Co-regulation of tracheal tone by cyclic AMP- and cyclic GMP-dependent mechanisms

The regulation of guinea pig tracheal muscle tone by cyclic AMP-dependent and cyclic GMP-dependent relaxant mechanisms was investigated by studying the tracheal relaxant activities of forskolin, nitroprusside, N6-2'-O-dibutyryl-cyclic AMP and 8-bromoguanosine-cyclic GMP. In carbachol (3 X 10(-6) M)-contracted isolated tracheal rings, N6-2'-O-dibutyryl-cyclic AMP and 8-bromoguanosine-cyclic GMP each caused biphasic relaxation responses, which consisted of an acute relaxation followed by a sustained but lesser degree of relaxation. The biphasic nature of this response is suggested to result from a functional counter-balancing of cyclic nucleotide-dependent relaxant mechanisms and the contractile mechanisms stimulated by carbachol. The sensitivity of carbachol-contracted tracheal rings to forskolin and nitroprusside (activators of adenylate and guanylate cyclase, respectively) was generally not influenced by N6-2'-O-dibutyryl-cyclic AMP or 8-bromoguanosine-cyclic GMP in concentrations that induced up to 50% relaxation of the trachea. Furthermore, the partial relaxation of tracheal tension with one cyclic nucleotide analog did not alter the sensitivity of the tracheal rings to the other. These results demonstrate that cyclic AMP- and cyclic GMP-dependent mechanisms induce relaxations of the trachea that are functionally additive, each neither potentiating nor depressing the effects of the other. In the presence of 3 X 10(-6) M carbachol, the effectiveness of cyclic AMP- and cyclic GMP-dependent relaxant mechanisms appears to be fixed, and independent of the amount of active tension being maintained by the tracheal muscle itself.     

Inhibition of adenylate cyclase attenuates adenosine receptor-mediated relaxation in coronary artery.

This study was designed to evaluate whether the adenylate cyclase inhibitor 2',5'-dideoxyadenosine (DDA) would attenuate the relaxation produced by adenosine analogs in order to provide functional evidence in support of the working hypothesis that adenosine receptor-mediated relaxation of coronary artery involves adenylate cyclase. Rings from porcine left anterior descending coronary artery were mounted in organ chambers for measurement of isometric force. Rings contracted with KCl (30 mM) relaxed in a concentration-dependent manner to 2-chloroadenosine (CAD), 5'-N-ethylcarboxamidoadenosine (NECA), isoproterenol, sodium nitroprusside (SNP) and forskolin. Treatment of coronary rings with DDA (50 microM) significantly attenuated the relaxation produced by CAD, NECA, forskolin and isoproterenol, but had no effect on the relaxation response to SNP. The nucleoside transport inhibitor dilazep (10 microM) completely reversed the inhibitory effect of DDA on the relaxation produced by forskolin and CAD, whereas dilazep only partially reversed the DDA inhibition of NECA-induced relaxation. In a membrane preparation from porcine coronary artery CAD, but not NECA, increased cyclic AMP production in a GTP-dependent manner. DDA significantly decreased basal cyclic AMP production and also decreased CAD-, forskolin-, GTP- and NaF-stimulated cyclic AMP production. These results provide functional and biochemical evidence in support of the working hypothesis that adenosine receptor-mediated coronary relaxation involves adenylate cyclase. Furthermore, the results from this study suggest that the signaling mechanisms responsible for adenosine receptor-mediated coronary relaxation are more complicated than a single receptor coupled with adenylate cyclase because 1) dilazep completely reversed the inhibitory effect of DDA on the CAD relaxation but not the NECA relaxation, and 2) NECA did not increase cyclic AMP production.     

