Effects of cyclic GMP and analogues on neurogenic transmission in the rat tail artery.

1. The effects of membrane permeable analogues of guanosine 3':5'-cyclic monophosphate (cyclic GMP), and of the NO donor, 3-morpholinosydnonimine-N-ethylcarbamide (SIN-1) were investigated on [3H]-noradrenaline release and neurogenic vasoconstriction in electrical field stimulated rat tail arteries. 2. Two 8-substituted analogues of cyclic GMP (8-bromoguanosine 3':5'-cyclic monophosphate; 8-bromo-cyclic GMP and 8-(4-chlorophenylthio)-guanosine 3':5'-cyclic monophosphate; 8-pCPT-cyclic GMP) concentration-dependently enhanced stimulation-induced [3H]-noradrenaline release. These prejunctional effects were antagonized by the cyclic AMP-dependent protein kinase (PKA) inhibitor N-[2-((3-(4-bromophenyl)-2-propenyl)-amino)-ethyl]-5 isoquinolinesulphonamide dihydrochloride (H-89; 100 nM) but not by the cyclic GMP-dependent protein kinase (PKG) inhibitors, Rp-8-bromoguanosine 3':5'-cyclic monophosphorothioate (Rp-8-bromo-cyclic GMPS; 10 microM) or Rp-8-(4-chlorophenylthio)-guanosine 3':5'-cyclic monophosphorothioate (Rp-8-pCPT-cyclic GMPS; 10 microM). 3. beta-Phenyl-1,N2-ethenoguanosine 3':5'-cyclic monophosphate (PET-cyclic GMP) had no effect on stimulation-induced [3H]-noradrenaline release but concentration-dependently decreased the stimulation-induced vasoconstriction. 4. The two 8-substituted cyclic GMP derivatives, PET-cyclic GMP and SIN-1, both decreased stimulation-induced vasoconstriction. In addition, SIN-1 relaxed rat tail arteries precontracted with phenylephrine (1 microM). The SIN-1 concentration-relaxation curve was shifted in parallel manner to the right by Rp-8-bromo-cyclic GMPS (10 microM) and Rp-8-pCPT-cyclic GMPS (10 microM) with no change in the maximum effect, showing that the relaxation was mediated by a cyclic GMP/PKG-dependent mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Urotensin I effects on intracellular content of cyclic AMP in the rat tail artery.

Rat tail artery strips were incubated in the presence of 4 x 10(-3) M theophylline and urotensin I (UI). At the concentrations of 1.50, 7.50 mU/ml but not of 0.75 mU/ml UI, the content of cAMP increased significantly. Cyclic AMP content and muscle tension were measured at 5--30 sec intervals during noradrenaline (NA)- and potassium chloride (KCl)-induced contractions and during subsequent urotensin I-induced relaxation. Cyclic AMP content declined initially after addition of NA but returned to baseline levels during the contraction. Significant dose-related increases in cAMP were noted upon addition of UI, prior to the onset of the relaxation response followed by a marked decrease during the relaxation response. An increase in cAMP was observed after initiation of the contractile response to KCl-contracted tissues, but no further increase in cAMP after addition of UI was detected. These findings suggest an involvement of cyclic AMP in the initial events leading to the relaxation response to urotensin I in the NA-contracted rat tail artery. Urotensin I-induced relaxation in KCl-contracted tissues appears to proceed by an alternative mechanism which does not require increased levels of cAMP.     

Role of cyclic AMP in the prejunctional α2-adrenoceptor modulation of noradrenaline release from the rat tail artery

