Adenylate Cyclase In Homogenates Research Articles

Capsaicin induced afferent denervation and receptor-linked responses to CGRP in the rat

We have examined the effect of neonatal capsaicin-induced destruction of primary afferent nerves in rats on the response to calcitonin gene-related peptide (CGRP) in vitro and in vivo. Denervation was confirmed by immunohistochemistry. Rat α-CGRP (rCGRP) activated adenylate cyclase in homogenates of rat spleen (basal activity 45 ± 8.27, δV max 75 ± 16 pmol cyclic AMP min −1 mg −1 protein; K act 2.04 ± 0.44 nM . A single specific binding site for [ 125I]hCGRP was demonstrated in homogenates of spleen ( K d = 4.84 ± 0.66 nM , B max = 1.43 ± 0.35 pmol mg −1 protein). Neither adenylate cyclase activation nor binding site characteristics were affected by capsaicin-induced denervation. In addition, hypotensive responses to intravenous boluses of rCGRP were examined in anaesthetized rats. Neither the basal blood pressure nor the blood pressure fall in response to rCGRP were altered by neonatal denervation by capsaicin. In conclusion, there is no evidence of denervation hypersensitivity of receptor mediated responses to CGRP in vivo or in vitro, following capsaicin-induced denervation in the rat. This suggests that CGRP is unlikely to exert a sustained tonic influence on cardiovascular regulation in the rat.

Effect of adenosine analogs on plasma corticosterone concentration in rats

Adenylate cyclase in homogenates of guinea pig ventricles was inhibited by the stable adenosine analogs N6-phenylisopropyl-adenosine (PIA) and 5′-N-ethylcarboxamidoadenosine (NECA). Inhibition required GTP and was enhanced by sodium ion. The maximum inhibition observed was 35.1±1.1%, the EC50 (95% confidence limits,n) for PIA and NECA were 0.20μM (0.17–0.25μM, 6) and 0.66 μM (0.26–1.7 μM, 4) respectively. 8-Phenyltheophylline (10 and 100 μM) and isobutylmethylxanthine (100 μM) antagonized the inhibitory effects of the adenosine analogs. These results indicate that adenosine receptors of the inhibitory type (RI or A1) are present in guinea pig myocardium and may mediate some of the cardiac responses to adenosine.

Synthesis and Octopaminergic Agonist Activity of 2-(Substituted benzylamino)-2-thiazolines.

2-(Substituted benzylamino)-2-thiazolines (SBAT) were synthesized by a hydrochloric acid-catalyzed cyclization of the corresponding thioureas, using a reaction of 2-methylthio-2-thiazoline with substituted benzylamines or by alkylating 2-amino-2-thiazoline. 2-(Alkylthio)-2-thiazolines were obtained by alkylating 2-mercaptothiazoline. Most of the SBAT compounds activated adenylate cyclase in homogenates of cockroach ventral nerve cords; the effect of introducing substituents at the phenyl of the SBAT compounds on octopaminergic agonist activity was not significant. 2-[β-(Substituted phenyl)ethylamino]-2-thiazolines and 2-(alkylthio)-2-thiazolines were not significant octopaminergic agonists. Washing removed nearly all of the activity of one of the SBAT compounds, suggesting that the SBAT compounds bound reversibly to the octopaminergic receptor.

Effects of chronic electroconvulsive shock on D 1 and D 2 dopamine receptor-mediated activity of adenylate cyclase in homogenates of striatum and limbic forebrain of rat

Stimulation of adenylate cyclase by dopamine in homogenates of the striatum was unaltered in rats which had received either a single or a series of 10 electroconvulsive shocks. However, stimulation by the selective D 1 dopamine receptor agonist SKF 38393 was significantly increased in homogenates of striatum from rats which had received chronic electroconvulsive shock, compared to those which received sham treatment. In homogenates of the limbic forebrain, stimulation by both 100 μM dopamine and by 4 μM SKF 38393 was significantly increased after chronic electroconvulsive shock. The activity of D 2 receptors, as measured by inhibition of forskolin-stimulated adenylate cyclase, in the presence of the d 1 receptor antagonist SCH 23390, was unaltered by chronic electroconvulsive shock in either area of the brain. The selective effect of chronic electroconvulsive shock in increasing the activity of D 1 receptors may account both for the increase, in dopamine-mediated behaviour, seen after chronic electroconvulsive shock and for the antiparkinsonian effects of this treatment.