Inhibitory effect of methacholine on drug-induced relaxation, cyclic AMP accumulation, and cyclic AMP-dependent protein kinase activation in canine tracheal smooth muscle

Functional antagonism between bronchoconstricting and bronchodilating pathways was examined in canine tracheal smooth muscle. Trachealis strips were contracted with either 0.3 microM (EC55) or 3.0 microM (EC80) methacholine before being relaxed by the cumulative addition of isoproterenol, prostaglandin E2, or forskolin. The EC50 for all three relaxants was increased 10-fold in tissues contracted with 3.0 microM methacholine vs. those contracted with 0.3 microM methacholine. Moreover, contracting tissues with the higher concentration of methacholine reduced the maximum relaxation induced by prostaglandin E2 and isoproterenol. Forskolin produced total relaxation regardless of the concentration of methacholine used and thus was a much more effective bronchodilator than either isoproterenol or prostaglandin E2. The inhibitory effect of methacholine on the relaxant response to these agents was paralleled by a reduction in drug-stimulated cyclic AMP-dependent protein kinase activity. Methacholine reduced the maximum activation of cyclic AMP-dependent protein kinase elicited by isoproterenol, prostaglandin E2 and submaximal concentrations of forskolin, which was a much more powerful enzyme activator than the other two agents. The ability of a maximum concentration of forskolin (30 microM) to activate cyclic AMP-dependent protein kinase was not inhibited by methacholine. Although methacholine also appeared to suppress drug-stimulated cyclic AMP accumulation, the inhibitory effect was only statistically significant in forskolin-treated tissues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacology and inotropic potential of forskolin in the human heart

We evaluated the effects of the diterpene compound forskolin in human myocardial adenylate cyclase preparations, isolated trabeculae and papillary muscles derived from failing human hearts, and acutely instrumented dogs. Forskolin was a potent, powerful activator of human myocardial adenylate cyclase and produced maximal effects that were 4.82 (normally functioning left ventricle) and 6.13 (failing left ventricle) fold greater than isoproterenol. In contrast to isoproterenol, forskolin retained full activity in membrane preparations derived from failing hearts. In cyclase preparations, forskolin demonstrated unique substrate and Mg2+ kinetic properties that could be distinguished from hormone receptor-coupled agonists or fluoride ion. The adenylate cyclase stimulatory effect of forskolin was synergistic with isoproterenol, apparently due to the location of forskolin activation being beyond the level of hormone receptor-agonist in the receptor-cyclase complex. Forskolin was a potent positive inotrope in failing human myocardium, producing a stimulation of contraction that was similar to isoproterenol. Finally, in open chest dogs forskolin was a positive inotropic agent that reduced preload and afterload. We conclude that forskolin belongs to a class of agents that may have therapeutic potential in the treatment of congestive heart failure.     

Involvement of endothelial cells in relaxation and contraction responses of the aorta to isoproterenol in naive and streptozotocin-induced diabetic rats

Experiments were designed to investigate the importance of the endothelium in the relaxation of isolated rat aorta caused by a beta adrenoceptor agonist. Mechanical removal of the endothelium attenuated the relaxation induced by isoproterenol (ISO) and did not affect the relaxation produced by forskolin and by sodium nitroprusside. High concentrations of ISO produced an increase in the resting tension of aortic strips with and without endothelium in a concentration-dependent manner. Mechanical removal of the endothelium or treatment with methylene blue enhanced the maximal contraction induced by ISO. Phentolamine antagonized the contractile responses induced by ISO. In the case of streptozotocin-induced diabetic rats, both aortic strips with and without endothelium generated concentration-response curves for ISO-induced relaxation that were shifted to the right. The relaxant responses to forskolin and sodium nitroprusside were not significantly different between vessels from diabetic and age-matched control rats. In both aortic strips with and without endothelium, the maximal contraction in response to high concentrations of ISO was significantly enhanced in strips from diabetic rats. These results suggest that ISO-induced relaxation of aortic strips with endothelium is mediated by beta adrenoceptors on both the endothelium and the smooth muscle, and high concentrations of ISO produce an increase in the resting tension through alpha adrenoceptors. It is further suggested that the decreased relaxant response of the aorta to ISO in diabetes may be due to decreased density or affinity of beta adrenoceptors on the smooth muscle.     

