytic component of this enzyme in rat brain. Regions described as being intensely labelled with [3H]forskolin (e.g. basal ganglia, hilus of the dentate gyrus and molecular layer of the cerebellum) were found to be associated with relatively low [3H]cyclic AMP binding levels. These findings suggest a marked difference between the localization of the two related enzyme entities. However, the distribution of the enzymes is indirectly correlated as high levels of particulate cyclic AMP-dependent protein kinase are present in the soma of neurons with high concentrations of adenylate cyclase in their terminals. Alternatively, it is possible that [3H]forskolin localizes only a subpopulation of adenylate cyclase.(ABSTRACT TRUNCATED AT 400 WORDS)     

Muscarinic receptor activation attenuates D2 dopamine receptor mediated inhibition of acetylcholine release in rat striatum: indications for a common signal transduction pathway

In the present investigations, we used a superfusion system to study the effect of simultaneous activation of D2 dopamine receptors and so-called muscarinic "autoreceptors" on the K(+)-evoked in vitro release of [3H]acetylcholine from rat striatal tissue slices. Activation of D2 receptors with the selective agonist LY 171555 (0.01-1 microM) clearly decreased the evoked release of [3H]acetylcholine. This effect was markedly attenuated in the presence of either the selective muscarinic receptor agonist oxotremorine (3 microM) or the cholinesterase inhibitor physostigmine (1 microM). Conversely, D2 receptor activation with LY 171555 (1 microM) completely abolished the muscarinic receptor mediated inhibition of evoked [3H]acetylcholine release induced by oxotremorine (0.03-10 microM). These results show that the inhibitory effects of D2 dopamine receptor and muscarinic receptor activation on striatal acetylcholine release are non-additive and therefore are interdependent processes. In addition, we investigated some aspects of the signal transduction mechanism by which the muscarinic receptor mediates inhibition of K(+)-evoked in vitro release of [3H]acetylcholine from rat striatal tissue slices. It appeared that the effect of muscarinic receptor activation was not significantly influenced either by a lowering of the extracellular Ca2+ concentration from the usual 1.2-0.12 mM or by an increase of the intracellular cyclic adenosine-3',5'-monophosphate content. However, increasing extracellular K+ strongly decreased the inhibition of evoked [3H]acetylcholine release mediated by activation of muscarinic receptors. This set of results indicates that the muscarinic "autoreceptor" mediates the decrease of depolarization induced [3H]acetylcholine release from rat striatum to a large extent through stimulation of K+ efflux (opening of K+ channels) in a cyclic adenosine-3',5'-monophosphate independent manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Presynaptic inhibition of acetylcholine release

High potassium (51 mM) has been shown to evoke release of acetylcholine ([3H]ACh and endogenous ACh) from cholinergic nerves in rat bronchial smooth muscle. The release of [3H]ACh was reduced by 85% when the Ca2+ concentration was changed from 2 to 0.1 mM. The veratridine-induced release was completely inhibited by tetrodotoxin, but tetrodotoxin did not reduce the potassium-evoked release. The muscarinic agonist, oxotremorine, reduced the potassium stimulated release of [3H]ACh, without affecting the basal release. In contrast, scopolamine substantially potentiated the potassium-evoked release. Adenosine had a dual effect in the rat bronchi. Adenosine inhibited the potassium-evoked release of [3H]ACh and this presynaptic effect of adenosine was antagonized by 8-phenyltheophylline. Adenosine also induced contraction of the bronchial smooth muscle and there was potentiation by adenosine of the ACh-induced contraction. The results indicate that cholinergic nerve terminals in the rat bronchi possess muscarinic receptors which inhibit the release of ACh. Adenosine may have analogous effects, e.g. presynaptic inhibition of transmitter release in addition to postsynaptic enhancement of bronchial smooth muscle contraction.     

Characterization of the actions of botulinum neurotoxin type E at the rat neuromuscular junction

Botulinum neurotoxin (BoTx) serotype E blocks spontaneous and evoked quantal release of acetylcholine at the rat neuromuscular junction. Increasing extracellular Ca2+ to 8 mmol l-1 or substituting Ca2+ with La3+ (0.1 and 1.0 mmol l-1) or depolarizing the nerve terminals by 20 mmol l-1 K+ markedly increases miniature end-plate potential frequency in normal muscle, but in BoTx-E poisoned preparations none of these ions, with the exception of 1 mmol l-1 La3+, was able to restore spontaneous quantal transmitter release to levels recorded at unpoisoned junctions. In absolute values the enhancement with La3+ was much less than that reported at normal junctions. Nerve stimulation in the presence of 3,4-diaminopyridine (10-20 mumol l-1) and high calcium (8 mmol l-1) evoked multiquantal end-plate potentials and muscle twitches. We conclude that the neuromuscular block produced by BoTx serotype E is similar to that previously described for BoTx serotype A but differs from that produced by BoTx serotypes B, D and F in not causing desynchronization of nerve impulse-evoked transmitter release. 3,4-Diaminopyridine might be useful in the treatment of poisoning by BoTx serotype E since it markedly enhanced synchronous transmitter release from poisoned motor nerve terminals.