Tissue Cyclic AMP Research Articles

Intrathecal Injection of Ropivacaine Reduces Cervical Resistance in Late-Pregnant Rats.

PurposeNeuraxial (spinal and epidural) anesthesia is the cornerstone of ensuring the satisfaction rate of painless delivery; however, whether it prolongs the first stage of labor remains controversial. Although current clinical research results tend to be negative, the conclusions are not convincing due to the lack of basic research. This study was conducted to provide a theoretical reference for this controversy through basic research.Materials and MethodsA spinal anesthesia model was established by the intrathecal injection of 0.1% ropivacaine in late-pregnant rats (day 22). The cervical resistance test was used to measure the tension of different groups of isolated cervical tissues. Western blotting and cervical tissue cyclic AMP (cAMP) enzyme-linked immunosorbent assay were performed to clarify the possible related mechanisms.ResultsCervical resistance experiments showed that the intrathecal injection of ropivacaine decreased the cervical resistance, and norepinephrine injection reversed this effect. Western blotting showed that α2A adrenergic receptor (α2A-AR) levels gradually increased over time in pregnant rats. The cAMP enzyme-linked immunosorbent assay revealed that the intrathecal injection of norepinephrine reversed the increase in cervical tissue cAMP concentration caused by ropivacaine injection.ConclusionRopivacaine relaxes the cervix. Further, α2-AR may be involved in the process of cervical contraction.

Effect of morphine on the tissue cyclic AMP and cyclic GMP content in two strains of mice.

The effect of morphine on the cyclic (c) AMP and cyclic (c) GMP concentrations in several organs, and its reversal by naloxone have been investigated in C57BL and DBA strains of mice. Morphine increased the cAMP contents in lungs and muscle, and the cGMP contents in lungs, intestine, heart, liver and muscle in a naloxone-reversible way in C57BL mice only. This is consistent with our previous observation that morphine increased plasma cyclic nucleotide levels in C57BL mice, whereas such an increase was marginal in the DBA strain. These results show that there is a strain difference in the effect of morphine on tissue cyclic nucleotide contents and the possible origin of the plasma cyclic nucleotides which are increased by morphine.

Evaluating the Role of the Serotoninergic System in the Control of Human Seminal Vesicle Smooth Muscle—An in Vitro Approach

It has been suggested that serotonin re-uptake inhibitors (SRIs) may retard the ejaculatory response by acting directly on the seminal vesicle (SV) and ductus deferens smooth muscle. However, until now, only a very few experimental studies have investigated such potential local (peripheral) effects. To elucidate the effects of serotonin (5-HT) and the SRIs clomipramine, fluoxetine and imipramine on the tension induced by norepinephrine (NE) of isolated human SV smooth muscle, as well as on the production of tissue cyclic AMP and cyclic GMP. To measure the inhibition exerted by serotonin and SRIs clomipramine, fluoxetine, and imipramine on the contractile response of isolated SV tissue. In addition, the effects of the drugs on the turn-over of cyclic nucleotides cAMP and cGMP were also elucidated. The effects of the cumulative addition of serotonin and the SRIs clomipramine, fluoxetine and imipramine (1 nM-10 microM) on the tension induced by the alpha(1)-adrenoceptor agonist NE (10 microM) of SV strip preparations were studied using the organ bath technique. Cyclic AMP and cyclic GMP were measured by means of specific radioimmunoassays. The tension induced by NE was dose-dependently reversed by the drugs tested. The rank order of efficacy was: imipramine > or = fluoxetine > or = clomipramine > serotonin. Mean reversion of tension was measured between 66 +/- 6.6% and 52 +/- 6.6%. These effects were paralleled by a 1.3-fold to 2.7-fold increase in tissue cAMP in response to exposure to the drugs. In contrast, no significant enhancement in cGMP was noted. The findings, for the first time, present evidence that SRIs may antagonize the sympathetic contraction of SV smooth muscle via stimulation of tissue cyclic AMP.

Evidence for a significant role of a Gs-triggered mechanism unrelated to the activation of adenylyl cyclase in the cyclic AMP-independent relaxant response of guinea-pig tracheal smooth muscle.

Cyclic AMP is a key molecule in the regulation of airway smooth muscle tone. Increased cyclic AMP leads to relaxation of this smooth muscle and its inhibition results in the muscle contraction. A constitutive role for cyclic AMP in the contraction and relaxation of airway muscle is supported by the observations that direct activators of adenylyl cyclase, such as forskolin and membrane-permeable cyclic AMP analogues, relax this smooth muscle potently. This traditional view of the role for cyclic AMP is the basis for the idea that relaxation of airway smooth muscle mediated through adenylyl cyclase-linked, G(s)-coupled receptors, including the beta(2)-adrenoceptor, is achieved mainly by the elevation of cyclic AMP content [cyclic AMP-dependent mechanism(s)]. However, recent pharmacological and biochemical evidence raises a fundamental question concerning the role of cyclic AMP; can G(s)-coupled receptor-mediated relaxation of tracheal smooth muscle be attributed exclusively to cyclic AMP-dependent mechanism(s)? In the present study, we show that cholera toxin (CTX, 5 microg/ml), an activator of the heterotrimeric guanine-nucleotide-binding protein G(s), relaxes guinea-pig tracheal smooth muscle. CTX also elevates tissue cyclic AMP content by about 30-fold and this is practically abolished by an adenylyl cyclase inhibitor, SQ 22,536 (100 microM). However, unexpectedly, the relaxant response to CTX is not affected by SQ 22,536. These results firstly show that activation of G(s) is able to produce a relaxation in tracheal smooth muscle independently of the elevation of cyclic AMP. G(s)-triggered, cyclic AMP-unrelated cellular mechanism(s) seem(s) to play a substantial role in smooth muscle relaxation mediated through adenylyl cyclase-linked receptors. This mechanism may account in part for the cyclic AMP-independent relaxant response of tracheal smooth muscle.