Influence of stimulators and inhibitors of cyclic nucleotides on lower esophageal sphincter

These studies were performed in vitro to investigate the nature of the second messenger for lower esophageal sphincter (LES) smooth muscle relaxation in response to electrical field stimulation (EFS) and vasoactive intestinal polypeptide (VIP). It was seen that VIP, permeant derivatives of the cyclic nucleotide 8-bromo cyclic GMP (BrcGMP) and 8-bromo cyclic AMP (8-BrcAMP), the guanylate cyclase stimulant sodium nitroprusside (SNP), the adenylate cyclase stimulant forskolin, M&B 22,948 (cGMP phosphodiesterase inhibitor) and SK&F 94,120 (cAMP phosphodiesterase inhibitor) caused dose-dependent and tetrocotoxin resistant fall in LES tension. Guanylate cyclase inhibitor methylene blue (MB) (3 x 10(-5) M), caused significant antagonism of fall in LES tension by SNP without modifying the inhibitory response of forskolin. The possible adenylate cyclase inhibitor N-ethylmaleimide (NEM) (1 x 10(-4) M), on the other hand, caused significant antagonism of fall in LES tension by forskolin without any effect on that caused by SNP. The inhibitory responses of 8-BrcGMP and 8-BrcAMP were not modified by MB or NEM. NEM (1 x 10(-4) M) and MB (3 x 10(-5) M) caused significant inhibition of the fall in LES tension with EFS. NEM also caused inhibition of fall in LES tension by VIP. Furthermore, SK&F 94,120 and not M&B 22,948 caused significant potentiation of fall in LES tension by EFS. From these results we conclude that: 1) cAMP and cGMP may act as second messengers for LES relaxation with EFS and VIP, and 2) VIP may act primarily via cAMP system and remains a strong possibility for one of the inhibitory neurotransmitters in the LES.     

Protein kinase A-dependent and -independent effects of isoproterenol in rat isolated mesenteric artery: interactions with levcromakalim

The effect of beta-adrenoceptor activation on levcromakalim-induced relaxation was investigated in myograph-mounted rat mesenteric arteries. The nonselective beta-adrenoceptor agonist isoproterenol (at a concentration causing approximately 30% relaxation of methoxamine-induced tone) potentiated relaxation to levcromakalim; higher concentrations exerted no additional effect. The modulatory and relaxant effects of isoproterenol were inhibited by the beta(1)-adrenoceptor antagonist atenolol, but the ATP-sensitive K(+) (K(ATP)) channel inhibitor glibenclamide did not inhibit relaxations to isoproterenol. The protein kinase A inhibitor Rp-adenosine 3',5'-cyclic monophosphothioate triethylamine (Rp-cAMPS) inhibited the ability of isoproterenol to modulate levcromakalim relaxation. However, neither Rp-cAMPS nor N-[2-(p-bromocinnamylamino)ethyl]-6-isoquinolinesulfonamide (H-89) (another protein kinase A inhibitor) markedly reduced isoproterenol-induced relaxation, although Rp-cAMPS inhibited relaxations induced by forskolin (an adenylyl cyclase activator). Iberiotoxin (50 nM), an inhibitor of large conductance Ca(2+)-activated K(+) channels (BK(Ca)), attenuated isoproterenol relaxation. Moreover, both Rp-cAMPS and H-89 caused inhibition of the effects of isoproterenol in the presence of iberiotoxin, whereas glibenclamide did not. We conclude that isoproterenol modulates the actions of levcromakalim through beta(1)-adrenoceptors and protein kinase A, even though K(ATP) channels do not contribute to its relaxant effects. However, the major relaxant mechanism for isoproterenol appears to be protein kinase A-independent activation of BK(Ca), with cyclic AMP-dependent mechanisms only being unmasked when the BK(Ca) mechanism is inhibited. Although direct G protein-mediated activation of BK(Ca) has been demonstrated previously in electrophysiological studies of single smooth muscle cells, this is the first time that such a mechanism has been shown to be functionally important in an intact blood vessel preparation.     

