Calcium and cyclic AMP as possible mediators of neurohormone action in the hindgut of the cockroach, Leucophaea maderae

Adenosine 3′,5′-monophosphate (cyclic AMP) (10⁻⁵ g/ml) often caused a gradual increase in spotaneous contractile activity of the hindgut of the cockroach, Leucophaea maderae, and on rare occasions it would evoke a hormone-like response. However, aminophylline (2·5 × 10⁻⁴ g/ml) was capable of mimicking the neurohormone, and a concentration of 2·5 × 10⁻⁵ g/ml potentiated the contractile response evoked by the neurohormone: these responses were blocked by either the presence of 1 mM manganous ion or in a high potassium solution (162 mM). Propranolol (10⁻⁶ g/ml) and dopamine (10⁻⁴ g/ml) suppressed both spontaneous contractile events and neurohormone action. Dopamine (5 × 10⁻⁶ g/ml) also blocked action potential generation as did propranolol at 10⁻⁴ g/ml.These results lead us to suppose that cyclic AMP might serve as a mediator of neurohormone action by increasing calcium transport across the surface membrane of muscle fibres. Caffeine (2·5 × 10⁻⁴ g/ml), like aminophylline, caused a hormone-like response in normal hindguts. Even when the visceral muscles of the hindgut were depolarized in 162 mM potassium solution (without calcium), caffeine was still capable of inducing a phasic response. However, the addition of 2 mM calcium to such potassium-depolarized preparations caused a gradual increase in muscle tonus and substantially potentiated the response to caffeine.Such findings clearly implicate calcium as the mediator of excitation-contraction coupling in visceral muscle. While the interactions between the neurohormone, cyclic AMP, and calcium seem to be largely associated with the surface membrane and action potential generation.     

Acetylcholine-induced initiation of thymic lymphoblast DNA synthesis and proliferation

Low (5 × 10⁻⁹ M to 10⁻⁷ M) acetylcholine concentrations cause a calcium-independent stimulation of the initiation of DNA synthesis and proliferation of lymphoblasts which are part of rat thymocyte populations suspended in vitro. A much higher (5 × 10⁻⁵ M) acetylcholine concentration also stimulates lymphoblast DNA synthesis and proliferation, but this action is calcium-dependent. This proliferogenic response to acetylcholine is however not clearly mediated by either cyclic GMP or cyclic AMP.     

Soluble cyclic AMP-dependent protein kinases from chick kidney: Effects of ions and heat-stable modulator

• 1.1. Two cyclic nucleotide-dependent protein kinases have been isolated from chick kidney cytosol on DEAE cellulose.
• 2.2. Both have an apparent K_m ATP of 5 × 10⁻⁶ M, are stimulated half maximally by 1.8 × 10⁻⁸ M cyclic AMP, are inhibited half maximally by 5 × 10⁻³ M inorganic phosphate, and are inhibited slightly by Tris and chloride ions.
• 3.3. The heat-stable modulator of protein kinase inhibited both activities in the presence of cyclic AMP or cyclic GMP and histone, or cyclic AMP and casein, but with cyclic GMP and casein one kinase was stimulated and the other inhibited.

     

Atrial natriuretic factor receptors and stimulation of cyclic GMP formation in normal and malignant osteoblasts

Synthetic rat atrial natriuretic factor (Ile-ANF-26) stimulated cyclic GMP formation by up to several hundred-fold in osteoblast-rich cultures from newborn rat calvaria and in clonal osteogenic sarcoma cells (UMR 106-01) which are phenotypically osteoblast. ANF had no effect on the cyclic AMP response to parathyroid hormone in the same cells. Specific, high-affinity binding sites for ANF were identified in both cell types, with K_d and receptor numbers in normal osteoblasts of 1.2 ± 0.1 × 10⁻¹⁰ M and 42 ± 4 × 10³ per cell, and in UMR 106-01 cells of 1.4 ± 0.1 × 10⁻¹⁰M and 22 ± 4 × 10³per cell.     

Rat pancreatic adenylate cyclase. IV. Effect of hormones and other agents on cyclic AMP level and enzyme release.