Glycogen autophagy in newborn rat hepatocytes.

Glycogen autophagy in newborn rat hepatocytes was studied by using enzyme determinations and electron microscopy. Cyclic AMP induced glycogen autophagy in these cells. Glycogen-hydrolyzing acid glucosidase activity increased whereas acid mannose 6-phosphatase activity decreased in the liver of these animals. Parenteral glucose, which prevents postnatal glucagon secretion and tissue cyclic AMP elevation, and propranolol which antagonizes cyclic AMP, inhibited glycogen autophagy. Glucosidase activity decreased and phosphatase activity increased. These findings raise the possibility that cyclic AMP-induced autophagic mechanisms in newborn rat hepatocytes are associated with changes in the activity of acid mannose 6-phosphatase.

Cellular mechanisms involved in rabbit carotid body excitation elicited by endothelin peptides

The present study evaluated the effects of endothelin (ET) peptides on carotid sinus nerve (CSN) activity, catecholamine (CA) release, and second messenger signaling pathways in rabbit carotid bodies superfused in vitro, and in dissociated chemosensory type I cells. ET-1 (1.0 μM) and ET-3 (1.0 μM) did not alter basal CSN activity and CA release, but they potentiated nerve activity ( P<0.05) and CA release ( P<0.05) evoked by hypoxia. Under basal conditions, ET-1 and ET-3 (1.0 μM each) elevated tissue cyclic AMP (cAMP) levels nearly 3-fold ( P<0.001, ET-1; P<0.05, ET-3) and inositol phosphate (IP n ) levels nearly 4-fold ( P<0.01, ET-1). Hypoxia evoked an increase in carotid body cAMP, and this response was also potentiated in the presence of 1.0 μM ET-1 ( P<0.01) or 1.0 μM ET-3 ( P<0.001). Patch-clamp studies of isolated type I cells showed that 100 nM ET-1 elevated the peak amplitude of voltage-sensitive (L-type) Ca 2+-currents by an average of 37.6% ( P<0.001). Fluorescent Ca 2+-imaging revealed that 100 nM ET-1 did not alter [Ca 2+] i under basal conditions, but that [Ca 2+] i-responses evoked by hypoxia were potentiated by 87% ( P<0.01). Our data indicate that ET augments chemoreceptor responses by activating second messenger signaling pathways which promote the phosphorylation of Ca 2+-channel protein, thereby enhancing stimulus-evoked intracellular Ca 2+ levels.

The mechanisms of α 2-adrenoceptor agonist-induced contraction in longitudinal muscle of the porcine uterus

The aim of the present study was to clarify the cellular mechanisms underlying the α 2-adrenoceptor-mediated contraction of porcine myometrium (nonvascular smooth muscle). Acetylcholine (3 nM–1 μM), clonidine (1 nM–10 μM) and 5-bromo- N-[2-imidazolin-2-yl]-6-quinoxalinamine (UK14304) (1 nM–10 μM) in Krebs solution caused a concentration-dependent contraction in the longitudinal muscles of the porcine uterus with similar EC 50 values and maximum responses. A lowered external Ca 2+ concentration and verapamil (10 nM–10 μM) decreased the contractile response to clonidine and UK14304 more markedly than the response to acetylcholine. However, in Kumagai solution, neither clonidine nor UK14304 caused contractile responses, but acetylcholine remained effective. The effects of α 2-adrenoceptor agonists on intracellular Ca 2+ concentration ([Ca 2+] i) and smooth muscle force were measured simultaneously using fura-PE3-loaded muscle preparations. Clonidine and UK14304 caused increases in [Ca 2+] i and force of the longitudinal muscle. The increases in [Ca 2+] i and muscle force were markedly inhibited by verapamil and in Ca 2+-free solution (EGTA, 1 mM). In the absence of external Ca 2+, clonidine caused only a small increase in [Ca 2+] i in Ca 2+-loaded preparations compared with those increases caused by carbachol, histamine, and oxytocin. Ca 2+ (2.5 mM) caused increases in [Ca 2+] i and force of the longitudinal muscles in a Ca 2+-free high K + solution. Clonidine concentration dependently potentiated the Ca 2+-induced contraction without significantly changing the increase in [Ca 2+] i, and this potentiation was inhibited by yohimbine. These results suggested that clonidine increases the Ca 2+ sensitivity of the contractile elements through activation of α 2-adrenoceptors. During the development of the contractile response to clonidine (1 μM, 0–5 min), tissue cyclic AMP levels did not change significantly. In vitro treatment with pertussis toxin (1 μg/ml for 2 h) significantly decreased the contraction induced by clonidine without affecting the responses to carbachol and high K +. The present results indicate that in porcine myometrium, α 2-adrenoceptor stimulation caused contraction of the longitudinal muscles by mechanisms largely dependent on the influx of extracellular Ca 2+, probably through voltage-dependent Ca 2+ channels (VDCCs), and that the potentiation of the Ca 2+ sensitivity of the contractile elements is another mechanism of the contractile responses. These actions involve a pertussis-toxin-sensitive G protein (probably G i type) in the signal transduction pathway.