Smooth muscle pharmacology in the isolated virgin and pregnant rat uterus and cervix

Uterine smooth muscle function is established, but comparatively little is known about cervical smooth muscle pharmacology. We performed a proof-of-principle experiment that smooth muscle was expressed in the cervix in both virgin and pregnant rats, using the uterus as a comparator. We tested whether all tissues were pharmacologically responsive to contractile and relaxant agonists. Immunohistochemistry revealed the expression of smooth muscle α-actin in all tissues. The isolated tissue bath was used to measure isometric contractility of uterine strips and whole cervices from virgin and pregnant (day 11 ± 2) female Sprague-Dawley rats. We tested classic activators of uterine smooth muscle contraction and relaxation in both uterus and cervix. All tissues contracted to the depolarizing agent potassium chloride, prostaglandin F2α, muscarinic cholinergic agonist carbachol [2-[(aminocarbonxyl)oxy]-N,N,N-trimethylethanaminium chloride], and 5-hydroxytryptamine. Unlike other tissues, the pregnant cervix did not contract to oxytocin, but the oxytocin receptor was present. Both cervix and uterus (virgin and pregnant) had concentration-dependent, near-complete relaxation to the adrenergic agonist norepinephrine and adenylate cyclase activator forskolin [(3R,4aR,5S,6S,6aS,10S,10aR,10bS)-6,10-10b-trihydroxy-3,4a,7,10a-pentamethyl-1-oxo-3-vinyldodecahydro-1H-benzo[f] chroment-5-yl acetate]. The β-adrenergic receptor agonist isoproterenol was less potent in pregnant cervix versus virgin by ～10-fold. All tissues, particularly the cervix, responded poorly to the nitric-oxide donor sodium nitroprusside, relaxing ～20% maximally. These findings support the importance of smooth muscle in the cervix, the use of the isolated cervix in pharmacological studies, and a similarity between smooth muscle pharmacology of the nonpregnant uterus and cervix. This work highlights the unappreciated smooth muscle function of the cervix versus uterus and cervical changes in pharmacology during pregnancy.     

The nicardipine-isoprenaline interaction in human and guinea-pig isolated airways

Summary— The effects of the dihydropyridine calcium antagonist nicardipine on the concentration-response curves of relaxant compounds acting through the adenylate-cyclase/cAMP system (isoprenaline, forskolin, adenosine and theophylline) or by the cGMP pathway (sodium nitroprusside) were studied on human isolated bronchus and guinea-pig isolated trachea. These effects were compared with those of nifedipine (a dihydropyridine derivative) and theophylline (a non-selective phosphodiesterase inhibitor). Nicardipine, in the range of 0.01 to 1 μM, significantly potentiated the relaxant effects of isoprenaline, forskolin, adenosine and theophylline, whereas the effects of sodium nitroprusside were significantly potentiated at 10 μM only. These results suggest that nicardipine behaves as an inhibitor of phosphodiesterases III and IV. One such effect may be involved in the potentiation of the isoprenaline relaxation of human and guinea-pig isolated airways.     

M2 muscarinic receptors inhibit isoproterenol-induced relaxation of canine airway smooth muscle.

Classification of muscarinic receptors in the central airways has revealed the coexistence of M2 and M3 muscarinic receptors in this tissue, with the M2 subtype being predominant. Although M3 muscarinic receptors have been linked to airway smooth muscle contraction, a functional role for the M2 subtype in this tissue has been unclear. In nonairway smooth muscle, stimulation of the M2 muscarinic receptor has been shown to be associated with inhibition of adenylyl cyclase. In the present study, characterization of muscarinic receptors in canine tracheal smooth muscle confirmed that the majority of these muscarinic receptors were of the M2 subtype (89 +/- 3%), with a minor population of M3 receptors (11 +/- 3%). In functional studies, both isoproterenol and forskolin cause a dose-dependent relaxation of precontracted airway smooth muscle. In tissues precontracted with methacholine, 11-([[2-(diethylamino)methyl]-1-piperidinyl]acetyl)5,11- dihydro-6H-pyrido[2,3-6][1,4]benzodiazepine-6-one (AF-DX 116), a selective M2 antagonist, shifted dose-response curves to both isoproterenol and forskolin significantly to the left. In contrast, AF-DX 116 did not alter relaxation induced by the K+ channel opener BRL 38227. Furthermore, the ability of AF-DX 116 to enhance isoproterenol-induced relaxation appears to be limited to smooth muscle precontracted with muscarinic agonists because AF-DX 116 had no effect on isoproterenol dose-response curves in muscle strips precontracted with histamine. Hexahydrosiladifenidol (HHSiD), a selective antagonist for M3 receptors, did not shift the isoproterenol dose-response curve in muscle precontracted with methacholine. This study demonstrates that stimulation of M2 muscarinic receptors in canine airway smooth muscle plays an important role in functional antagonism by reducing the relaxation caused by agents such as isoproterenol and forskolin.     