1. The effects of secretin and pancreozymin-C-octapeptide and phosphodiesterase inhibitors on the concentration of adenosine 3',5'-cyclic monophosphate (cyclic AMP) and on the release of enzymes from rat pancreas have been studied. 2. In determininging cyclic AMP by means of the saturation assay of Brown et al. ((1971) Biochem. J. 121, 561-563) it is found essential to purify the pancreatic tissue extract by ion-exchange chromatography prior to the assay. 3. Injection of synthetic secretin or pancreozymin-C-octapeptide in anaesthetized rats in a secretory active dose (0.1 nmol) has no effect on the pancreatic cyclic AMP level. 4. Incubation for up to 10 min of pancreatic slices in Krebs-Ringer bicarbonate glucose medium containing 10(-2) M theophylline as phosphodiesterase inhibitor does not result in an increase of the cyclic AMP level. With 10(-2) M 1-methyl-3-isobutylxanthine as phosphodiesterase inhibitor the level is more than doubled after the first min of incubation and remains constant thereafter. 5. Addition of 3-10(-7) M secretin to slices incubated in the presence of 10(-2) M theophylline causes 84% increase of the cyclic AMP level above control, whereas the addition of 3-10(-7) M pancreozymin-C-octapeptide has no significant effect. In the presence of 10(-2) M 1-methyl-3-isobutylxanthine the latter hormone causes significant increases of up to 34% above control during 10 min of incubation. Secretin in this condition augments the cyclic AMP level by up to 296% above control during a 10 min incubation period. Addition of secretin and pancreozymin-C-octapeptide together has no greater effect than of secretin alone. 6. A broken cell fraction of rat pancreas contains adenylate cyclase activity which can be stimulated to 457 and 600% above the basal activity by 3-10(-7) M pancreozymin-C-octapeptide and secretin, respectively. Incubation of pancreatic slices with either hormone has no effect on the cyclic AMP phosphodiesterase activity in the homogenate of these slices. 7. Pancreozymin-C-octapeptide, dibutyryl cyclic AMP, 1-methyl-3-isobutylxanthine and carbamylcholine cause an elevated release of chymotrypsin from pancreatic slices incubated for 2 h in Krebs-Ringer bicarbonate medium, containing 10 mM glucose, while secretin, cyclic AMP and butyric acid have no significant effect. The release of the cytoplasmic enzyme lactate dehydrogenase is also elevated by dibutyryl cyclic AMP, 1-methyl-3-isobutylxanthine and carbamylcholine, but not significantly by pancreozymin-C-octapeptide. 8. The results support the role of cyclic AMP in the action of secretin, and do not exclude a mediating function of this nucleotide in the actions of pancreozymin in rat pancreas.     

Effects of VA-045, a novel apovincaminic acid derivative,on isolated blood vessels: Cerebroarterial selectivity

We investigated the effects of VA-045, an apovincaminic acid derivative, on isolated blood vessels. VA-045 (10⁻⁷−10⁻⁵M) and vinpocetine (10⁻⁷−10⁻⁵M) inhibited the 64 mM KCl-induced and 10⁻⁶M norepinephrine (NE)-induced contraction of rat aortic strips. VA-045 (10⁻⁷−10⁻⁴M) and vinpocetine (10⁻⁷−10⁻⁴M) inhibited the activity of cyclic AMP and cyclic GMP phosphodiesterase in porcine coronary artery. VA-045 (3×10⁻⁹−3×10⁻⁶M) relaxed the 64 mM KCl-induced contraction of the canine basilar artery without affecting the peripheral arteries. These results indicate that VA-045 selectively dilates canine cerebral artery, and that it may be a useful agent for the treatment of cerebrovascular diseases such as stroke.     

Interaction of thyroid hormones and cyclic AMP in the stimulation of hepatic gluconeogenesis

