Increased interleukin-8 release by beta-adrenoceptor activation in human transformed bronchial epithelial cells

1. The effect of beta-adrenoceptor activation on release of the neutrophil chemoattractant, interleukin-8 (IL-8), was examined in human transformed bronchial epithelial cells (16HBE cells). 2. The combined beta 1- and beta 2-adrenoceptor agonist, isoprenaline, time- (100 nM, 2-18 h) and concentration- (1-30 nM) dependently increased IL-8 protein content in the cell culture supernatant as measured by an enzyme immunosorbent assay standardized for DNA by fluoro-colorimetry. 3. Isoprenaline (1-100 nM, 15 min) increased cyclic AMP concentration-dependently. 4. The effect of isoprenaline (100 nM) was inhibited by the beta-adrenoceptor blocker propranolol (10 microM). The maximum magnitude of IL-8 increase caused by beta-adrenoceptor activation was 40% of that caused by the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha 100 ng ml-1). 5. The selective beta 2-adrenoceptor agonist salbutamol (1 microM), increased IL-8 protein similarly to isoprenaline and the cyclic AMP analogue, dibutyryl cyclic AMP (1 mM) produced a corresponding effect. 6. Pretreatment with isoprenaline (100 nM) followed by TNF-alpha (20 ng ml-1) increased IL-8 additively. 7. In conclusion, beta-adrenoceptor stimulation increased the release of the neutrophil chemoattractant, IL-8 in 16HBE cells, via an increase in intracellular cyclic AMP. beta-adrenoceptor stimulation adds to the IL-8 increase caused by the pro-inflammatory cytokine TNF-alpha. If this mechanism exists in vivo, beta-adrenoceptor activation may increase neutrophil chemotaxis into the airways.     

Paradoxical facilitation of acetylcholine release from parasympathetic nerves innervating guinea-pig trachea by isoprenaline

1. Previous studies have provided evidence that activation of beta-adrenoceptors on cholinergic nerve terminals can inhibit neurotransmission in the airways. However, in most cases, this conclusion has been based on indirect evidence obtained from mechanical experiments where changes in airways smooth muscle tone were measured. 2. We have assessed whether modulation of cholinergic neurotransmission by beta-adrenoceptor agonists is due to a pre- or post-junctional action by investigating the effect of isoprenaline on contractile responses evoked by exogenous acetylcholine (ACh) and electrical field stimulation (EFS; 4 Hz, 40 V, 0.5 ms pulse width every 15 s), and on EFS-induced ACh release from cholinergic nerves innervating guinea-pig and human trachea. Furthermore, the subtype of beta-adrenoceptor which modulates neurotransmission and the potential role of cyclic AMP in this response were evaluated. 3. In guinea-pig trachea, isoprenaline (1 nM-1 microM) inhibited the contractile response evoked by exogenous ACh (1 microM) to a similar extent to that evoked by EFS (EC50 = 19.9 and 23 nM, respectively). 4. In epithelium-denuded guinea-pig strips treated with indomethacin (10 microM), isoprenaline significantly enhanced EFS-induced ACh release from cholinergic nerve terminals (by 36% at 0.3 microM). This effect was blocked by propranolol and ICI 118, 551 (each 0.1 microM). In contrast, isoprenaline failed to affect EFS-induced ACh release from parasympathetic nerves innervating human trachea. 5. To evaluate the role of cyclic AMP in the beta-adrenoceptor-induced facilitation of cholinergic neurotransmission, the effects of various cyclic AMP elevating drugs on ACh release were studied. Forskolin (10 microM) significantly augmented (by 17%) EFS-induced ACh release, an effect which was not reproduced by 1,9-dideoxyforskolin (10 microM) which does not activate adenylyl cyclase. Similarly, the cyclic AMP analogue, 8-bromo-cyclic AMP (1 mM) and cholera toxin (1 microgram ml-1) facilitated ACh output by 22 and 47% respectively, whereas prostaglandin E2 (PGE2, 0.1 nM-1 microM) inhibited this response (by 67% at 1 microM). 6. Zardaverine (10 microM), a dual inhibitor of the phosphodiesterase (PDE)3 and PDE4 isoenzyme families, did not affect EFS-induced ACh release and failed to facilitate the actions of either isoprenaline or PGE2. Similarly, neither SK&F 94120 (10 microM) nor rolipram (10 microM), selective inhibitors of PDE3 and PDE4 respectively, significantly affected the release of ACh in response to EFS. 7. The result of this study suggests that isoprenaline facilitates cholinergic neurotransmission in guinea-pig, but not human, trachea by activation of pre-junctional beta 2-adrenoceptors, an effect that may be mediated via activation of the cyclic AMP/cyclic AMP-dependent protein kinase cascade. Furthermore, the data presented herein illustrate the need to undertake direct measurements of neurotransmitter release when examining the effect of agents purported to act pre-junctionally.     