Opposing effects on infarction of delta and kappa opioid receptor activation in the isolated rat heart: implications for ischemic preconditioning.

Delta-opioid receptors are known to participate in the protection found following ischemic preconditioning (IPC), but the role of kappa-receptors in IPC is currently controversial. Langendorff-perfused rat hearts received 35 min regional ischemia and 2 h reperfusion. PC (2 cycles 5 min global ischemia) substantially reduced infarct size. Pharmacological PC with the delta-agonist DADLE (10 nmol/L) had similar protective effects. However, higher dose DADLE (1 micromol/L) had a less beneficial effect, and in conjunction with the delta-antagonist naltrindole unexpectedly increased infarct size (61.5 +/- 2.0%, p<0.05 v 45.9 +/- 2.4% in controls) suggesting a non-delta effect. The universal kappa-opioid agonist bremazocine (30 nmol/L) increased infarct size (61.3 +/- 1.6%, p<0.05 v controls), an effect abrogated by the selective kappa1-antagonist nor-binaltorphimine (BNI). Since opiates are known to have anti-adrenergic effects, which hypothetically may help to mediate IPC, cyclic AMP levels were measured in DADLE and in bremazocine-treated hearts. Decreased levels of cyclic AMP at the start of the regional ischemic period were found in low dose DADLE hearts (0.485 +/- 0/020, n = 8, vs controls, 0.654 +/- 0.025 nmol/g wet weight, p<0.001), but not in high dose DADLE nor in bremazocine treated hearts. Thus, in the isolated rat heart kappa1-opioid receptor activation exacerbates infarct size through an as yet unknown mechanism, suggesting that there could be an "antipreconditioned state". In contrast, delta-activity mediates protection which may be associated with a reduction of tissue cyclic AMP levels.

The effects of levosimendan on the left ventricular function and protein phosphorylation in post-ischemic guinea pig hearts.

The widely accepted theories for the decreased function in the stunned myocardium relate to Ca2+ desensitization and free radical-mediated tissue damage of the myofilaments. The aim of the present study was to examine whether the depressed contractile function and Ca2+ responsiveness of the stunned myocardium may be restored by a new Ca2+ sensitizer (levosimendan), which has been shown to improve the Ca2+ response of the myofilaments. The effects of levosimendan on the left ventricular function and the in vivo protein phosphorylation were examined in both the non-ischemic and the stunned myocardium. Myocardial stunning was induced in Langendorff-perfused guinea pig hearts by suspending the circulation for 8 min, followed by a 20-min reperfusion period. Perfusion of post-ischemic guinea pig hearts with levosimendan (0.03-0.48 microM, 6 min) was associated with dose- and time-dependent increases in both dP/dtmax (contractility) and dP/dtmin (speed of relaxation). When the effectiveness of levosimendan was compared in non-ischemic and post-ischemic hearts, no significant differences were noted in the relative stimulatory effects on contractility and relaxation, at any given time point (time-response curve) or concentration (dose-response curve). Perfusion of the guinea pig hearts with a high (0.3 microM) levosimendan concentration did not reveal any qualitative or quantitative difference in the phosphodiesterase inhibitory potential of the compound (elevation of tissue cyclic AMP levels and characteristics of protein phosphorylation) between the non-ischemic and the post-ischemic myocardium. However, when isoproterenol was administered to induce maximal in vivo phosphorylation of cardiac phosphoproteins, an attenuation of the 32P-incorporation into troponin I was noted in the post-ischemic hearts. The decrease in isoproterenol-induced 32P-incorporation into troponin I was associated with similar alterations in the tissue level of this protein. We conclude that the Ca2+ sensitizer levosimendan exerts dose- and time-dependent positive inotropic and lusitropic effects on the post-ischemic myocardium, lending support to the hypothesis tha Ca2+ desensitization of the myofibrils is involved in myocardial stunning.