The possible interaction of l-3,3′,-5-triiodthyronine (T₃) and cycli AMP on hepatic gluconeogenesis was investigated in perfused livers isolated from hypothyroid rats starved for 24 h. T₃ (1·10^?6) and cyclic AMP (2·10^?4 M) increased hepatic gluconeogenesis from alanine within 30–60 min perfusion time (+85%/ + 90%), both were additive in their action (+191%). Concomitantly, α-amino[¹⁴C]isobutyric acid as well as net alanine uptake and urea production were elevated by T₃ and by cyclic AMP. T₃ increased the oligomycin-sensitive O₂ consumption and the tissue ‘overall’ ATP/ADP ratio, whereas cyclic AMP showed only a minor effect on cellular energy metabolism. As was observed recently for cyclic AMP, the stimulating action of T₃ on hepatic gluconeogenesis was independent of exogenous Ca²⁺ concentration. T₃ by itself affected neither the total nor the protein-bound hepatic cyclic AMP contents, pyruvate kinese (v:0.15 mM) activation nor the tissue levels of gluconeogenic intermediates. In contrast, cyclic AMP itself — although less effective than in euthyroid livers — decreased pyruvate kinase activity in hypothyroid livers with a concomitant increase in hepatic phosphoenolpyruvate concentration. This resulted in a ‘crossover’ between pyruvate and phosphoenolpyruvate. Cyclic AMP action was not affected by the further addition of T₃. Glucagon (1·10^?8 M) was less effective in hypo-than in euthyroid livers in increasing endogenous cyclic AMP content, deactivating pyruvate kinase and stimualting glucose production; this is normalized by the further addition of 1-methyl-3-isobutylxanthine (50 μM). It is concluded that T₃ stimulats hepatic gluconeogenesis by a cyclic-AMP-independent mechanism. In addition, the stimulatory action of cyclic AMP and glucagon with respect to hepatic gluconeogenesis is reduced in hypothyroidism. This may be explained by an increase in hepatic phosphodiesterase activity.     

Action of formamidine pesticides on the octopamine receptors on locust fat body

1. The action of two formamidines, chlordimeform (CDM) and desmethylchlordimeform (DCDM), on isolated fat body of locusts has been examined.2. Treatment with DCDM resulted in a dose-dependent stimulation of lipid release while CDM was ineffective at 10⁻⁴M.3. The action of DCDM was blocked by the α-adrenergic receptor blocker phentolamine, but not by the β-blocker propranolol.4. DCDM (5 × 10⁻⁶ M) in the presence of a phosphodiesterase inhibitor caused a 4-fold elevation of cyclic AMP in the fat body; this response was blocked by phentolamine but not by propranolol. CDM (10⁻⁵ M) did not alter cyclic AMP levels in the fat body.5. The effects of octopamine (10⁻⁵ M) and DCDM (10⁻⁵ M) on cyclic AMP level were not additive.6. It is concluded that DCDM (or its metabolites) act upon octopamine receptors on locust fat body while CDM has no effect. In view of the positive action of CDM on other octopamine receptors this indicates there may be differences between octopamine receptors of different tissues.     

The influence of low and high doses of theophylline on spontaneous motility and cyclic 3',5' AMP content in isolated rat uterus

It was found in isolated rat uterus that 5 × 10^?4 N theophylline inhibited spontaneous contractions which were restituted by increasing extracellular calcium 4-fold. Tissue level of cyclic 3′, 5′ AMP was not affected. On the other hand, 10^?2 M theophylline elevated cyclic 3′, 5′ AMP by 170 % for at least 60 minutes. The concomitant inhibition of spontaneous uterine motility could neither be restituted by increasing calcium up to 40-fold nor by washing. It was suggested that cyclic 3′, 5′ AMP was involved in theophylline-induced uterine relaxation when the drug was administrated in high amounts able to inhibit phosphodiesterase. Small doses of theophylline (5 × 10^?4 M) were supposed to initiate relaxing effects by a calcium-antagonistic intrinsic activity.     

Effects of isoproterenol,theophylline and carbachol on cyclic nucleotide levels and relaxation of bovine tracheal smooth muscle