Age-related changes in relaxant response of vascular smooth muscles to atrial natriuretic peptide

The effect of aging on responses of vascular smooth muscles to atrial natriuretic peptide (ANP) and other vasodilator substances was investigated in isolated rat aortae, rat renal arteries and monkey renal arteries which were precontracted with norepinephrine. There was no significant difference in the ANP-induced maximum relaxation between young and old rat aortae. However, the concentration of agonists causing a 50% relaxation (ED50) value for the old rats was 7.3 times greater than that for the young ones. In rat and monkey renal arteries, the ED50 ratios were 6.2 and 3.8, respectively. The relaxant responses of the rat aortae to isoproterenol and acetylcholine also decreased with increasing age. The ED50 ratios for isoproterenol and acetylcholine were more than 40 and 17, respectively. The maximum relaxation induced by 10(-5) M isoproterenol also decreased significantly in the aortae from the older rats. On the other hand, the ED50 for nitroprusside, nifedipine- and potassium-induced relaxation was not affected by increasing age. These results suggest that ANP-induced relaxation of vascular smooth muscles is reduced with increasing age in rat aortae, rat renal arteries and monkey renal arteries. The mechanisms by which the ANP-induced relaxation decreased in association with the aging process may be quite different from those in acetylcholine-induced and beta adrenoceptor-induced relaxation.     

Phorbol ester modulation of cyclic AMP accumulation in a primary culture of rat aortic smooth muscle cells

Over the past few years, the importance of calcium and cyclic AMP in the regulation of vascular smooth muscle tone has been well documented. We used a primary culture of rat aortic myocytes to study the effect of protein kinase C on isoproterenol- and forskolin-stimulated cyclic AMP production. Addition of the protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA) to these cells, but not an inactive analog, increased the stimulation of cyclic AMP production induced with isoproterenol or forskolin without changes in the apparent affinity of these compounds but did not affect the basal cAMP level. TPA also enhanced the cholera toxin-stimulated cyclic AMP accumulation. Isoproterenol and cholera toxin increased the forskolin apparent potency suggesting that interaction of activatory GTP-dependent protein with the catalytic subunit of adenylate cyclase facilitates forskolin interaction to the catalytic subunit. Treatment of myocytes with pertussis toxin had no effect on the basal level of cyclic AMP production and did not significantly modify isoproterenol- and forskolin-induced stimulation. Pertussis toxin treatment of cells did not affect the TPA-enhanced isoproterenol or forskolin stimulations suggesting that pertussis toxin and TPA actions would not share a common target of myocyte adenylate cyclase system. Our data would be in agreement with a possible direct interaction of protein kinase C with the catalytic subunit of adenylate cyclase system.     

The time course of changes in force and cyclic AMP levels produced by isoproterenol and forskolin in isolated rabbit detrusor muscle

The effects of forskolin and isoproterenol on contractile force and cyclic AMP (cAMP) levels were compared in rabbit detrusor. Both forskolin and isoproterenol produced relaxation of rabbit detrusor and an increase in cAMP levels in the tissue. Although the relaxant response to forskolin was similar to that of isoproterenol, the increase in cAMP levels induced by forskolin was significantly larger than that induced by isoproterenol. These results suggest that there is a discrepancy in the relaxation responses and cAMP levels in response to forskolin and isoproterenol.     