Summary Experiments were designed to evaluate the effect of cyclic AMP on the electrically-induced release of noradrenaline from vascular sympathetic nerve terminals. The possible implication of the inhibition of adenylate cyclase in the negative feed-back control by prejunctional α₂-adrenoceptors of neurotransmitter release was also investigated. Rat isolated tail arteries were preincubated with [³H]-noradrenaline; the preparations were subsequently perfused/superfused with [³H]-noradrenaline-free medium and their perivascular nerves were field stimulated with 24 pulses at 0.4 Hz (0.3 ms, 200 mA). 2 compounds known to enhance the intracellular concentration of cyclic AMP, namely the membrane permeant analogue 8-Br-cAMP (10–300 μmol/l) and forskolin (0.3–10 μmol/l), an activator of adenylate cyclase, concentration-dependently enhanced the stimulation-evoked tritium overflow. The 1,9-dideoxy derivative of forskolin, which does not stimulate adenylate cyclase, was ineffective. Exposure to the cyclic AMP phosphodiesterase inhibitor rolipram 30 μmol/l produced a moderate increase (about 20%) in tritium overflow. However, in the presence of rolipram the facilitatory effect of forskolin was significantly more pronounced than in its absence. Whereas 8-Br-cAMP produced a slight concentration-dependent enhancement of the stimulation-induced vasoconstriction, forskolin and rolipram depressed it. The α₂-adrenoceptor agonist B-HT 933 (3–30 μmol/l) concentration-dependently inhibited the tritium overflow. The effect of B-HT 933 30 μmol/l was slightly, but significantly reduced in the presence of 8-Br-cAMP 100 and 300 μmol/l, but was not changed in the presence of forskolin 3 μmol/l The facilitatory effect of rauwolscine 1 μmol/l was enhanced in the presence of 8-Br-cAMP 100 μmol/l. During perfusion with 8-Br-cAMP 100 μmol/l, the current strength and frequency were decreased to 150 mA and 0.2 Hz, respectively in order to obtain similar amounts of tritium overflow to those observed in the absence of the cyclic AMP analogue with the initial stimulation parameters. Under these conditions, the inhibition of the overflow by B-HT 933 30 μmol/l and the facilitation by the α₂-adrenoceptor antagonist rauwolscine 1 μmol/l were unaltered as compared to controls under initial stimulation conditions. It is concluded that, in the rat tail artery, the terminals of perivascular sympathetic nerves are endowed with an adenylate cyclase system. Cyclic AMP is able to modulate noradrenaline release, but does not appear to play a role in the initiation of the release process itself. In addition, the results do not support the hypothesis that prejunctional α₂-adrenoceptors depress noradrenaline release through the inhibition of adenylate cyclase. Send offprint requests to B. Bucher at the above address     

Effects of urotensin I on the isolated rat tail artery.

Urotensin I (UI) was found to elicit dose-related relaxation responses in isolated helical strips of the rat tail artery. The responses were not prevented by adrenergic, cholingergic or histaminergic blocking agents. Competitive and non-competitive components of antagonism were observed to noradrenaline-, 5-hydroxytryptamine-, and arginine vasopressin-induced contractions. Atropine caused a direct relaxation of the isolated vascular tissues, as well as a significant potentiation of UI responses.     

An amplifying effect of exogenous and neurally stored 5-hydroxytryptamine on the neurogenic contraction in rat tail artery.

1. The interactions between sympathetic neuroeffector transmission and 5-hydroxytryptamine (5-HT) were investigated in segments of rat isolated tail artery. 2. Contractile responses to field stimulation of the artery segments were abolished by tetrodotoxin (3 x 10(-7) M). A subthreshold concentration of acutely applied exogenous 5-HT (10(-8) M) markedly enhanced the contractions induced by sympathetic nerve stimulation, through an action on postjunctional 5-HT2-receptors. 3. The amplifying effect of 5-HT involved an enhanced influx of extracellular calcium into the smooth muscle cells. In contrast, the neurogenic contractions in vessels not exposed to 5-HT were not dependent on extracellular calcium. 4. The adrenergic component of the amplified response involved postjunctional alpha 1- but not alpha 2- adrenoceptor activation. 5. Exposure of the vessels to 5-HT (5 x 10(-7) M) for 30 min resulted in uptake of the amine into the perivascular sympathetic nerves, as demonstrated by immunohistochemistry. After chemical sympathectomy with 6-hydroxydopamine in vitro or in vivo, or surgical sympathectomy, there was little or no uptake. 6. Exposure to 5-HT followed by repeated washing resulted in an enhancement of the neurogenic contraction, which was still fully tetrodotoxin-sensitive. The enhanced response was blocked by ketanserin (10(-8) M) and prevented by the presence of the 5-HT uptake blocker, paroxetine (3 x 10(-8) M), during the period of exposure to 5-HT.     

Effects of flavonoids on cyclic AMP phosphodiesterase and lipid mobilization in rat adipocytes.