Pharmacological Characterization of Effects of UK-1745, a Novel Cardiotonic Agent withβ-Adrenoceptor-blocking Action, in Aequorin-loaded Canine Right Ventricular Muscle

UK-1745, a derivative of furoindolinone, is a novel cardiotonic agent that was designed to have bothβ-adrenoceptor-blocking and cardiotonic activity. The aim of this study was to clarify the mode of action of UK-1745 in the canine and rabbit myocardium. UK-1745 elicited a weak but definite concentration-dependent positive inotropic effect in association with a decrease in the total duration of contraction: in particular, a decrease in the relaxation time in isolated canine right ventricular trabeculae. The maximum positive inotropic effect of UK-1745 was achieved at 3×10−5m and amounted to approximately 15% of the maximum response to isoproterenol. The EC50for the positive inotropic effect of UK-1745 was 3.3×10−6m . Carbachol, a muscarinic receptor agonist, at 3×10−6m completely inhibited the positive inotropic effect of UK-1745. UK-1745 shifted the concentration-response curve for isoproterenol to the right with pA2value of 5.70. By contrast, UK-1745 at 3×10−7to 3×10−5m shifted the concentration-response curve for forskolin to the left. In aequorin-loaded ventricular trabeculae, UK-1745 induced a positive inotropic effect that was accompanied by an increase in Ca2+transients. It did not affect the relationship between the amplitude of Ca2+transients and peak force as compared with that associated with elevation of the extracellular concentration of Ca2+ions ([Ca2+]o). The level of cyclic AMP in tissue was not significantly increased at 3×10−5m UK-1745. The present results indicate that UK-1745 exerts a positive inotropic effect mainly via a cyclic AMP-dependent mechanism but, in addition, it hasβ-adrenoceptor-blocking activity over the same range of concentrations. A drug with such a pharmacological profile might have the potential advantage of avoiding Ca2+overload and superfluous oxygen consumption, which may contribute to the unfavorable effects of novel cardiotonic agents that act purely by inhibition of phosphodiesterase III.

Mechanisms of 5-hydroxytryptamine-induced inhibition in the porcine myometrium.

The present experiments were designed to clarify the mechanisms of the inhibitory response of 5-hydroxytryptamine (5-HT) in the porcine uterine circular muscle. Inhibitory responses induced by 5-HT (1 nM-1 microM) were not affected by apamin (1 microM), charybdotoxin (100 nM) or glibenclamide (20 microM) but were significantly attenuated by 4-aminopyridine (3 mM) and tetraethylammonium (3 mM). Imidazole (100 microM) decreased but 3-isobutyl-1-methylxanthine (30 microM), milrinone (30 microM) and Ro 20-1724 (10 and 30 microM) potentiated the 5-HT-induced inhibition. On the other hand, zaprinast (3-30 microM) had no significant effect on the inhibitory response of 5-HT. 5-HT caused a time (0-5 min)-and concentration (1 nM-1 microM)-dependent increase in the tissue cyclic AMP level, but had no effect on the tissue cyclic GMP level. A significant correlation (P < 0.05) was observed between the inhibition of contraction and tissue cyclic AMP level. The effect of 5-HT on contractile force and cytosolic Ca2+ level ([Ca2+]i) was investigated using fura-PE3-loaded myometrial strips. A low concentration of 5-HT (< or = 10 nM) inhibited the spontaneous contraction without changing the amplitude of the spontaneous [Ca2+]i increase, but a higher concentration of 5-HT (> or = 100 nM) decreased the resting [Ca2+]i and inhibited both the spontaneous [Ca2+]i increase and spontaneous contraction. High-K+ (50 mM) caused increases in muscle contractile force and [Ca2+]i. 5-HT concentrationdependently inhibited the high-K(+)-induced contraction (EC50, 45 nM) with only a small decrease in [Ca2+]i increase. Carbachol also caused increases in muscle contractile force and [Ca2+]i. 5-HT significantly decreased both the carbachol-induced contraction and [Ca2+]i increase, but was more potent at inhibition of contractile force than [Ca2+]i. In Ca(2+)-loaded myometrial strips, carbachol, but not caffeine, caused a transient increase in [Ca2+]i and contraction in the absence of external Ca2+ (EGTA, 1 mM). 5-HT inhibited both the carbachol-induced increases in [Ca2+]i release and contractile force. In the beta-escin permeabilized myometrium, 5-HT significantly inhibited the Ca(2+)-induced contraction. The present results indicate that 5-HT stimulates tissue cyclic AMP production, and inhibits the porcine uterine muscle contractility by a reduction in [Ca2+]i and in Ca2+ sensitivity of the contractile elements. Activation of K+ channels might be partially involved in 5-HT-induced inhibition of the myometrial contractility.

Regulation of phosphoinositide turnover in neonatal rat cerebral cortex by group I- and II- selective metabotropic glutamate receptor agonists.

