Inhibition of the low km cyclic AMP phosphodiesterase in intact canine trachealis by SK&F 94836: mechanical and biochemical responses

The mechanical and biochemical responses of the canine trachealis to SK&F 94836 [2-cyano-1-methyl-3-[4-(4-methyl-6-oxo- 1,4,5,6-tetrahydropyridazine-3-yl)phenyl]guanidine], a selective inhibitor (ki = 1-3 microM) of the low km cyclic AMP (cAMP) phosphodiesterase, were assessed. Time course studies indicated that SK&F 94836-induced relaxation of trachealis strips contracted with 0.1 microM methacholine was accompanied by an activation of cAMP-dependent protein kinase (cAMP-PK). In subsequent experiments, trachealis strips were contracted with three concentrations of methacholine (0.1, 1.0 or 3.0 microM) or two concentrations of histamine (10 or 300 microM) before being relaxed by the cumulative addition of SK&F 94836. The relaxant response to SK&F 94836 (EC50 = 1-10 microM) decreased progressively as tissues were contracted with higher concentrations of methacholine. In parallel with its inhibitory effect on SK&F 94836-induced relaxation, methacholine suppressed the ability of SK&F 94836 to activate cAMP-PK. Interestingly, the inhibition of cAMP-PK activity was not accompanied by a significant inhibition of SK&F 94836-stimulated cAMP accumulation. Unlike the results with methacholine, the concentration of histamine used to contract tissues had no effect on SK&F 94836-induced relaxation or cAMP-PK activation. To determine the effect of SK&F 94836 on the mechanical and biochemical responses to the beta adrenoceptor agonist isoproterenol, tissues were first contracted with 3.0 microM methacholine and then incubated with 0, 0.3, 3.0 or 30 microM SK&F 94836 before being relaxed by the cumulative addition of isoproterenol. In these experiments, SK&F 94836 potentiated isoproterenol-induced relaxation, cAMP accumulation and cAMP-PK activation in a concentration-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional antagonism in canine tracheal smooth muscle: inhibition by methacholine of the mechanical and biochemical responses to isoproterenol

The biochemical basis for the functional interaction between bronchoconstricting and bronchodilating pathways was investigated. Contracting canine trachealis strips with increasing concentrations of methacholine resulted in a progressive shift to the right of isoproterenol concentration-response curves. Thus, the EC50 for the relaxant response to isoproterenol was nearly 500-fold higher in preparations exposed to 3.0 microM methacholine than in tissues exposed to 0.03 microM methacholine. The maximum relaxation produced by isoproterenol was also dependent upon the initial muscarinic cholinergic tone. For example, isoproterenol reversed completely the contraction induced by 0.03 microM methacholine but did not relax trachealis strips contracted with 30 microM methacholine. To identify the molecular mechanism responsible for this functional antagonism, experiments were conducted to determine the effect of methacholine on isoproterenol-stimulated cyclic AMP accumulation and cyclic AMP-dependent protein kinase activation. Methacholine did not alter basal cyclic AMP content but did reduce cyclic AMP accumulation in response to isoproterenol. Furthermore, the ability of isoproterenol to activate cyclic AMP-dependent protein kinase was inhibited by methacholine in a concentration-dependent manner. This inhibition paralleled the decrease in mechanical responsiveness to isoproterenol. These results suggest that muscarinic cholinergic stimulation of canine tracheal smooth muscle functionally antagonizes the relaxant responses to beta adrenergic agonists and that a portion of this antagonism may be due to a suppression of catecholamine-stimulated cyclic AMP accumulation and cyclic AMP-dependent protein kinase activation.     

Differential effects of methacholine and leukotriene D4 on cyclic nucleotide content and isoproterenol-induced relaxation in the opossum trachea