Thirty-one flavonoids were tested for their effects on low Km phosphodiesterase with cyclic AMP as the substrate. Quercetin, luteolin, scutellarein, phloretin and genistein showed inhibitory potencies comparable to or greater than 3-isobutyl-2-methylxanthine (EC50 30-50 microM). Only four compounds namely, catechin, epicatechin, taxifolin and fustin stimulated the enzyme activity (stimulatory EC50 130-240 microM). The most potent phosphodiesterase (PDE) inhibitors were aglycones that had a C2.3 double bond, a keto group at C4 and hydroxyls at C3' and/or C4'. However, when the C-ring is opened then the requirement for the C2.3 double bond is eliminated. The same series of flavonoids were also tested for their lipolytic activity. The structural features required for effective synergistic lipolysis (with epinephrine) were generally similar to that required for potent PDE inhibition except that, for lipolytic activity, an intact C-ring was necessary. Fisetin and quercetin having the above-mentioned structure showed a dose- and time-dependent increase in lipolysis which was synergistic with epinephrine. Only butein and hesperetin showed inhibition of epinephrine-induced lipolysis, and their effect was dose-dependent. A time-course study indicated that hesperetin was able to delay the lipolytic action of epinephrine. It is most likely that the lipolytic effects of these compounds were not a result of PDE inhibition, as the orders of potency for the two activities had poor correlation. Apparently, the effective lipolytic flavonoids were also potent PDE inhibitors but not all the PDE inhibitors were able to induce lipolysis.     

Vasoconstrictor effects of various neuropeptide Y analogues on the rat tail artery in the presence of phenylephrine.

1. The increase in perfusion pressure induced by neuropeptide Y (NPY), peptide YY (PYY) and related peptides were compared in the perfused rat tail artery precontracted by a submaximal concentration (1 microM) of the vasoconstrictor, phenylephrine. 2. NPY, PYY, [Leu31,Pro34]NPY, [Glu16,Ser18,Ala22,Leu28,31]NPY (ESALL-NPY) and the centrally truncated and stabilized analogues [D-Cys5,8-aminooctanoic acid7-20, Cys24]-NPY (D-Cys5-NPY) and [D-Cys7, 8-aminooctanoic acid8-17,Cys20]-NPY (D-Cys7-NPY) produced a concentration-dependent enhancement of the vasoconstrictor response induced by 1 microM phenylephrine. PYY was two times more potent than NPY and [Leu31,Pro34]NPY while ESALL-NPY, D-Cys7-NPY and D-Cys5-NPY were approximately 3, 5 and 16 times less potent than NPY respectively. NPY, D-Cys5-NPY and D-Cys7-NPY gave similar maximal responses whereas those observed for PYY, [Leu31,Pro34]NPY and ESALL-NPY were much greater than that of NPY. 3. NPY 13-36 and [des-Ser3,Lys4,Cys2,8-aminooctanoic acid3-24, D-Cys27]-NPY ([es-Ser3,Lys4]Cys2-NPY) were practically inactive at concentrations up to 3 microM, whereas [des-Ser3,Lys4,D-Cys2,8-aminooctanoic acid3-24,Cys27]-NPY ([des-Ser3,Lys4]D-Cys2-NPY), which differs from [des-Ser3,Lys4]Cys2-NPY in the disulphide bridge (a D-Cys in position 2 for [des-Ser3,Lys4]D-Cys2-NPY instead of an L-CYs for [des-Ser3,Lys4]Cys2-NPY) was a weak agonist the maximal effect of which could not be ascertained. 4. The contractile effects of [des-Ser3,Lys4]D-Cys2-NPY were additive with those of NPY and [Leu31,Pro34]NPY demonstrating that it is not a partial agonist but may simply not interact competitively with the receptor binding site for NPY.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of alniditan on neurogenic oedema in the rat dura mater and on contraction of rat basilar artery.