Comparison of the acute actions of amine-depleting drugs and dopamine receptor antagonists on dopamine function in the brain in rats

The effects of the monoamine depleting drugs oxypertine, tetrabenazine and reserpine were compared with those of the dopamine receptor antagonists, chlorpromazine and trifluoperazine, on behavioural and biochemical indices of dopamine function in the brain. Oxypertine (0.625–20 mg/kg, i.p.), chlorpromazine (0.625–20 mg/kg i.p.) and trifluoperazine (0.0625–2.0 mg/kg i.p.), administered to rats 1 hr previously, inhibited stereotyped behaviour induced by both amphetamine (5.0 mg/kg i.p.) and apomorphine (1.0 mg/kg, s.c.) in a dose-dependent manner. Tetrabenazine (0.625–20 mg/kg i.p., l hr previously) inhibited stereotypy induced by amphetamine but not that induced by apomorphine. Reserpine (0.1–10 mg/kg i.p., 6 hr previously) did not inhibit, but in larger doses, tended to enhance the stereotyped responses to both amphetamine and apomorphine. Oxypertine (10 mg/kg, i.p., 1 hr previously), tetrabenazine (5 mg/kg i.p., 1 hr previously) and reserpine (2.5 mg/kg i.p., 6 hr previously) reduced the content of dopamine in the striatum but increased the concentrations of homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC). Chlorpromazine (5 mg/kg i.p.) and trifluoperazine (0.5 mg/kg i.p.), given 1 hr previously, did not alter concentrations of dopamine in the striatum but increased those of HVA and DOPAC. Oxypertine, chlorpromazine and trifluoperazine weakly inhibited dopamine-stimulated adenylate cyclase in homogenates of the striatum in the rat. Tetrabenazine and reserpine had no effect. Similarly, trifluoperazine and chlorpromazine displaced the specific binding of [ 3H]piflutixol to membranes from the striatum. Oxypertine also was weakly effective, but tetrabenazine and reserpine were without effect. Trifluoperazine, chlorpromazine and oxypertine displaced specific binding of [ 3H]spiperone and [ 3 H]N,n- propylnorapomorphine (NPA) to preparations of the striatum. Tetrabenazine was only weakly active and reserpine was without effect. In conclusion, oxypertine, like chlorpromazine and trifluoperazine, appears to cause blockade of post-synaptic dopamine receptors. Reserpine and tetrabenazine appear to act selectively by altering release of monoamines.

Timolol inhibits adenylate cyclase activity in the iris‐ciliary body and trabecular meshwork of the eye and blocks activation of the enzyme by salbutamol

The enzymatic activity of adenylate cyclase in homogenates and membrane-rich fractions prepared from rabbit iris-ciliary bodies and bovine trabecular meshwork was found to be inhibited by timolol. Treatment of iris-ciliary body homogenates with Triton X-305 resulted in abolition of the inhibitory effect of the drug on the activity of the enzyme. The stimulatory effect of salbutamol on the enzyme was also susceptible to blockade by timolol. It is suggested that the hypotensive action of timolol on intraocular pressure results from structural and functional changes induced on the plasma membranes of the iris-ciliary body and trabecular meshwork by the thiadiazole group of the molecule, and, also, from the occupation of the adrenergic receptors of the iris-ciliary body by the tert-butylamino-2-hydroxypropoxy part of the compound.