Bronchodilator mechanisms in bullfrog lung: differences in response to isoproterenol, theophylline and papaverine

The effects of bronchodilators and smooth muscle relaxants on mechanical responses and lung cyclic nucleotide levels in the isolated hemilung of Rana catesbeiana demonstrate striking differences in intensity and time course of drug action in an unstimulated preparation of airway smooth muscle. Isoproterenol, nitroprusside and nitroglycerin elicit a fast onset relaxation (minutes) with ceiling effects at 20, 22 and 43%, respectively, of maximal relaxation. Theophylline, dibutyryl cyclic AMP and papaverine produce maximal or near maximal relaxation, but require 8 to 32 hr for peak effect. Papaverine-induced relaxation is accompanied by a slow increase in lung cyclic AMP and cyclic GMP and is markedly accelerated by isoproterenol. Theophylline (10(-3) M) produces no change in cyclic nucleotide levels and its relaxant effect is not accelerated by isoproterenol. The hierarchy of relaxant responses suggests drug action at discrete loci in a highly compartmentalized effector chain, with cyclic AMP-dependent mechanisms separable into at least two components. The first is activated by isoproterenol and elicits a rapid, limited response, presumably reflecting an increase in cyclic AMP in a relatively restricted pool. The second is activated by papaverine and elicits a very slow, but complete relaxation, presumably reflecting a more pervasive or diffuse accumulation of cyclic AMP secondary to phosphodiesterase inhibition. The major portion of theophylline-induced relaxation in this preparation appears to be independent of changes in cyclic nucleotide metabolism.     

Mechanism of cytokine-induced modulation of beta-adrenoceptor responsiveness in airway smooth muscle.

To elucidate the role of specific proinflammatory cytokines in regulating airway responsiveness, we examined the effects and mechanisms of action of IL-1beta, TNF-alpha, and IL-2 on the beta-adrenoceptor- and postreceptor-coupled transmembrane signaling mechanisms regulating relaxation in isolated rabbit tracheal smooth muscle (TSM) segments. During half-maximal isometric contraction of the tissues with acetylcholine, relaxation responses to isoproterenol, PGE2, and forskolin were separately compared in control (untreated) TSM and tissues incubated for 18 h with IL-1beta (10 ng/ml), TNF-(alpha (100 ng/ml), or IL-2 (200 ng/ml). Relative to controls, IL-1beta- and TNF-alpha-treated TSM, but not IL-2-treated tissues, depicted significant attenuation of their maximal relaxation and sensitivity (i.e., -log dose producing 50% maximal relaxation) to isoproterenol (P < 0.001) and PGE2 (P < 0.05); whereas the relaxation responses to direct stimulation of adenylate cyclase with forskolin were similar in the control and cytokine-treated tissues. Further, the attenuated relaxation to isoproterenol and PGE2 was ablated in the IL-1beta-treated TSM that were pretreated with either the muscarinic M2-receptor antagonist, methoctramine (10(-6) M), or pertussis toxin (100 ng/ml). Moreover, Western immunoblot analysis demonstrated that: (a) Gi protein expression was significantly enhanced in membrane fractions isolated from IL-1beta-treated TSM; and (b) the latter was largely attributed to induced enhanced expression of the Gi alpha2 and Gi alpha3 subunits. Collectively, these observations provide new evidence demonstrating that IL-lbeta and TNF-alpha induce impaired receptor-coupled airway relaxation in naive TSM, and that the latter effect is associated with increased muscarinic M2-receptor/Gi protein-coupled expression and function.     

Pharmacological characterization of muscarinic receptors mediating inhibition of adenylate cyclase activity in the rat retina

Incubation of the rat retina with acetylcholine resulted in about a 20 to 30% decrease of basal cyclic AMP accumulation. Oxotremorine, arecoline, [4-hydroxy-2-butynyl]trimethylammonium chloride, m-chlorocarbanilate and carbachol also inhibited cyclic AMP accumulation. Nicotine had no effect. The response was blocked by atropine and pirenzepine but not gallamine. Intraocular injection of pertussis toxin 72 hr before testing also blocked the response to acetylcholine. The presence of forskolin exaggerated the response to acetylcholine. Intraocular injection of the cholinotoxin AF64A resulted in apparent supersensitivity of the response to acetylcholine. Our results suggest that rat retina contains muscarinic M1 receptors that are coupled negatively to adenylate cyclase.     

Mechanisms of impaired beta-adrenoceptor-induced airway relaxation by interleukin-1beta in vivo in the rat.