The effect of theophylline and isoproterenol on bovine tracheal smooth muscle tension and cyclic AMP levels was investigated. Concentrations of isoproterenol (4 × 10^?6 M) and theophylline (10 mM) that relaxed carbachol-contracted tracheal muscle by 85–95% did not significantly elevate control levels of cyclic AMP. In the absence of carbachol, several-fold increases in cyclic AMP were caused by isoproterenol although no elevations by theophylline were measurable. However, when isoproterenol and theophylline were administered together, theophylline potentiated the rise in cyclic AMP caused by isoproterenol. Phosphodiesterase studies in tracheal muscle showed the presence of a high and a low K_m enzyme which were inhibited by theophylline. Cyclic GMP levels were elevated in muscles contracted by carbachol as well as in carbachol-contracted muscles that had been relaxed by theophylline. In non-tension studies, in which the tracheal muscle was not under isometric tension, carbachol or theophylline alone increased cyclic GMP and together they synergistically elevated cyclic GMP. Atropine blocked the elevation caused by carbachol but not that caused by theophylline. In contrast to theophylline, isoproterenol did not elevate cyclic GMP, and in carbachol-contracted muscles that had been relaxed by isoproterenol, cyclic GMP levels were no different from control. Also, in non-tension studies, isoproterenol decreased basal cyclic GMP and antagonized the increase in cyclic GMP due to carbachol.The results indicate that whole-tissue levels of cyclic AMP and cyclic GMP do not correlate with the state of tracheal smooth muscle tension. Cyclic GMP levels do not clearly correlate with either contraction or relaxation. The inhibition by carbachol of increases in cyclic AMP due to isoproterenol and the inhibition by isoproterenol of increases in cyclic GMP due to carbachol provide evidence for a reciprocal cholinergic-adrenergic antagonism of cyclic AMP and cyclic GMP levels. The antagonism did not appear to be due to either cyclic nucleotide affecting the elevation of the other since the levels of both cyclic nucleotides were depressed.     

Functional receptors for vasoactive intestinal peptide in cultured vascular smooth muscle cells from rat aorta

Specific binding sites for vasoactive intestinal peptide (VIP), a potent vasodilatory polypeptide, and its effect on formation of intracellular cyclic AMP levels were studied in cultured vascular smooth muscle cells (VSMC) from rat aorta. Specific binding of ¹²⁵I-labeled-VIP to cultured VSMCs was time- and temperature-dependent. Scatchard analysis of binding studies suggested the presence of two classes of high and low affinity binding sites for VIP; the apparent K_d and the number of maximal binding capacity were ∼8×10⁻⁹ M and 60,000 sites/cell (high-affinity sites) and ∼4×10⁻⁸ M and 140,000 sites/cells (low-affinity sites), respectively. Unlabeled VIP competitively inhibited the binding of ¹²⁵I-labeled-VIP to its binding sites, whereas neither peptides structurally related to VIP, nor other vasoactive substances affected the binding. VIP stimulated formation of intracellular cyclic AMP in cultured VSMCs in a dose-dependent manner; the stimulatory effect of VIP on cyclic AMP formation was not blocked by propranolol and was additive with isoproterenol. The present study first demonstrates the presence of specific receptors for VIP in VSMCs functionally coupled to adenylate cyclase system. It is suggested that VIP exerts its vasodilatory effect through its specific receptors distinct from β-adrenergic receptors.     

1-Isoamyl-3-isobutylxanthine: a remarkably potent agent for the potentiation of norepinephrine, histamine, and adenosine-elicited accumulations of cyclic AMP in brain slices.

1-Isoamyl-3-isobutylxanthine (EC₅₀ t 5 μM) potentiates by 2 to 6-fold the accumulations of cyclic AMP elicited in guinea pig cerebral cortical slices by norepinephrine, histamine, and adenosine. In addition, the xanthine derivative causes a 2 to 3-fold elevation of basal levels of cyclic AMP. 1-Isoamyl-3-isobutylxanthine has no effect on accumulations of cyclic AMP elicited by histamine or adenosine in the presence of a potent phosphodiesterase inhibitor, ZK 62771. The xanthine derivative retards the disappearance of cyclic AMP after a prior stimulation by adenosine. The results indicate that 1-isoamyl-3-isobutylxanthine is an extremely potent and effective inhibitor of phosphodiesterases involved in the regulation of cyclic AMP levels in guinea pig cerebral cortical slices. The 1-benzyl, 1-isoamyl, and 1-isobutyl derivatives of 3-isobutylxanthine potentiate the accumulation of cyclic AMP elicited by adenosine, while the 1-methyl derivative and 1-isoamyl-3-methylxanthine are inhibitory undoubtedly because of blockade of adenosine-receptors by these compounds. Xanthines with bulky 1- and 3- substituents appear to be relatively weak adenosine-antagonists and relatively specific and potent agents for inhibition of phosphodiesterases involved in cyclic AMP metabolism in brain tissue.     