The effects of leukotriene D4 and methacholine on cyclic nucleotide content and isoproterenol-induced relaxation were examined in the isolated opossum trachea. Although leukotriene D4 (-log EC50 = 6.70) was a more potent contractile agent than methacholine (-log EC50 = 5.78), the maximal response to leukotriene D4 was only 65% of the maximum response to methacholine. Contraction of tracheal strips with leukotriene D4 was accompanied by a 3-fold increase in cyclic GMP accumulation. Methacholine-induced contraction was not associated with an increase in cyclic GMP. Neither agent altered basal cyclic AMP content. Additional experiments were carried out to examine functional inhibitory interactions between bronchoconstricting and bronchodilating pathways. In these studies, cumulative isoproterenol concentration-response curves were constructed in tracheal strips contracted with three different concentrations of methacholine and in tissues contracted with three corresponding equieffective concentrations of leukotriene D4. Although the relaxant response to isoproterenol decreased as tissues were contracted with higher concentrations of either agent, the inhibitory effect of methacholine on isoproterenol-induced relaxation was much greater than the inhibitory effect of leukotriene D4. Previous studies from our laboratory suggested that a potential explanation for the greater inhibitory effect of methacholine on the mechanical response to isoproterenol was that methacholine may inhibit isoproterenol-stimulated cyclic AMP accumulation whereas leukotriene D4 may not. However, neither methacholine nor leukotriene D4 inhibited isoproterenol-stimulated cyclic AMP accumulation in the opossum trachea. The results of this study indicate that the sensitivity of airway smooth muscle to beta adrenoceptor agonists is influenced both by the initial contractile state of the tissue and by the type of agent used to induce tone.     

A re-evaluated role for cyclic AMP in uterine relaxation. Differential effect of isoproterenol and forskolin

Our previous observations suggested that beta adrenergic-mediated relaxation of the rat myometrium could not be ascribed solely to cyclic AMP. The present study examines the relationships between relaxation and cyclic AMP accumulation in the myometrium in response to isoproterenol, forskolin and the combination of both. The diterpene enhanced cyclic AMP generation and potentiated the rises in cyclic AMP due to isoproterenol and prostaglandin (PG) E2. Isoproterenol-induced relaxation of a carbachol-contracted myometrium was associated with modest increments in cyclic AMP (6-12 pmol/mg of protein) in contrast to forskolin whose relaxing effect could be expressed only when associated with large increases in cyclic AMP (80-180 pmol/mg of protein). PGE2, although elevating cyclic AMP to the same extent as isoproterenol, caused contractions which were antagonized by isoproterenol and forskolin, respectively, associated with low and high cyclic AMP concentrations. Both PGE2 and forskolin, by virtue of their stimulatory effect on cyclic AMP generation, enhanced the efficiency of isoproterenol to cause relaxation. Likewise, the greater efficacy of forskolin to relax a PGE2- as opposed to a carbachol-contracted myometrium, was ascribed to its potentiated cyclic AMP response when combined with PGE2. It is proposed that the beta adrenoceptor-linked relaxation results from the concerted effects of both a cyclic AMP-dependent (sensitive to low cyclic AMP) and a cyclic AMP-independent process; the latter is postulated to operate at the membrane level with an ultimate reduction in cytosolic Ca++. On the other hand, cyclic AMP, provided it reached a critical concentration essential to mediate intracellular Ca++ sequestration, would be the sole determinant for forskolin-elicited relaxation.     

Regional differences in relaxation of electric field-stimulated canine airway smooth muscle by verapamil, isoproterenol and prostaglandin E2

Regional differences in contraction produced by methacholine and electric field stimulation (EFS) and in relaxation produced by isoproterenol, prostaglandin E2 and verapamil were studied in isolated canine airway smooth muscle in vitro. Low-frequency EFS (3 Hz, 0.5 msec, 50 V) contracted thoracic trachealis to 43% of maximal EFS response, whereas cervical trachealis contracted to only 14% of maximum. EFS at 10 Hz produced 75% of the maximal response in both regions of the trachea. These EFS responses were abolished by 0.1 microM tetrodotoxin and 1.0 microM atropine. Contraction produced by EFS was also matched in each tissue by contraction with methacholine. The concentrations of methacholine that matched EFS at 10 Hz were 52 +/- 7, 378 +/- 84 and 66 +/- 11 nM for cervical and thoracic trachealis and lobar bronchi, respectively. Both EFS and matched methacholine contractions of cervical trachealis and lobar bronchi were completely relaxed by isoproterenol, whereas thoracic trachealis relaxed maximally to only 60% of induced tone. When verapamil was used to relax EFS and matched methacholine contractions, cervical trachealis was completely relaxed whereas thoracic trachealis relaxed to 15% of induced tone. Although there was a regional difference in the relaxant potency of isoproterenol and, to some extent, verapamil, there was no difference in isoproterenol or verapamil EC50 values for EFS vs. matched methacholine contractions within each region. In contrast, EFS contractions of thoracic trachealis were more sensitive to prostaglandin E2-induced relaxation than were matched methacholine contractions. These data demonstrate marked differences in cholinergic and beta adrenergic receptor-mediated responses between regions of the tracheobronchial tree.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipoxygenase inhibitors and cyclic AMP-mediated insulin secretion caused by forskolin, theophylline and dibutyryl cyclic AMP