We studied the in vivo mechanism of beta-adrenergic receptor (beta-AR) hyporesponsiveness induced by intratracheal instillation of interleukin-1beta (IL-1beta, 500 U) in Brown-Norway rats. Tracheal and bronchial smooth muscle responses were measured under isometric conditions ex vivo. Contractile responses to electrical field stimulation and to carbachol were not altered, but maximal relaxation induced by isoproterenol (10(-6)-10(-5) M) was significantly reduced 24 h after IL-1beta treatment in tracheal tissues and to a lesser extent, in the main bronchi. Radioligand binding using [125I]iodocyanopindolol revealed a 32+/-7% reduction in beta-ARs in lung tissues from IL-1beta-treated rats, without any significant changes in beta2-AR mRNA level measured by Northern blot analysis. Autoradiographic studies also showed significant reduction in beta2-AR in the airways. Isoproterenol-stimulated cyclic AMP accumulation was reduced by IL-1beta at 24 h in trachea and lung tissues. Pertussis toxin reversed this hyporesponsiveness to isoproterenol but not to forskolin in lung tissues. Western blot analysis revealed an IL-1beta-induced increase in Gi(alpha) protein expression. Thus, IL-1beta induces an attenuation of beta-AR-induced airway relaxation through mechanisms involving a reduction in beta-ARs, an increase in Gi(alpha) subunit, and a defect in adenylyl cyclase activity.     

Relaxation of vascular smooth muscle by isoproterenol, dibutyryl-cyclic AMP and theophylline

There is evidence that the relaxation of vascular smooth muscle produced by isoproterenol or cyclic AMP is mediated by membrane hyperpolarization. The current study investigates the possibility that this hyperpolarization, and hence the relaxation, may be produced by activation of the electrogenic sodium pump. Rat and pig tail artery strips were placed in a 1.0-mM potassium solution for 15 min. This procedure results in a decrease in the activity of the sodium pump. The strips were then made to contract in response to norepinephrine. Two minutes later, the concentration of potassium was increased to 6.0 mM and a relaxation occurred. The amplitude of this relaxation reflects the activity of the sodium pump. Either isoproterenol or dibutyryl-cyclic AMP causes an enhancement (time or degree) of potassium-induced relaxation. Theophylline potentiated potassium-induced relaxation in pig arteries but not in rat arteries. The relaxant action of isoproterenol on 1.0 mM barium contractures of rat arteries was inhibited by treatment with ouabain or with potassium-free solution. Ouabain inhibited the relaxant action of isoproterenol in pig arteries contracted with depolarizing potassium solution but not in rat tail arteries. Dibutyryl-cyclic AMP, theophylline and nitroprusside caused relaxation of serotonin-induced contractions; however, in rat arteries these responses were not inhibited by ouabain or by the absence of potassium. Similar studies on tail arteries from baboons, dogs, pigs and cats showed that relaxation by dibutyryl-cyclic AMP or by theophylline had some dependency on the activity of the sodium pump. These observations are consistent with the following conclusions: 1) isoproterenol and cyclic AMP potentiate the electrogenic pumping of sodium and potassium responsible for potassium-induced relaxation; 2) the relaxing action of isoproterenol, dibutyryl-cyclic AMP and theophylline are dependent upon experimental conditions and the species from which the vascular tissue is obtained; and 3) there is a component of isoproterenol- and cyclic AMP-induced relaxation which is not altered by inhibition of the electrogenic sodium pump in the rat.     

Evidence that parathyroid hormone-mediated calcium transport in rat brain synaptosomes is independent of cyclic adenosine monophosphate.