1. The interactive effects of different metabotropic glutamate (mGlu) receptor subtypes to regulate phosphoinositide turnover have been studied in neonatal rat cerebral cortex and hippocampus by use of agonists and antagonists selective between group I and II mGlu receptors. 2, The group II-selective agonist 2R,4R-4-aminopyrrolidine-2,4-dicarboxylate (2R,4R-APDC; 100 microM) had no effect on basal total inositol phosphate ([3H]-InsPx) accumulation (in the presence of Li+) in myo-[3H]-inositol pre-labelled slices, but enhanced the maximal [3H]-InsPx response to the group I-selective agonist (S)-3,5-dihydroxyphenylglycine (DHPG) by about 100% in both hippocampus and cerebral cortex. In cerebral cortex the enhancing effect of 2R,4R-APDC occurred with respect to the maximal responsiveness and had no effect on EC50 values for DHPG (-log EC50 (M): control, 5.56+/-0.05; +2R,4R-APDC, 5.51+/-0.08). 2R,4R-APDC also caused a significant enhancement of the DHPG-stimulated inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) mass response over an initial 0-300 s time-course. 3. The enhancing effects of 2R,4R-APDC on DHPG-stimulated [3H]-InsPx accumulation were observed in both the presence and nominal absence of extracellular Ca2+, and irrespective of whether 2R,4R-APDC was added before, simultaneous with, or subsequent to DHPG. Furthermore, increasing the tissue cyclic AMP concentration up to 100 fold had no effect on DHPG-stimulated Ins(l,4,5)P3 accumulation in the absence or presence of 2R,4R-APDC. 4. 2R,4R-APDC and (2S, 1'R, 2'R, 3'R)-2-(2,3-dicarboxylcyclopropyl)glycine (DCG-IV), the latter agent in the presence of MK-801 to prevent activation of NMDA-receptors, each inhibited forskolin-stimulated cyclic AMP accumulation by about 50%, with respective EC50 values of 1.3 and 0.04 microM (-log EC 50 (M): 2R,4R-APDC, 5.87+/-0.09; DCG-IV, 7.38+/-0.05). In the presence of DHPG (30 microM), 2R,4R-APDC and DCG-IV also concentration-dependently increased [3H]-InsPx accumulation with respective EC50 values of 4.7 and 0.28 microM (-log EC50 (M): 2R,4R-APDC, 5.33+/-0.04; DCG-IV, 6.55+/-0.09) which were 3-7 fold rightward-shifted relative to the adenylyl cyclase inhibitory responses. 5. The group II-selective mGlu receptor antagonist LY307452 (30 microM) caused parallel rightward shifts in the concentration-effect curves for inhibition of forskolin-stimulated adenylyl cyclase, and enhancement of DHPG-stimulated [3H]-InsPx accumulation, by 2R,4R-APDC yielding similar equilibrium dissociation constants (KdS, 3.7+/-1.1 and 4.1+/-0.4 microM respectively) for each response. 6. The ability of 2R,4R-APDC to enhance receptor-mediated [3H]-InsPx accumulation appeared to be agonist-specific; thus although DHPG (100 microM) and the muscarinic cholinoceptor agonist carbachol (10 microM) stimulated similar [3H]-InsPx accumulations, only the response to the former agonist was enhanced by co-activation of group II mGlu receptors. 7. These data demonstrate that second messenger-generating phosphoinositide responses stimulated by group I mGlu receptors are positively modulated by co-activation of group II mGlu receptors in cerebral cortex and hippocampus. The data presented here are discussed with respect to the possible mechanisms which might mediate the modulatory activity, and the physiological and pathophysiological significance of such crosstalk between mGlu receptors.

Cadmium disrupts the signal transduction pathway of both inhibitory and stimulatory receptors regulating chloride secretion in the shark rectal gland.

The heavy metal cadmium causes nephrotoxicity and alters the transport function of epithelial cells. In the shark rectal gland, chloride secretion is regulated by secretagogues and inhibitors acting through receptors coupled to G proteins and the cyclic AMP-protein kinase A pathway. We examined the effects of cadmium on the response to the inhibitory peptide somatostatin (SRIF), and to the stimulatory secretagogues forskolin and vasoactive intestinal peptide (VIP). In control experiments, SRIF (100 nM) entirely inhibited the chloride secretory response to 10 microM forskolin (maximum chloride secretion with forskolin 1984 +/- 176 microEq/h/g; with forskolin + SRIF 466 +/- 93 microEq/h/g, P < 0.001). Cadmium (25 microM) entirely reversed the inhibitory response to SRIF (chloride secretion 2143 +/- 222 microEq/h/g) and caused an overshoot (2917 +/- 293 microEq/h/g) that exceeded the response to forskolin (P < 0.01). Cadmium also enhanced forskolin-stimulated chloride secretion (2628 +/- 418 vs. 1673 +/- 340 microEq/h/g, P < 0.02) and reversed the declining phase of the forskolin response. Cadmium had a concentration-dependent, biphasic effect on the response to VIP. Cd (10-100 microM) increased both chloride secretion and tissue cyclic AMP content, whereas higher concentrations (1 mM) inhibited chloride secretion and cyclic AMP accumulation. Our findings provide evidence that Cd disrupts the signal transduction pathways of both inhibitory receptors and secretagogues regulating cAMP mediated transport in an intact epithelia. The results are consistent with direct effects of cadmium on adenylate cyclase and/or phosphodiesterase activity in this marine epithelial model.

Stimulation of the hypothalamo-pituitary-adrenal axis in the rat by three selective type-4 phosphodiesterase inhibitors: in vitro and in vivo studies.