Forskolin caused a marked and a concentration-dependent elevation of cyclic AMP content in isolated pancreatic islets (EC50, 10 microM). Cyclic AMP level reached a plateau within 30 min after the addition of 10 microM forskolin. In a low glucose (3.3 mM) medium, forskolin induced slight but significant insulin secretion in a concentration-dependent manner (EC50, 0.3 microM). When the glucose concentration was increased to 5.5 mM, marked enhancement of insulin secretion was observed with forskolin (EC50, 0.5 microM). Lipoxygenase inhibitors, such as nordihydroguaiaretic acid, 3-amino-1-(trifluoromethylphenyl)-2-pyrazoline and 1-phenyl-3-pyrazolidinone failed to affect the forskolin-stimulated cyclic AMP generation. The selective cyclooxygenase inhibitor indomethacin also had no influence on forskolin-stimulated cyclic AMP generation. Insulinotropic effects of forskolin, however, were suppressed by these lipoxygenase inhibitors but not by indomethacin. Both nordihydroguaiaretic acid and 1-phenyl-3-pyrazolidinone also prevented the insulinotropic effects of theophylline and dibutyryl cyclic AMP, whereas indomethacin failed to inhibit them. It seems conceivable that a lipoxygenase product(s) is involved in the insulin secretory process distal to cyclic AMP generation, or that alternatively a lipoxygenase product(s) is permissively involved in the insulin secretory process independently from the cyclic AMP-mediated process.     

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.     

Relaxation of vascular smooth muscle by HA-1004, an inhibitor of cyclic nucleotide-dependent protein kinase

A newly synthesized compound, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA-1004), was shown to be a potent inhibitor of two cyclic nucleotide-dependent protein kinases, cyclic GMP-dependent protein kinase and cyclic AMP-dependent protein kinase and the Ki values were 1.4 and 2.3 microM, respectively. HA-1004 relaxed rabbit aortic strips contracted by various agonists and with similar ED50 values. Phenotolamine, propranolol and atropine did not affect this HA-1004-induced relaxation, thereby suggesting that this compound does not act through these membrane receptor associated mechanisms. HA-1004 shifted the dose-response curve for CaCl2 to the right in a competitive manner in depolarized rabbit renal arterial strips. This compound also relaxed the A-23187 and phenylephrine-induced contractions elicited in Ca++-free solution. These findings suggest that HA-1004 exerts its action at the intracellular or submembranal level. This vasodilator has little effect on actomyosin adenosine triphosphatase and Ca++-calmodulin-dependent myosin light chain kinase. Studies using its derivatives with various lengths of alkyl chain (C0-C6) indicated that the potencies of these compounds, as vasorelaxants, correlated well with their potential to inhibit cyclic nucleotide-dependent protein kinase. HA-1004 should be a useful tool for investigating in smooth muscle, regulatory mechanism(s) by second messengers, cyclic AMP and cyclic GMP.     

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.     

Role of cyclic AMP protein kinase in decreased arterial cyclic AMP responsiveness in hypertension

A decreased relaxation responsiveness to isoproterenol and forskolin is manifest in aortic smooth muscle isolated from spontaneously hypertensive rats (SHR) when compared with normotensive Wistar Kyoto (WKY) rats. Inasmuch as the effector of cyclic AMP (cAMP) is cAMP-dependent protein kinase, we sought to determine if alterations in this enzyme might be responsible for this decreased responsiveness to cAMP-increasing vasodilators. The concentration of cAMP protein kinase activity in aortic, carotid and caudal arteries (approximately 300 pmol/mg of protein per min) was similar in both WKY and SHR. Activity in femoral arteries from SHR and WKY rats was greater (approximately 600 pmol/mg/min); branches of the femoral artery from SHR had less protein kinase activity (660 pmol/mg/min) than their WKY counterparts (1000 pmol/mg/min). There were no differences between WKY and SHR in isozymic distribution of soluble cAMP protein kinase in any of these sources of arterial smooth muscle. Concentration and temporal-related relaxation of KCl-contracted aortic muscle strips by forskolin was associated with concomitant activation of cAMP protein kinase in both groups. The rate and extent of kinase activation was similar for both groups even though the rate and extent of relaxation was markedly less in SHR. These findings show that neither the concentration, isozymic distribution nor activation of cAMP-dependent protein kinase are different in aortic smooth muscle isolated from SHR when compared with WKY animals. Thus, decreased relaxation responsiveness to cAMP-increasing vasodilators is probably not related to events proximal to and including activation of arterial cAMP-dependent protein kinase.     