9-Isoleucine]ACTH1-24, a competitive antagonist of ACTH1-24 induced cyclic AMP and corticosterone production.

Substitution of tryptophan⁹ in ACTH_1–24 by isoleucine results in complete loss of biological activity. A dose of 3.4 × 10^?5 M per ml fails to stimulate corticosterone and cyclic AMP production. This analogue inhibits cyclic AMP production and corticosterone production induced by ACTH_1–24 in isolated adrenal cortex cells. The I₅₀ values for corticosterone and cyclic AMP inhibition are 2.3 × 10^?6 M and 3.4 × 10^?6 M respectively.     

Effect of catecholamines and ovarian hormones on cyclic AMP accumulation in rat hypothalamus

Effect of different monoamines and estradiol were studied on cyclic AMP (cAMP) accumulation in hypothalami from 21 day old female rats. Incubation for 5 min with 10^?4M epinephrine, norepinephrine or dopamine resulted in an increase in cAMP accumulation in the hypothalamus. Incubation of hypothalamic tissue with estradiol (4 × 10^?7M to 2 × 10^?5M) also resulted in an increase in cyclic AMP levels. The increase caused by estradiol was observed only after 50 min of incubation period. The estradiol induced increase in cyclic AMP accumulation was abolished by both α and β blockers. These results suggest that the estradiol-induced increase in cyclic AMP may be mediated by a prior increase in catecholamines in the hypothalamic tissue.     

Stimulation by octopamine of the sodium efflux in barnacle muscle fibres

1. The behaviour of the ouabain-insensitive Na efflux in barnacle muscle fibres towards dl-octopamine has been investigated.2. It is found that these fibres are quite often sensitive to external application of octopamine. A concentration as low as 10⁻⁹ M is effective.3. The kinetic results indicate that the stimulatory response develops within 5–10 min of exposure of the fibre to octopamine and is transitory in nature.4. The response to octopamine is greater in size in the presence of l-isoamyl-3-isobutylxanthine (IAX) than in the presence of l-propyl-3-methyl-7-(5-hydroxyhexyl)-xanthine (PMX). But neither IAX nor PMX stops the response from decaying.5. The response to octopamine is highly dependent on the presence of external Ca²⁺.6. The provisional conclusion is that the barnacle muscle fibre may be a useful preparation for studying the hormonal function of octopamine.     

Restorative effects of cyclic AMP on complex bioelectric activities of cultured fetal rodent CNS tissues after acute CA++-deprivation

Explants of fetal mouse spinal cord and cerebral cortex generate organotypic slow-wave and repetitive-spike discharges in vitro which can be abolished by agents which reduce the concentration of Ca⁺⁺ available to the tissue. Synaptically mediated discharges are rapidly blocked in Ca⁺⁺-free balanced salt solution (BSS), or in regular BSS after addition of 10^?3 M EGTA, 5–10 × 10^?3 M Mg⁺⁺, or 10^?4 M xylocaine, but simple spike potentials can still propagate. When low concentrations of cyclic AMP or dibutyryl cyclic AMP (2 × 10^?6 M) are added to the Ca ⁺⁺-free BSS or Ca⁺⁺-antagonist-BSS, a temporary (1–20 min) restoration of characteristic complex bioelectric activities occurs (or the onset of depression is delayed if cyclic AMP is initially added). Phosphodiesterase inhibitors, e.g. 10^?3 M caffeine, are also effective in restoring these blockades, whereas 5′AMP and ATP are not. Application of 10^?6 M cyclic AMP or 10^?3 M caffeine in regular BSS greatly enhances excitability of some CNS explants, resembling convulsive effects observed in CNS in situ. The data suggest that cyclic AMP can mobilize Ca⁺⁺ from membranebound Ca pools within neurons in CNS explants so as to permit Ca⁺⁺-dependent release of neurotransmitter during Ca⁺⁺ deficits. Thus, it may also be that under normal conditions, cyclic AMP can regulate the availability of Ca⁺⁺ for synaptic transmission in the central nervous system, thereby modulating the efficacy of synaptic functions.     