Previous studies in our laboratory have shown that the synthetic xanthine analogue denbufylline, a selective type 4 phosphodiesterase (PDE‐4) inhibitor, is a potent activator of the hypothalamo‐pituitary‐adrenal (HPA) axis when given orally or intraperitoneally (i.p.) to adult male rats. This paper describes the results of experiments in which well established in vivo and in vitro methods were used to compare the effects of denbufylline on HPA function with those of two other selective PDE‐4 inhibitors, rolipram and BRL 61063 (1,3‐dicyclopropylmethyl‐8‐amino‐xanthine). For comparison, parallel measurements of the immunoreactive‐ (ir‐) luteinising hormone (LH) were made where appropriate. When injected intraperitoneally, rolipram (40 and 200 μg kg−1, P&lt;0.005), denbufylline (0.07–0.6 μg kg−1, P&lt;0.05) and BRL 61063 (30 μg kg−1, P&lt;0.005) each produced marked rises in the serum ir‐corticosterone concentrations. However, lower doses of rolipram (1.6 and 8 μg kg−1) and BRL 61063 (0.25–6 μg kg−1) were without effect (P&gt;0.05). By contrast, intracerebroventricular (i.c.v.) injection of rolipram (8 ng–1 μg kg−1) or denbufylline (50 ng–1 μg kg−1) failed to influence the serum ir‐corticosterone concentration. BRL 61063 (8–120 ng kg−1, i.c.v.) was also ineffective in this regard although at a higher dose (1 μg kg−1, i.c.v.) it produced a small but significant (P&lt;0.05) increase in ir‐corticosterone release. Denbufylline also increased the serum ir‐LH concentration when given peripherally (0.2–0.6 μg kg−1, i.p., P&lt;0.05) or centrally (100 ng kg−1, i.c.v., P&lt;0.05) but rolipram (1.6–200 μg kg−1, i.p. or 8 ng–1 μg kg−1, i.c.v.) and BRL 61063 (0.25–30 μg kg−1, i.p. or 1 ng–1 μg kg−1, i.c.v.) did not (P&gt;0.05). In vitro rolipram (10 μM, P&lt;0.01), denbufylline (1 mM, P&lt;0.001) and BRL 61063 (1 and 10 μM, P&lt;0.05) stimulated the release of corticotrophin releasing hormone (ir‐CRH‐41) but lower concentrations of the drugs were without effect as also was BRL 61063 at 100 μM (P&gt;0.05); the rank order of potency was thus BRL 61063&gt;rolipram&gt;denbufylline. The adenylyl cyclase activator forskolin (100 μM, P&lt;0.01) also stimulated the release of ir‐CRH‐41, producing effects which were additive with those of rolipram and denbufylline but not with those of BRL 61063. The secretory responses to forskolin (100 μM) were accompanied by a highly significant increase in the cyclic AMP content of the hypothalamic tissue (P&lt;0.005). Rolipram (10 μM) also significantly (P&lt;0.05) elevated the hypothalamic cyclic AMP but denbufylline (10 mM) and BRL 61063 (10 μM) did not. However, all three PDE‐inhibitors potentiated the rise in cyclic AMP induced by forskolin (P&lt;0.05). None of the drugs tested, alone or in combination, modified the release of arginine vasopressin (ir‐AVP) from the hypothalamus. Rolipram (100 μM), denbufylline (100 μM) and BRL 61063 (100 μM) stimulated the release of corticotrophin (ir‐ACTH) from pituitary tissue in vitro (P&lt;0.05) but in lower concentrations they were without significant effect. In addition, rolipram (10 μM, P&lt;0.05), denbufylline (0.1 μM, P&lt;0.05) and BRL 61063 (10 μM, P&lt;0.05) potentiated the significant (P&lt;0.05) rises in ir‐ACTH secretion induced by forskolin (100 μM). Forskolin (100 μM) also produced a highly significant increase (P&lt;0.01) in the tissue cyclic AMP content which was further potentiated by rolipram (10 μM), denbufylline (10 μM) and BRL 61063 (10 μM) which, alone did not affect the cyclic AMP content of the tissue. Since both denbufylline and BRL 61063 possess significant adenosine A1 receptor blocking activity, further studies examined the potential influence of these receptors on the secretion in vitro of CRH‐41, AVP and ACTH. The release of ir‐CRH‐41 was increased significantly by adenosine deaminase (ADA, 5 u ml−1, P&lt;0.05) and the A1‐receptor antagonist, 1,3‐dicyclopropyl‐8‐cyclopentylxanthine (DPCPX, 0.1–10 nM, P&lt;0.05). The responses to ADA were abolished by the A1 receptor agonist N6‐cyclo‐hexyladenosine (CHA, 100 nM, P&lt;0.05) which alone had no significant effect on ir‐CRH‐41 release. ADA (0.1–10 u ml−1) and DPCPX (1 nM) had weak stimulant and inhibitory effects, respectively, on the release of ir‐ACTH from the pituitary gland while CHA (0.1–10 nM) was without effect. Ligand binding studies with [3H]‐DPCPX as a probe demonstrated the presence of specific high affinity A1 binding sites in the hypothalamus (Kd=0.7 nM; Bmax=367±32 fmol mg−1 protein) and in the hippocampus (Kd=1 nM; Bmax=1165±145 fmol mg−1 protein). In both tissues binding of the ligand was displaced by CHA (IC50=1 nM (hypothalamus) and 2 nM (hippocampus)), BRL 61063 (IC50=80 nM (hypothalamus) and 100 nM (hippocampus)) and denbufylline (IC50=5 μM (hypothalamus) and 9 μM (hippocampus)) but not by rolipram. The results suggest that rolipram, denblufylline and BRL 61063 stimulate the HPA axis in the rat, acting at the levels of both the hypothalamus and the pituitary gland. Their actions may be explained, at least in part, by inhibition of PDE‐4 but additional actions including blockade of hypothalamic adenosine A1 receptors by denbufylline and BRL 61063 cannot be excluded.