Mechanisms of beta-adrenoceptor mediated increase in delayed rectifier potassium current

Experiments were carried out in single ventricular cells of the guinea-pig heart. Isoproterenol, forskolin, intracellularly applied cyclic AMP and 3-isobutyl-1-methylxanthine increased the delayed rectifier potassium current (IK). The effect of isoproterenol was abolished by intracellularly applied guanosine 5'-O-(3-thio-triphosphate). These results indicate that isoproterenol stimulates beta-adrenoceptors to activate adenylate cyclase by mediation through the stimulatory GTP-binding protein, and causes an increase in intracellular cyclic AMP levels. Then IK is probably increased by phosphorylation of the IK-channel protein by cyclic AMP-dependent protein kinase.     

Differential inhibitory effects of forskolin, isoproterenol, and dibutyryl cyclic adenosine monophosphate on phosphoinositide hydrolysis in canine tracheal smooth muscle.

A characteristic feature of airway smooth muscle is its relative sensitivity to relaxant effects of beta adrenergic agonists when contracted by inflammatory mediators, such as histamine, vs. resistance to these relaxant effects when contracted by muscarinic agonists. Because contractions presumably depend upon the hydrolysis of membrane phosphoinositides (PI) and the generation of inositol phosphates (IP), our goal was to test for the effects of forskolin, isoproterenol, and dibutyryl cAMP on histamine- vs. methacholine-induced IP accumulation in canine tracheal smooth muscle. Methacholine (10(-3) M) was a more effective stimulant of IP accumulation (9.6 +/- 2.1-fold increase) than equimolar histamine (3.6 +/- 0.5-fold increase) in this tissue. When responses to equieffective methacholine (4 x 10(-6) M) and histamine (10(-3) M) were compared, neither forskolin, isoproterenol, nor dibutyryl cAMP significantly decreased IP accumulation in response to methacholine. In contrast, each of these three agents significantly decreased responses to histamine (by 56 +/- 9, 52 +/- 2, and 61 +/- 2%, respectively). We concluded that, in canine tracheal smooth muscle, increased cAMP is associated with inhibition of PI hydrolysis in response to histamine but not methacholine. The findings suggest a novel mechanism for selective modulation by cAMP of receptor-mediated cellular activation.     

Potentiation of cyclic adenosine monophosphate production by thrombin in the human erythroleukemia cell line, HEL.

The human erythroleukemia cell line (HEL) has been used as a model system for studying signal transduction processes as they might relate to platelet/megakaryocyte function. We were interested in examining the role of thrombin in the regulation of adenylyl cyclase in this cell line. As opposed to its predominantly inhibitory effects on cyclic AMP production in platelets or in membranes from HEL cells, our initial experiments in intact HEL cells revealed that thrombin markedly potentiated the cyclic AMP response to prostaglandin E1 (2.9 +/- 0.2-fold), prostacyclin (1.9 +/- 0.2-fold) and carbacyclin (2.5 +/- 0.5-fold), measured either by radioimmunoassay or by the [3H]adenine preloading procedure. Thrombin, although ineffective alone, also potentiated cyclic AMP production stimulated by vasoactive intestinal peptide (1.6 +/- 0.2-fold), cholera toxin (3.0 +/- 0.6-fold) and AIF4- (2.3 +/- 0.6-fold), but not by forskolin (0.9 +/- 0.1-fold). The thrombin effect 1) produced an increase in the efficacy of the prostaglandins with no change in potency; 2) was long-lived; 3) required the proteolytic activity of thrombin; 4) was insensitive to pertussis toxin; and 5) was at least partially mimicked by trypsin, extracellular ATP and UTP, platelet activating factor and activators of protein kinase C. Down-regulation of protein kinase C or pre-exposure to the protein kinase inhibitor staurosporine blocked the potentiating effect. Together, these results suggest that in HEL cells, the mechanism of thrombin potentiation of cyclic AMP production may involve alterations in the interaction between stimulatory guanine nucleotide binding protein and the catalytic subunit of adenylyl cyclase, possibly involving protein kinase C-mediated phosphorylation.     