The non-indole 5-HT receptor agonist, alniditan (R 91274), was tested and compared to sumatriptan in an in vivo model of neurogenic inflammation within the meninges of rats and in rat basilar artery in a Mulvany-Halpern chamber in vitro. Alniditan dose dependently attenuated the neurogenic inflammation and was more potent than sumatriptan. The alniditan response was blocked by the 5-HT(1B/D) receptor antagonist, GR 127935 (2'-methyl-4'-(5-methyl-[1,2, 4]oxadiazol-3-yl)-biphenyl-4-carboxylic acid [4-methoxy-3-(4-methyl-piperazin-1-yl)-phenyl]-amide), but not by ketanserin, indicating that the effect is mediated through 5-HT(1B/D) receptors. Alniditan did not attenuate substance P-induced inflammation, suggesting that the mediating receptors are located prejunctionally. In vitro alniditan exhibited less vasoconstrictive effects on the rat basilar artery than did sumatriptan, although at a very high concentration (1 mM), alniditan caused intensive constriction, most likely through a mechanism independent from 5-HT receptor activation.     

Effects of prostaglandins and cyclic AMP on cytokine production in rat leukocytes

Prostaglandins E₁, prostaglandin E₂, 3-oxa-methano-prostaglandin I₁ (SM-10906), a stable prostaglandin I₂ analog, and dibutyryl cyclic AMP suppressed the production of tumor necrosis factor and interleukin-1 in lipopolysaccharide-stimulated rat pleural resident monocytic cells, whereas they enhanced the production of interleukin-6 and cytokine-induced neutrophil chemoattractant (CINC), a rat interleukin-8-like chemokine, in these cells. SM-10906 also inhibited the in vivo production of tumor necrosis factor and interleukin-1 in pleural exudates, when injected into the rat pleural cavity concomitantly with carrageenin. The cyclic AMP (cAMP) level in the lipopolysaccharide-stimulated resident cells was increased when the cells were incubated in the presence of prostaglandin E₁, prostaglandin E₂ or SM-10906. Prostaglandin I₂ showed only slight effects. The addition of pentoxifylline, a phosphodiesterase inhibitor, to the incubation mixture increased the cAMP level and also enhanced the effect of prostaglandins, indicating that these regulating actions of prostaglandins may be exerted partly through a mechanism involving an increased intracellular cAMP level.     

Effects of isoprenaline on the time course of the cyclic AMP level in rat uterus.

Isoprenaline markedly relaxed rat uterine muscle and increased its content of cyclic AMP. The cyclic AMP concentration reached its highest value, about ten times the control after 3 min., after which it declined rather quickly at first but later more slowly. After 60 min. the cyclic AMP content reached twice the prestimulation value. The muscle remained relaxed all the time. If the incubation medium from the uterus treated with isoprenaline for 60 min. was added to a fresh muscle, a normal response was elicited. Addition of a new dose of isoprenaline to a muscle previously incubated with isoprenaline for 60 min. only gave a minimal rise in cyclic AMP after 3 min. The presence of catechol or corticosterone, which both decreased the inactivation of isoprenaline, showed no effect on the decrease of the isoprenaline-stimulated cyclic AMP level. Nor could the decrease in the cyclic AMP response be prevented in the presence of GTP,F-, adenosine or in Ca2+-deficient medium. The addition of theophylline and puromycin both lead to a slower decline of cyclic AMP content, but after 60 min. the value returned to near that seen ater only isoprenaline. Our studies on isoprenaline-induced desensitization of rat uterus have led us to the conclusion that the change in the capacity of the preparations to accumulate cyclic AMP may be a result of either changes in the beta-adrenoceptors or in the cyclase-system.     

Effects of alternative transmitter amines on cyclic AMP formation in rat brain tissue.

Aromatic amines and related compounds, some of which are taken up and released from nerve terminals, might act at brain receptors ordinarily stimulated by traditional amine neurotransmitters. Several of these compounds were evaluated for their ability to stimulate or impede synthesis of cyclic AMP in rat striatal homogenates and cortical slices. In contrast to catecholamines, most had no effect, consistent with their possible role as false transmitters.     

Effects of vasopressin on noradrenaline-induced cyclic AMP accumulation in rat brain slices

Addition of arginine vasopressin (AVP) or 1-desamino-8-D-arginine vasopressin (DDAVP) to rat cortical slices resulted in significant inhibition of the rise in cyclic AMP produced by incubation with 50 microM noradrenaline. A single injection of DDAVP (20 micrograms/rat) produced a reduced response to noradrenaline in derived cortex and caudate slices. In animals pretreated at day 5 of life with IP desipramine and intracisternal 6-hydroxydopamine (6-OHDA), both acute and chronic treatments with DDAVP resulted in a reduction in response in derived cortical, caudate and hippocampal slices. The 6-OHDA pretreated animals also showed reduced open-field behavioural activity after both acute and chronic DDAVP, while animals which were not pretreated responded to acute treatment only. The relationship between the effects of vasopressin on noradrenaline-induced cyclic AMP accumulation and its action on learning and memory is discussed.     