Effect of the antitumor drug caracemide on the neurochemistry of murine neuroblastoma cells (clone N1E-115)

Because the antitumor drug caracemide causes neuropsychiatric effects in patients, we investigated its effects on the neurochemistry of cultured neuroblastoma cells (murine clone N1E-115). The drug caused a transient elevation in the level of [ 3H]cyclic GMP that was not blocked by receptor antagonists or by desensitization of histamine or muscarinic receptors. The EC 50 for the response to caracemide was 635 μM. Preincubation of cells with caracemide led to the inhibition of muscarinic receptor-mediated [ 3H]cyclic GMP formation with an ic 50 of 450 μm. Caracemide inhibited basal guanylate cyclase activity in homogenates noncompetitively with a K i , value of 162 μM. The drug also inhibited sodium nitroprusside-stimulated guanylate cyclase in homogenates. Caracemide did not inhibit basal adenylate cyclase activity in either intact cells or homogenates, but inhibited adenylate cyclase activated by prostaglandin E 1 (PGE 1) or forskolin. The muscarinic receptor-mediated reduction of PGE 1-stimulated [ 3H]cyclic AMP formation was not affected. The K i , for the inhibition of PGE 1-activated adenylate cyclase in homogenates was 110 μM. Caracemide was a competitive inhibitor of acetylcholinesterase with a K 1, value of 8 μM. The drug did not inhibit, but slightly stimulated, monoamine oxidase activity in N1E-115 cells. The results indicate that caracemide can affect several neurochemical systems in neural cells in culture in a way that correlates with its neuropsychiatric effects. The N1E-115 clone thus appears to be useful for evaluating some of the molecular pharmacological effects of drugs interacting with the nervous system.

Prostaglandin endoperoxide analogues which are both thromboxane receptor antagonists and prostacyclin mimetics.

Two prostaglandin endoperoxide analogues, EP 035 and EP 157, behave as specific thromboxane receptor antagonists on isolated smooth muscle preparations such as rabbit aorta, dog saphenous vein and guinea-pig trachea. However, in human platelet-rich plasma (PRP) they produce an unsurmountable block of aggregation induced by a wide range of agents (ADP, platelet-activating factor, thrombin); this inhibitory profile is typical of that seen with either prostaglandin I2 (PGI2) or PGD2. EP 035 and EP 157 induce large increases in cyclic AMP levels (up to 20 times basal) in human PRP. Simultaneous exposure to PGE1 markedly reduces their effect on cyclic AMP; exposure to PGD2 is much less effective in this respect. The adenylate cyclase inhibitor SQ 22,536 opposes the inhibitory action of EP 035, EP 157, iloprost (a stable PGI2 analogue) and PGD2 on platelet aggregation. However, the xanthone derivative AH 6809 blocks the inhibitory action of PGD2 but does not affect EP 035, EP 157 and PGI2 and its structural analogues. EP 035 and EP 157 displace [3H]-iloprost binding to the PGI2 receptor on human platelet membranes. Displacing ability is ranked as follows: iloprost greater than 6a-carba PGI2 greater than EP 157 greater than EP 035 greater than EP 164 (alpha-dinor derivative of EP 157). This order of potency is the same as that found for activation of adenylate cyclase in homogenates of washed human platelets and for inhibition of aggregation in washed human platelets. The activities of EP 035 and EP 157 were studied in two other systems containing PGI2 receptor-adenylate cyclase complexes, the NCB-20 cell line and human lung tissue. In both cases stimulation of adenylate cyclase was found but maximum rates were below that achieved with iloprost. These effects of EP 035 and EP 157 could be correlated with their abilities to displace [3H]-iloprost binding. 5 These results indicate that EP 035 and EP 157 inhibit the aggregation of human platelets by acting as agonists at the PGI2 receptor linked to adenylate cyclase. They represent a class of compound with both thromboxane receptor blocking activity and prostacyclin mimetic activity.

A study of the H2-receptor for histamine stimulating adenylate cyclase in homogenates of guinea-pig lung parenchyma.

The effect of forskolin and several H2-agonists was investigated on the activity of adenylate cyclase in homogenates of guinea-pig lung parenchyma. Histamine, 0.1 microM to 1 mM, dimaprit, 1 microM to 10 mM, 4-methyl histamine, 0.1 microM to 10 mM, impromidine, 10 nM to 10 microM and forskolin, 1 nM to 100 microM, all produced a dose-dependent stimulation of adenylate cyclase activity above the basal level. The histamine H1-receptor antagonist mepyramine, 10 microM, and beta-adrenoceptor antagonist propranolol, 10 microM, had no effect on the stimulation by histamine of adenylate cyclase. The dose-response curve for stimulation by histamine of adenylate cyclase was shifted to the right in a dose-dependent manner by increasing concentrations of several H2-antagonists. Schild plots constructed for each H2-antagonist produced straight lines with slopes not significantly different from unity. The equilibrium dissociation constants obtained for the H2-antagonists in this study were similar to those previously reported for inhibition of dimaprit-induced relaxation of the pre-contracted lung strip, inhibition of [3H]-tiotidine binding to homogenates of guinea-pig lung parenchyma and inhibition of histamine-stimulated adenylate cyclase in guinea-pig gastric mucosa.