Physiological approach of beta receptor coupling to adenylate cyclase in rat airways: ontogenical modification and functional antagonism

The relaxant effects of isoproterenol, forskolin and sodium nitroprusside were studied on tracheal pieces and lung parenchymal strips of Sprague-Dawley and Wistar rats according to age and functional antagonism with carbachol applied previously to induce the contraction. The beta receptor-related maximal relaxant effect of isoproterenol decreased from 4 to 11 weeks in Sprague-Dawley rat airways contracted with 10(-6) M carbachol. This maximal relaxant effect did not change with age in the Wistar strain. When lower carbachol concentrations were applied to Wistar trachea, the maximal relaxant effect of isoproterenol raised with a large decrease of the EC50 values. In the Sprague-Dawley strain, a similar diminution of carbachol concentration also allowed to increase the maximal amplitude of relaxation, but a smaller decrease of EC50 was observed as referred to the Wistar strain. These results suggest that the decrease with age of the maximal relaxation of Sprague-Dawley airways by isoproterenol might be linked to impaired functional antagonism between beta adrenergic and muscarinic stimulation in this rat strain. This hypothesis was strengthened by the observation of the effects of forskolin, an activator of adenylate cyclase, and sodium nitroprusside, a cyclic GMP-related relaxant drug, that did not show any modified effect in function of age in both rat strains. A modified regulation of adenylate cyclase complex with ontogenesis and with rat strain is suggested.     

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.     

Relationship between cyclic guanosine monophosphate accumulation and relaxation of canine trachealis induced by nitrovasodilators.

The role of cyclic GMP (cGMP) in mediating relaxation of canine trachealis produced by nitrovasodilators (NVDs), compounds that activate guanylate cyclase, was examined. Sodium nitroprusside (SNP) produced a concentration-dependent relaxation of the canine trachealis that was accompanied by a concentration-related increase in cGMP content. In time course studies, relaxation of isolated trachealis strips induced by 30 microM SNP was paralleled by an increase in cGMP that reached a maximum of 18-fold above basal levels within 2 min. Zaprinast, an inhibitor of the cGMP-specific phosphodiesterase, potentiated both SNP-induced relaxation and cGMP accumulation. A cell-permeable analog of cGMP, 8-bromo-cGMP, mimicked the relaxant effects of SNP. Also assessed were the effects of methylene blue, an agent that inhibits soluble guanylate cyclase activity, and hemoglobin, an agent that competitively binds NO-containing compounds. In these experiments, tissues were pretreated with the above agents for 10 min, contracted with 1 or 3 microM methacholine, and then relaxed by the cumulative addition of SNP or two other NVDs, S-nitroso-N-acetyl-penicillamine (SNAP) and glyceryl trinitrate (GTN). Tissues were flash-frozen after adding the final concentration of the various NVDs and assayed for cGMP. Methylene blue and hemoglobin suppressed both cGMP accumulation and relaxation in response to SNAP and GTN. in contrast, methylene blue and hemoglobin inhibited SNP-induced cGMP accumulation but, paradoxically, potentiated SNP-induced relaxation. The results of this study generally support a role for cGMP in NVD-induced relaxation of airway smooth muscle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Subtype-selective regulation of beta adrenergic receptor-adenylyl cyclase coupling by phorbol esters in 3T3-L1 fibroblasts

To study the epigenetic regulation of beta adrenergic receptor subtypes, we examined the effects of phorbol esters on beta adrenergic receptor coupling to adenylyl cyclase in 3T3-L1 fibroblasts, which express both beta-1 and beta-2 adrenergic receptor subtypes. Pretreatment of intact 3T3-L1 cells with the protein kinase C activator phorbol dibutyrate caused a dose- and time-dependent decrease in subsequent cyclic AMP (cAMP) accumulation mediated by the beta adrenergic agonist isoproterenol. This effect was selective for beta-adrenergic receptor-mediated responses because there was a potentiation of cAMP accumulation caused by other activators such as prostaglandin E1, forskolin or cholera toxin. The inactive phorbol, alpha-phorbol dibutyrate was ineffective at 1 microM in attenuating isoproterenol stimulation, and 25 nM of the protein kinase C inhibitor staurosporine blocked the effects of phorbol ester on beta adrenergic agonist responses. Stimulation of cAMP accumulation by isoproterenol occurred through a greater proportion of beta-2 adrenergic receptors in phorbol dibutyrate-treated cells than in control cells. This was demonstrated using the beta-1 adrenergic selective antagonist ICI 89.406 and the beta-2 adrenergic selective antagonist ICI 118.551 to inhibit competitively isoproterenol-stimulated cAMP accumulation. Beta-2 adrenergic receptor number and subtype in these cells are regulated by glucocorticoids and butyrate. Decreasing the proportion of beta-1 adrenergic receptors and concomitantly increasing beta-2 adrenergic receptors with either glucocorticoids or butyrate decreased the ability of phorbol ester pretreatment to attenuate cAMP accumulation by isoproterenol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of protease-activated receptor (PAR) 1 and PAR4 signaling in human platelets by compartmentalized cyclic nucleotide actions