Vasoactive intestinal polypeptide: increased tone, enhancement of acetylcholine release, and stimulation of adenylate cyclase in intestinal smooth muscle

Vasoactive intestinal polypeptide (VIP) was examined $\text{in vitro}$ for effects on tone and neuronal release mechanisms in intestinal smooth muscle since this is a site of high peptide concentration. VIP contracted the guinea pig ileum and rabbit jejunum in concentrations ranging from 10^?9 to 10^?7 M. Increased tone in the guinea pig ileum was partially antagonized by the anticholinergic agent, atropine (4.38 × 10^?6 M) suggesting that one component of the contractile response was due to the indirect release of acetylcholine. The H₁ receptor antagonist, pyrilamine, did not alter the increased tone produced by VIP indicating that histamine release did not contribute to the ileal contractile response and that VIP exerted a selective effect to enhance neuronal release of acetylcholine. The ability of VIP to modulate acetylcholine release was confirmed in field stimulated ileal preparations where VIP increased the force developed to endogenously released acetylcholine without altering the direct response to acetylcholine. In rabbit jejunum and ileal smooth muscle, VIP related cyclic AMP levels. However, inhibition of phosphodiesterase with papaverine did not potentiate either the VIP-induced ileal contraction or enhancement of the field stimulated response. This raises the possibility that increases in intestinal cyclic AMP may be involved more in VIP-induced alterations in ion transport or secretory phenomenon than in intestinal motility. These studies describing the ability of VIP to modulate acetylcholine release and to increase ileal tone are consistent with the proposed role of VIP in intestinal patholgies involving excessive mucous secretion and motility.     

Inhibiton and gamma-aminobutyric acid-induced conductance on locust (Schistocerca gregaria) extensor tibiae muscle fibres

• 1.1. Increases in membrane conductance (g_m) were induced by GABA in distal bundles 32, 33 and 34 of extensor tibiae muscles of the locust (Schistocerca gregaria).
• 2.2. Bath application of GABA (10⁻⁵−5 × 10⁻³ M) induced reductions in muscle fibre space constant (λ).
• 3.3. GABA (5 × 10⁻³ M) induced additional membrane conductance of 2.21 ± 0.03 × 10⁻⁶ S/mm, 0.38 ± 0.03 × 10⁻⁶ S/mm and 0.29 ± 0.06 × 10⁻⁶ S/mm on muscle bundles 34, 33 and 32 respectively. The greater sensitivity of muscle fibres in bundle 34 to GABA is due at least in part to a larger number of GABA receptors on bundle 34 muscle fibres.
• 4.4. The decrement of electrotonic potentials in the presence of GABA were measured over distances of both half fibre length and whole fibre length. Good agreement was obtained between changes in space constant produced by GABA using half fibre length and whole fibre length data.
• 5.5. By taking into account changes in space constant induced by GABA it was possible to demonstrate that presynaptic GABA receptors were involved in the inhibition of slow excitatory postsynaptic potentials by GABA.
• 6.6. “Slow” excitatory postsynaptic potentials recorded under current clamp were inhibited in a dose-dependent manner by GABA. This inhibition was not dependent on muscle-fibre GABA sensitivity and could not be completely accounted for by GABA-induced changes in the cable properties of the muscle fibres.

     

The relaxing effect of SCH 23390 in the molluscan smooth muscle

1. SCH 23390 (SCH, R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine) produced the relaxation of ACh-induced contraction in the anterior byssus retractor muscle of Mytilus edulis (ABRM) in a dose-dependent manner between 10⁻⁹-10⁻⁶M.2. The dose-response curve of SCH was shifted in parallel to the right by ketanserin (KET) with pA₂ value of 5.14 ± 0.08 and by 1-(1-naphthyl)piperazine (NAP) with that of 5.06 ± 0.01, but not by cyproheptadine (CYP), mianserin (MIA), butaclamol (BUTA), ICS 205–930 (ICS) and MDL 72222 (MDL) at 3 × 10⁻⁵ M.3. α-Methyl-serotonin (α-Me-5-HT), a selective 5-HT₂ receptor agonist dose-dependently relaxed the ACh-induced contraction of ABRM. The dose-response curve of α-Me-5-HT was shifted in parallel to the right by KET with pA₂ value of 5.01 ± 0.02, but not by BUTA, CYP, MIA, ICS and MDL at 3 × 10⁻⁵ M.4. These findings suggest that 5-HT₂-like receptors exist in the ABRM, and that the relaxation induced by SCH is mediated through these receptors.