Multiple defects in the activity of adenylate cyclase from the Drosophila memory mutant rutabaga.

Adenylate cyclase in homogenates of Drosophila melanogaster is heterogeneous with respect to its affinity toward MgATP and its subcellular distribution. Km values for MgATP range, under similar assay conditions, from approximately 10(-5) M to approximately 10(-3) M, depending on the body region and on the subcellular localization of the enzyme. The majority of the enzyme in whole-body preparations is particulate, but various body regions differ in the relative proportion of the soluble enzyme. The memory mutant rutabaga lacks up to 35% of the total particulate activity. Even ligands that stimulate directly the catalytic subunit are incapable of bringing the activity of the mutant's enzyme to normal levels. The defect is differentially pronounced in various body parts and is associated with an altered responsiveness of the enzyme to Mg2+, to Ca2+, and to forskolin. It is suggested that rutabaga is lesioned in a subpopulation, or a functional state, of adenylate cyclase, which may play a role in memory formation.

Inhibition of adenylate cyclase in rat adrenal glomerulosa cells by angiotensin II.

Adenylate cyclase in homogenates of rat adrenal glomerulosa cells was inhibited in a concentration dependent manner by angiotensin II (AII). The maximum inhibition of basal activity was 34.5 +/- 2.7% and the EC50 value for AII was 1.1 +/- 0.4 nM (n = 6). Similar maximum inhibitions were produced by the precursor decapeptide, AI, and the heptapeptide, AIII [(des asp) AII]. The EC50 values for these two peptides were respectively 72 +/- 8 nM and 25 +/- 5 nM (n = 6). The antagonist compound (1-sarcosine, 8-isoleucine)-AII, reversed the effect of AII. Inhibition of the adrenal enzyme required guanosine triphosphate and monovalent cations as has been described for adenylate cyclase inhibition in other tissues. Maximum inhibition was observed at 10(-5) M guanosine triphosphate and 150 mM Na+ or Li+ ion. Both basal adenylate cyclase activity and activity stimulated by adrenocorticotrophic hormone were inhibited. These results demonstrate the presence in rat adrenal glomerulosa cells of angiotensin receptors coupled to adenylate cyclase inhibition and show that their properties are similar to those of adrenal angiotensin receptors described previously.

Demonstration of R A — Adenosine receptors in rat renal papillae

Adenylate cyclase in homogenates of rat renal papillae was stimulated by adenosine agonist compounds. Adenosine agonists neither stimulated nor inhibited cyclase activity in cortex or medulla. The rank order of agonist potencies was 5′N-ethylcarboxamide adenosine ⪢ 2-chloroadenosine > L-N 6-phenylisopropyladenosine = cyclohexyladenosine > D-N 6-phenylisopropyladenosine. Stimulation of adenylate cyclase by 5′N-ethylcarboxamide adenosine was competitively antagonised by the methylxanthines, 1,3-diethyl-8-phenylxanthine and 8-phenyltheophylline; 1,3-diethyl-8-phenylxanthine being approximately 10 times more potent than 8-phenyltheophylline. These results demonstrate adenosine receptors of the R A-type localized to the papilla of the rat kidney.