In vivo PTH administration to rats resulted in increased brain synaptosomal Ca++ transport, while parathyroidectomy (PTX) resulted in decreased transport. To determine the mechanism of action of PTH on Ca++ transport in rat brain synaptosomes, we performed transport studies by the Na-Ca exchanger and also measured cAMP generation in synaptosomes from PTX rats. Ca++ transport was studied after in vivo additions of either bovine (b)PTH, cAMP, or forskolin, and adenylate cyclase activity was assessed after additions of either bPTH, forskolin, sodium fluoride (NaF), or isoproterenol. In the presence of 1-34 bPTH [10(-7) M], Ca++ uptake was significantly increased by 55% (P less than 0.001) above control, while 3-34 bPTH [10(-7) M] had no effect on uptake. Both 8br,cAMP [10(-6) M] and dibut,cAMP [10(-6) M] also significantly increased (P less than 0.001) Ca++ uptake above control by 63 and 44%, respectively. Similarly, forskolin [10(-5) M], the adenylate cyclase activator, increased Ca++ uptake by 41%. We next evaluated Ca++ efflux, and found that 1-34 bPTH [10(-7) M], 1-84 bPTH [10(-7) M], and forskolin [10(-5) M] also increased Ca++ efflux by 50, 73, and 120%, respectively, above control. Since Ca++ transport was increased by either PTH, cAMP, or forskolin, we decided to determine if PTH action on Ca++ transport in synaptosomes was dependent on cAMP. This was investigated by measuring cAMP production during the conversion of 32P-ATP to 32P-cAMP in the presence of an ATP regenerating system (30 micrograms creatine phosphokinase, 10 mM creatine phosphate), and the cyclic nucleotide phosphodiesterase inhibitor (1 mM IBMX). Whereas forskolin [10(-4) M] and NaF [100 mM] significantly increased (P less than 0.001) adenylate cyclase activity in synaptosomes by eight- and fourfold, respectively, neither 1-34 bPTH nor 1-84 bPTH increased synaptosomal cyclase activity. However, in canine renal cortical plasma membranes (CRCPM), we observed significant increases in cAMP production with either forskolin, NaF, or PTH. Finally, to determine if synaptosomes contain an intact adenylate cyclase system, we measured cAMP production in the presence of the beta adrenergic agent, isoproterenol. Isoproterenol significantly increased adenylate cyclase activity in both synaptosomes (90%) and CRCPM (50%). These data suggest that although there is an intact adenylate cyclase system in rat brain synaptosomes, PTH-stimulated calcium transport in synaptosomes appears to be independent of this system.     

Dissociation between muscarinic receptor-mediated inhibition of adenylate cyclase and autoreceptor inhibition of [3H] acetylcholine release in rat hippocampus

Activation of muscarinic cholinergic receptors (mAChRs) in the central nervous system reduces the catalytic activity of membrane-bound adenylate cyclase and attenuates depolarization-dependent release of acetylcholine (ACh). Inasmuch as reports have indicated that these mAChR-mediated responses exhibit pharmacological profiles similar to the M2 subclass of mAChR, the present studies were undertaken to ascertain whether attenuation of presynaptic adenylate cyclase activity [and concurrent reduction of intraneuronal cyclic AMP (cAMP) levels] underlies mAChR-mediated autoinhibition of electrically evoked ACh release. In [3H]choline-prelabeled rat hippocampal slices, the mAChR agonists oxotremorine (EC50 = 15 microM) and carbachol (EC50 = 80 microM) caused atropine-reversible inhibition of [3H]ACh release up to a maximum of 80% reduction. The rank order of potency for antagonist reversal of this inhibitory action (N-methylatropine = atropine greater than scopolamine much greater than pirenzepine) was generally consistent with an M2 mAChR-mediated response although pirenzepine was ineffective up to 1 mM. Under these assay conditions, forskolin (1-10 microM) and 8-bromo-cAMP (30-300 microM) enhanced electrically evoked [3H]ACh release maximally by 50 to 60%; however, neither agent significantly reversed mAChR agonist-induced inhibition of [3H]ACh release. Additional studies were undertaken to determine the consequences of chemically uncoupling mAChR from their G protein-adenylate cyclase effector system in this tissue. Whereas brief pretreatment with the sulfhydryl alkylating agent N-ethylmaleimide (30 microM) or pertussis toxin (1 microgram/ml) markedly attenuated carbachol inhibition of adenylate cyclase activity in hippocampal tissue, there was no concurrent reduction of carbachol-inhibited [3H] ACh release.(ABSTRACT TRUNCATED AT 250 WORDS)