Effects of selective phosphodiesterase inhibition on cyclic AMP hydrolysis in rat cerebral cortical slices.

1. The effects of selective inhibition of phosphodiesterase activities on the concentration and rate of hydrolysis of adenosine 3':5' cyclic-monophosphate (cyclic AMP) in rat cerebral cortical slices has been studied. 2. Isoprenaline caused a rapid, concentration-dependent increase in cyclic AMP concentration to new steady-state levels (basal: 7.1 +/- 0.7; 10 microM isoprenaline: 14.3 +/- 1.4 pmol mg-1 protein). Addition of a beta-adrenoceptor antagonist to isoprenaline-stimulated cerebral cortical slices caused a rapid decrease in cyclic AMP concentration to basal levels (t1/2: 58 +/- 18 s). 3. Preincubation of slices for 30 min with the phosphodiesterase inhibitors 1-methyl-3-isobutylxanthine, denbufylline, rolipram or Ro20,1724 caused concentration-dependent increases in basal and isoprenaline-stimulated cyclic AMP concentrations and decreased the rate of cyclic AMP hydrolysis measured after addition of a beta-adrenoceptor antagonist. However, SKF 94120 and zaprinast had none of these effects. 4. The results are discussed with respect to previous studies of phosphodiesterase isozymic activities isolated from cerebrum and it is suggested that the Ca2+/calmodulin-independent, low Km cyclic AMP phosphodiesterase isozyme, which is selectively inhibited by denbufylline, rolipram and Ro20,1724, and is present in cerebrum is of critical importance to the regulation of cyclic AMP concentration in this tissue.     

Effect of ethanol on the cyclic AMP system in rat brain.

The effects of ethanol on adenylate cyclase and phosphodiesterase activity in vitro, and on cyclic AMP, ATP and adenosine levels in vivo were studied. Ethanol appeared to affect the cyclic AMP system indirectly, probably through its effect on neurotransmitter release or on adenosine formation.     

Effects of alpha beta methylene ATP on membrane potential, neuromuscular transmission and smooth muscle contraction in the rat tail artery

alpha beta methylene ATP (meATP, 400 nM-1 microM) caused depolarization and constriction of the smooth muscle of the rat tail artery, and block of the excitatory junction potential (e.j.p.). A similar depolarization caused by 25 mM K did not block the e.j.p. Contractions caused by nerve-released noradrenaline acting on alpha-adrenoceptors were potentiated by meATP but this was probably due to the depolarization as 25 mM K had a similar effect. MeATP blocked e.j.p.s recorded in the presence of 3 microM tetrodotoxin, suggesting that meATP was not blocking e.j.p.s by a local anaesthetic action.     

Plasma cyclic AMP in the morphine-tolerant rat

Effects of morphine on plasma cyclic AMP leveis in the rat were investigated. Morphine (20 $\text{mg}\text{kg}$, s.c. + 4 $\text{mg}\text{kg/hr}$, i.v.) produced an increase in plasma cyclic AMP concentrations at 1hr and sustained elevated levels for several hours, but the levels returned to normal levels at 48 hr. the 48 hr increase in plasma cyclic AMP elicited by morphine (20 $\text{mg}\text{kg}$, s.c.) was far smaller than that of 1 hr; the plasma concentration of morphine was at the same level as the 1 hr. This indicated the development of tolerance to the morphine induced increase in plasma cyclic AMP. At 48 hr an epinephrine challenge increased plasma cyclic AMP to the same extent as in naive rats. Therefore, desensitization of peripheral tissues as being responsible for the increase in the plasma cyclic AMP could be ruled out. the development of tolerance by the sympatho-adrenal reflex elicited by morphine was suggested. In naioxone-precipitated abstinence, plasma cyclic AMP showed a 4-fold increase but. on spontaneous withdrawal, it showed on a slight but significant increase. It was concluded that plasma cyclic AMP is a sensitive index of tolerance to and dependence on morphine.