Biochemical evidence for the 5-HT agonist properties of PAT (8-hydroxy-2-(di-n-propylamino)tetralin) in the rat brain

In vitro investigations revealed that PAT (8-hydroxy-2-(n-dipropylamino)tetralin) interacted with postsynaptic 5-HT receptors in the rat brain: the drug stimulated 5-HT-sensitive adenylate cyclase in homogenates of colliculi from new-born rats (KAapp 8.6 μM) and inhibited the specific binding of [3H]5-HT to 5-HT1 sites. The PAT-induced inhibition of [3H]5-HT binding showed marked regional differences compatible with a preferential interaction of PAT (IC50 2 nM) with the 5-HT1A subclass. As previously seen with 5-HT agonists, the efficacy of PAT for displacing [3H]5-HT bound to hippocampal membranes was markedly increased by Mn2+ (1 nM) and reduced by GTP (0.1 nM). PAT also affected presynaptic 5-HT metabolism since it inhibited competitively (Ki 1.4 μM) [3H]5-HT uptake into cortical synaptosomes and reduced (in the presence of the 5-HT uptake inhibitor fluoxetine) the K+-evoked release of [3H]5-HT previously taken up or newly synthesized from [3H]tryptophan in cortical or striatal slices. This latter effect was prevented by 5-HT antagonists (methiothepin, metergoline) suggesting that it was mediated by the stimulation of presynaptic 5-HT autoreceptors by PAT. Like 5-HT, PAT counteracted the stimulatory effect of K+-induced depolarization on the synthesis of [3H]5-HT from [3H]tryptophan in cortical slices. It is concluded that PAT is a potent 5-HT agonist acting on both post- and presynaptic 5-HT receptors in the rat brain.

Fluoride-stimulated adenylate cyclase activity in rat brain following chronic treatment with psychotropic drugs

Chronic treatment with imipramine and reserpine increased fluoride-stimulated activity of adenylate cyclase in homogenates of cerebral cortex and “limbic” forebrain of the rat. Concomitant treatment with lithium counteracted this effect, while lithium alone had no effect on the activity of adenylate cyclase.

Abnormal activity of adenylate cyclase in the Drosophila memory mutant rutabaga

The activity of adenylate cyclase in homogenates prepared from the Drosophila memory mutant rutabaga is lower than normal. The effect is most pronounced in washed membranes prepared from the abdomen, in which the enzyme displays both a lower V max and a higher K m . Analysis of the effect of divalent cations suggests a lesion in the responsiveness of the enzyme to Mg 2+. Studies of adenylate cyclase in rutabaga may potentially pinpoint a subpopulation of the enzyme which may prove relevant to molecular mechanisms underlying conditioning.

Mesulergine and its 1,20-N,N-bidemethylated metabolite interact directly with D 1- and D 2-receptors

Mesulergine (CU 32-085), an 8α-aminoergoline, has been reported to influence striatal dopamine turnover in a time-dependent biphasic manner, suggesting initial dopamine antagonistic and late dopamine agonistic effects. To clarify whether these opposing in vivo effects are due to a metabolic conversion in vivo or reflect mixed antagonist/agonist effects expressed at different dose levels, mesulergine and a 1,20-N,N-bidemethylated metabolite, identified in rat urine, were investigated in functional dopamine receptor models. Dopamine-sensitive adenylate cyclase in homogenates of rat striatum and modulation of electrically evoked tritium overflow from rat striatal slices previously labelled with [ 3H]choline were used as tests for D 1- and D 2-receptors, respectively. Mesulergine was found to antagonise D 1-receptor responses at micromolar, and D 2-receptor responses at nanomolar concentrations. In contrast, the bidemethylated metabolite of mesulergine stimulated both D 1- and D 2-receptors at micromolar and nanomolar concentrations, respectively. These in vitro results suggest that at dopamine receptors, mesulergine has antagonistic effects and that the late agonistic effects seen in vivo are mostly due to metabolic conversion into a potent dopaminomimetic drug.