Vascular Smooth Muscle Relaxation by α-Adrenoceptor Blocking Action of Dobutamine in Isolated Rabbit Aorta

We investigated the mechanism of vascular relaxation produced by dobutamine, a positive inotropic agent with beta 1-adrenergic action. Dobutamine concentration-dependently (10 nM-10 microM) relaxed ring segments of rabbit aorta partially precontracted with 1 microM phenylephrine (PE) but did not relax those precontracted with 40 mM K+ or 5 microM prostaglandin F2 alpha (PGF2 alpha). The relaxation was not completely inhibited by pretreatment with 10 microM propranolol. Dobutamine did not significantly increase tissue cyclic AMP levels concomitantly with relaxation as does isoproterenol (ISO) in rabbit aorta. Dobutamine produced a parallel rightward shift in concentration-response curves to PE. The Schild plot analysis resulted in a linear regression of a slope of 1.077 +/- 0.077, which was not significantly different from unity. The pA2 value of dobutamine as compared with PE in rabbit aorta was 6.81 +/- 0.03. Dobutamine causes arterial dilatation mediated not only through a beta-adrenergic action but also through an alpha-adrenergic blocking action in rabbit aorta.

Regulation of phospholamban and troponin-I phosphorylation in the intact rat cardiomyocytes by adrenergic and cholinergic stimuli: roles of cyclic nucleotides, calcium, protein kinases and phosphatases and depolarization.

Protein phosphorylation was investigated in [32P]-labeled cardiomyocytes isolated from adult rat heart ventricles. The beta-adrenergic stimulation (by isoproterenol, ISO) increased the phosphorylation of inhibitory subunit of troponin (TN-I), C-protein and phospholamban (PLN). Such stimulation was largely mediated by increased adenylyl cyclase (AC) activity, increased myoplasmic cyclic AMP and increased cyclic AMP dependent protein kinase (A-kinase)-catalyzed phosphorylation of these proteins in view of the following observations: (a) dibutyryl-and bromo-derivatives of cyclic AMP mimicked the stimulatory effect of ISO on protein phosphorylation while (b) Rp-cyclic AMP was found to attenuate ISO-dependent stimulation. Unexpectedly, 8-bromo cyclic GMP was found to markedly increase TN-I and PLN phosphorylation. Both beta 1- and beta 2-adrenoceptors were present and ISO binding to either receptor was found to stimulate myocyte AC. However, the stimulation of the beta 2-AR only marginally increased while the stimulation of beta 1-AR markedly increased PLN phosphorylation. Other stimuli that increase tissue cyclic AMP levels also increased PLN and TN-I phosphorylation and these included isobutylmethylxanthine (non-specific phosphodiesterase inhibitor), milrinone (inhibits cardiotonic inhibitable phosphodiesterase, sometimes called type III or IV) and forskolin (which directly stimulates adenylyl cyclase). Cholinergic agonists acting on cardiomyocyte M2-muscarinic receptors that are coupled to AC via pertussis toxin(PT)-sensitive G proteins inhibited AC and attenuated ISO-dependent increases in PLN and TN-I phosphorylation. The in vivo PT treatment, which ADP-ribosylated Gi-like protein(s) in the myocytes, markedly attenuated muscarinic inhibitory effect on PLN and TN-I phosphorylation on one hand and, increased the beta-adrenergic stimulation, on the other. Controlled exposure of isolated myocytes to N-ethyl maleimide, also led to the findings similar to those seen following the PT treatment. Exposure of myocytes to phorbol, 12-myristate, 13-acetate (PMA) increased the protein phosphorylation, augmenting the stimulation by ISO, and such augmentation was antagonized by propranolol suggesting modulation of the beta-adrenoceptor coupled AC pathway by PMA. Okadaic acid (OA) exposure of myocytes also increased protein phosphorylation with the results supporting the roles for type 1 and 2A protein phosphatases in the dephosphorylation of PLN and TN-I. Interestingly OA treatment attenuated the muscarinic inhibitory effect which was restored by subsequent brief exposure of myocytes to PMA. While the stimulation of alpha adrenoceptors exerted little effect on the phosphorylation of PLN and TN-I, inactivation of alpha adrenoceptors by chloroethylclonidine (CEC), augmented beta-adrenergically stimulated phosphorylation. KCl-dependent depolarization of myocytes was observed to potentiate ISO-dependent increase in phosphorylation (incubation period 15 sec to 1 min) as well as to accelerate the time-dependent decline in this phosphorylation seen upon longer incubation. Verapamil decreased ISO-stimulated protein phosphorylation in the depolarized myocytes. Depolarization was found to have little effect on the muscarinic inhibitory action on phosphorylation. Prior treatment of myocytes with PMA, was found to augment ISO-stimulated protein phosphorylation in the depolarized myocytes. Such augmented increases were completely blocked by propranolol. Forskolin also stimulated PLN and TN-I phosphorylation. Prior exposure of myocytes to forskolin followed by incubation in the depolarized and polarized media showed that PLN was dephosphorylated more rapidly in the depolarized myocytes. The results support the view that both cyclic AMP and calcium signals cooperatively increase the rates of phosphorylation of TN-I and PLN in the depolarized cardiomyocytes during beta-adrenergic stimulation. (ABSTRACT TRUNCATED)