Thrombin potently regulates human platelets by the G protein-coupled receptors protease-activated receptor (PAR) 1 and PAR4. Platelet activation by thrombin and other agonists is broadly inhibited by prostacyclin and nitric oxide acting through adenylyl and guanylyl cyclases to elevate cAMP and cGMP levels, respectively. Using forskolin and YC-1 [3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole] to selectively activate the adenylyl and guanylyl cyclases, respectively, and the membrane-permeable analogs N(6),2'-O-dibutyryladenosine-3'-5'-cAMP (dibutyryl-cAMP) and 8-(4-parachlorophenylthoi)-cGMP (8-pCPT-cGMP), we sought to identify key antiplatelet steps for cyclic nucleotide actions in blocking platelet activation by PAR1 versus PAR4. Platelet aggregation by PAR1 or PAR4 was inhibited with similar EC(50) of 1.2 to 2.1 microM forskolin, 31 to 33 microM YC-1, 57 to 150 microM dibutyryl-cAMP, and 220 to 410 microM 8-pCPT-cGMP. There was a marked left shift in the inhibitory potencies of forskolin and YC-1 for alpha-granule release and glycoprotein IIbIIIa/integrin alphaIIbbeta3 activation (i.e., EC(50) of 1-60 and 40-1300 nM, respectively) that was not observed for dibutyryl-cAMP and 8-pCPT-cGMP (i.e., EC(50) of 200-600 and 40-140 microM, respectively). This inhibition was essentially instantaneous, and measurements of cyclic nucleotide levels and kinase activities support a model of compartmentation involving the cyclic nucleotide effectors and regulators and the key molecular targets for this platelet inhibition. The different sensitivities of PAR1 and PAR4 to inhibition of calcium mobilization and dense granule release identify key antiplatelet steps for cyclic nucleotide actions and are consistent with the signaling models for these receptors. Specifically, PAR4 inhibition depends on the regulation of both calcium mobilization and dense granule release, and PAR1 inhibition depends predominantly on the regulation of dense granule release.     

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.     

Vascular reactivity of isolated thoracic aorta of the C57BL/6J mouse

We characterized the thoracic aorta from the C57BL/6J mouse, a strain used commonly in the generation of genetically altered mice, in response to vasoactive substances. Strips of aorta were mounted in tissue baths for measurement of isometric contractile force. Cumulative concentration-response curves to agonists were generated to observe contraction, or relaxation in tissues contracted with phenylephrine or prostaglandin F(2alpha) (PGF(2alpha)). In endothelium-denuded strips, the order of agonist contractile potency (-log EC(50) [M]) was norepinephrine > phenylephrine = 5-hydroxytryptamine > dopamine > PGF(2alpha) > isoproterenol > KCl. Angiotensin II and endothelin-1 were weakly efficacious (15% of maximum phenylephrine contraction), as were UK14,304, clonidine, histamine, and adenosine. In endothelium-intact strips, agonists still caused contraction and both angiotensin II and endothelin-1 remained ineffective. In experiments focusing on angiotensin II, angiotensin II-induced contraction was abolished by the AT(1) receptor antagonist losartan (1 microM) but was not enhanced in the presence of the AT(2) receptor antagonist PD123319 (0.1 microM), tyrosine phosphatase inhibitor orthovanadate (1 microM) or when angiotensin II was given noncumulatively. Prazosin abolished isoproterenol-induced contraction and did not unmask isoproterenol-induced relaxation. Angiotensin II and endothelin-1 did not cause endothelium-dependent or -independent relaxation in phenylephrine- or PGF(2alpha)-contracted tissues. Acetylcholine but not histamine, dopamine, or adenosine caused an endothelium-dependent vascular relaxation. These experiments provide information as to the vascular reactivity of the normal mouse thoracic aorta and demonstrate that the mouse aorta differs substantially from rat aorta in response to isoproterenol, angiotensin II, endothelin-1, histamine, and adenosine.