Decreased response with age of the cardiac catecholamine sensitive adenylate cyclase system

The cardiac β-adrenergic coupled adenylate cyclase system was examined in young and old male Wistar rats. The concentration of binding sites for (−) 3H-DHA in membranes prepared from cardiac ventricles was 21.1 ± 2.78 (SD) fmoles/mg protein in 3–4 month old rats (young rats) and 31.2 ± 2.20 fmoles/mg protein in 24 month old rats (old rats). The dissociation constant, K D was 4.3 ± 1.8 nM and 6.7 ± 1.7 nM for young and old rats, respectively. Various compounds were used to study the characteristics of activation of adenylate cyclase in homogenates from cardiac ventricles. Basal adenylate cyclase was reduced 30% in old animals compared to young (6.1 pmoles/min/mg protein in 24 month vs. 8.6 pmoles/min/mg protein in 3–4 month). (−)Isoproterenol (10 −5M) alone stimulated adenylate cyclase greater than two-fold in young rats (10.6 pmoles/min/mg protein above basal) and this stimulation was 34% lower in old animals. GppNHp (100 μM), fluoride (10 mM), and forskolin (100 μM) activation of adenylate cyclase above basal was reduced 38, 37, and 34%, respectively, in the old animals. No significant changes between the two groups were noted in the apparent affinity of GppNHp either alone or in the presence of (−)isoproterenol nor in the affinities of catecholamine agonists for activation of cyclase. These results suggest a reduction in the amount of functional regulatory protein or possibly cyclase in 24 month old rat ventricular tissue compared to 3–4 month old tissue. However, this data does not rule out the possibility of altered molecular interactions of a full complement of regulatory protein (s) with β-adrenergic receptor and/or catalytic adenylate cyclase.

Coupling of dopamine D1 recognition sites with adenylate cyclase in nuclei accumbens and caudatus of schizophrenics.

Sodium fluoride, guanylimidodiphosphate, and the D1 dopamine receptor agonist SKF 38393 elicited a greater activation of adenylate cyclase in homogenates of caudate nucleus in schizophrenic than in nonschizophrenic subjects used as controls. Similarly, a greater activation of adenylate cyclase by sodium fluoride was observed in the nucleus accumbens of schizophrenics. These findings suggest that the coupling of dopamine D1 recognition sites with adenylate cyclase is more efficient in the brain of the schizophrenic, presumably because of an increased affinity of the G/F protein for guanosine 5'-triphosphate.

Opioid activities and structures of alpha-casein-derived exorphins.

Exorphins, peptides with opioid activity, have previously been isolated from pepsin hydrolysates of alpha-casein [Zioudrou, C., Streaty, R. A., & Klee, W. A. (1979) J. Biol. Chem. 254, 2446-2449]. Analysis of these peptides shows that they correspond to the sequences 90-96, Arg-Tyr-Leu-Gly-Tyr-Leu-Glu, and 90-95, Arg-Tyr-Leu-Gly-Tyr-Leu, of alpha-casein. These peptides, as well as two of their analogues Tyr-Leu-Gly-Tyr-Leu-Glu (91-96) and Tyr-Leu-Gly-Tyr-Leu (91-95), have now been synthesized and characterized. Their opioid activity was examined by three different bioassays: (a) displacement of D-2-alanyl[tyrosyl-3,5-3H]enkephalin-(5-L-methioninamide) and [3H]dihydromorphine from rat brain membranes; (b) naloxone-reversible inhibition of adenylate cyclase in homogenates of neuroblastoma x glioma hybrid cells; (c) naloxone-reversible inhibition of electrically stimulated contractions of the mouse vas deferens. The synthetic peptide of sequence 90-96 was the most potent opioid in all three bioassays and its potency was similar to that of the isolated alpha-casein exorphins. The synthetic peptides were totally resistant to hydrolysis by trypsin and homogenates of rat brain membranes, but were partially inactivated by chymotrypsin and subtilisin. The difference in opioid activity of alpha-casein exorphins may be related to differences in conformational flexibility observed by NMR spectroscopy.

Adenylate Cyclase Activation Shifts the Phase of a Circadian Pacemaker

Forskolin, a highly specific activator of adenylate cyclase, produced both delay and advance phase shifts of the circadian rhythm recorded from the isolated eye of the marine mollusk Aplysia. The phase dependence of the response to forskolin was identical to that with serotonin, which also stimulates adenylate cyclase in the eye. The ability of agents to activate adenylate cyclase in homogenates was correlated with their ability to shift the phase of the circadian oscillator. These results along with earlier findings show that adenosine 3',5'-monophosphate mediates the effect of serotonin on the rhythm and regulates the phase of the circadian pacemaker in the eye of Aplysia.