5-Hydroxytryptamine induces endothelium-independent relaxations of sheep pulmonary vein: role of cyclic nucleotide

5-Hydroxytryptamine produced concentration-dependent relaxations in isolated sheep pulmonary vein, which were insensitive to removal of the endothelium. 5-Hydroxytryptamine stimulated concentration-dependent increases of cyclic AMP levels in the pulmonary vein, and there was a significant linear correlation between relaxations elicited by 5-hydroxytryptamine and tissue cyclic AMP formations. The soluble guanylate cyclase inhibitor methylene blue (10 μM) failed to block 5-hydroxytryptamine-induced relaxations. The results suggest that 5-hydroxytryptamine-induced relaxations of sheep pulmonary vein are mediated, at least in part, by increases of tissue cyclic AMP levels.

Increased responsiveness of left ventricular apical myocardium to adrenergic stimuli.

The aim was to determine whether left ventricular apical myocardium has mechanisms to compensate for sparse sympathetic innervation. Contractile and metabolic responses to various adrenergic stimuli and beta adrenergic receptor density were compared between left ventricular basal and apical regions in 26 anaesthetised mongrel dogs, weight 12-28 kg. Regional contractile changes in response to graded cardiac sympathetic nerve stimulation were compared among three basal (anterior, middle, and posterior) regions, and between basal middle and apical regions. There were significant differences in contractile changes among the three basal regions with distinct regions of innervation from right and/or left sided sympathetic ganglia, but not between apical and basal regions. Constant infusion of noradrenaline (0.2-0.4 microgram.kg-1.min-1) produced a greater response in normalised end systolic length in the apical myocardium than in the basal region, at 9.86(SEM 0.06) mm v 10.14(0.04) mm (n = 5, p < 0.025), and a greater increase in tissue cyclic AMP: 1.04(0.20) v 0.60(0.08) pmol.mg-1 (n = 5, p < 0.05). Giving a forskolin derivative (30 micrograms.kg-1, n = 5) produced a greater increase in cyclic AMP in the apical region than in the basal region: 1.26(0.18) v 0.88(0.19) pmol.mg-1 (p < 0.02). beta Adrenergic receptor density in the apical region was greater than in the basal region: 455(45) v 341(35) fmol.mg-1 protein (n = 5, p < 0.05). Greater beta adrenergic receptor density and/or increased myocardial responsiveness to adenylate stimulation in apical myocardium compensates, at least in part, for its sparse sympathetic innervation.

Insulin Counters the Glycogenolytic Effect of Arginine Vasotocin in Liver Pieces from the Axolotl, Ambystoma mexicanum, Cultured in Vitro

In organ cultures of liver tissue from the axolotl, Ambystoma mexicanum, 1 nmol/l arginine vasotocin (AVT) increased tissue cyclic AMP (cAMP) concentration, activated glycogen phosphorylase, and caused glycogen breakdown and glucose release. Addition of 10 nmol/l insulin had no effect on any of these parameters. Addition of glucagon together with AVT caused a further increase in tissue cAMP but not in glucose release. Ten nanomoles per liter of insulin added to the cultures 5 min before 1 nmol/liter AVT inhibited all the above actions of AVT. This inhibitory action of insulin was not apparent in the presence of the cAMP phosphodiesterase inhibitor isobutylmethylxanthine (IBMX), which indicates that insulin activates cAMP phosphodiesterase and so reduces the concentration of cAMP in the tissue. This cannot occur in the presence of IBMX. These findings confirm previous reports that AVT causes hepatic glycogenolysis in the axolotl via an increase in tissue cAMP level.

Epidermal growth factor modulates cholera toxin induced mammary gland development.

Ovariectomized mice were either sham operated or sialoadenectomized and injected daily for 18 days with saline, estradiol + progesterone, cholera toxin or estradiol + progesterone+cholera toxin. Mammary development score and DNA were increased by estradiol + progesterone, but not by cholera toxin alone. In combination with estradiol + progesterone, cholera toxin increased mammary development score and mammary DNA. Sialoadenectomy reduced the ability of estradiol, progesterone and cholera toxin to induce mammary development. In other experiments, mice were primed with estradiol + progesterone for 10 days, and mammary tissue removed for in vitro culture with various combinations of insulin, aldosterone, cholera toxin and epidermal growth factor. In combination with insulin and aldosterone, cholera toxin increased mammary development in vitro. Sialoadenectomy reduced the ability of cholera toxin to induce mammary development in vitro. The effect of sialoadenectomy on mammary development was alleviated by adding epidermal growth factor to culture medium. Biochemical studies indicated that sialoadenectomy reduced the ability of estrogen and progesterone to induce cyclic AMP dependent protein kinase levels in mammary tissue, and also the ability of cholera toxin to induce accumulation of cyclic AMP in tissues. These effects of sialoadenectomy were reversed by addition of EGF to culture media.