Alpha 2-adrenergic receptors regulate generation of cyclic AMP in the pineal gland, but not in cerebral cortex of chick

A beta-adrenoceptor agonist isoprenaline potently stimulated cyclic AMP formation in chick cerebral cortical slices. L-Noradrenaline (10-1000 microM) also increased cortical nucleotide synthesis, the effect being antagonized by beta-adrenoceptor blocker propranolol, and not affected by alpha 1- and alpha 2-adrenoceptor blockers, prazosin and yohimbine, respectively. Clonidine, a selective alpha 2-agonist, had no effect on cerebral cyclic AMP production stimulated by both isoprenaline and forskolin. However, clonidine (0.001-10 microM) concentration-dependently suppressed forskolin-driven cyclic AMP synthesis in intact chick pineal glands. In living chicks clonidine suppressed the nocturnal activity of cyclic AMP-dependent serotonin N-acetyltransferase, a rate-limiting enzyme in melatonin biosynthesis, the effect being prevented by yohimbine. The data suggest that the cyclic AMP generating system of the pineal gland, but not that of cerebral cortex in chick, is negatively regulated by alpha 2-adrenergic receptor-mediated signal.     

Use of D-myo inositol 1,2,6 trisphosphate to inhibit contractile activity in rat ventricular cardiomyocytes induced by neuropeptide Y and other cardioactive peptides through phospholipase C

1. D-Myo inositol 1,2,6 trisphosphate (alpha-trinositol, pp56), an isomer of the second messenger substance, inositol 1,4,5 trisphosphate, has an interesting pharmacological profile that includes antagonism of a number of neuropeptide Y (NPY)-mediated cellular processes. The ability of pp56 to inhibit selectively the myocardial contraction mediated by NPY in relation to the responses to other cardioactive peptides, including endothelin-1, calcitonin gene-related peptide (CGRP), secretin and vasoactive intestinal peptide (VIP), was assessed. In order to investigate the possible interaction of pp56 with mechanisms of inositol phosphate signalling generated in heart muscle cells by activation of the beta-isoenzyme of phospholipase C (PLC beta), noradrenaline was used as a positive control, and isoprenaline and forskolin were included as negative controls. 2. Ventricular cardiomyocytes, isolated from the hearts of adult rats, were stimulated to contract at 0.5 Hz in the presence of calcium ion (2 mM). The concentrations of agonists used were in the region of their maximally effective concentrations for myocyte contraction and the concentration of pp56 was in the range of 1-100 microM. Contractile activity was monitored by video microscopy and maximum shortening determined by image analysis. 3. In the absence of agonist, contractile amplitudes following 20 min preincubation with pp56 were not different from that observed in the absence of pp56. Pp56 (1-100 microM) inhibited significantly the positive contractile response to noradrenaline (5 microM) in the presence of propranolol (500 nM), such that the response was almost completely attenuated at the highest concentration of the inhibitor. Pp56 did not inhibit the positive contractile responses to forskolin (40 microM) or isoprenaline (100 nM). 4. NPY alone does not influence the basal level of contraction of cardiomyocytes, but can attenuate isoprenaline-stimulated contraction and can increase contractile amplitude from basal when the transient outward current is blocked with 4-aminopyridine. In the presence of isoprenaline (100 nM), the negative response to NPY (100 nM) was attenuated significantly by pp56 (1-100 microM). With 4-aminopyridine, the positive contractile response to NPY (200 nM) was decreased by pp56, although this was not statistically significant. 5. Pp56 inhibited the positive contractile responses to CGRP (1 nM) and endothelin-1 (20 nM) completely, but did not affect the responses to secretin (20 nM) or VIP (20 nM). 6. In conclusion, these data challenge the previously obtained selectivity of pp56 as an antagonist of NPY-mediated cellular processes, since responses to CGRP and endothelin-1 were at least equally sensitive. Furthermore, as pp56 discriminated clearly in its inhibition of responses to alpha-adrenoceptor by comparison with beta-adrenoceptor/adenylate cyclase stimulation, it appears that pp56 may be a useful pharmacological agent with which to distinguish between PLC beta-dependent and PLC beta-independent coupling mechanisms. On this basis, further evidence has been obtained that, in rat cardiomyocytes, the contractile responses to NPY, CGRP and endothelin-1 are attributable to the activation of PLC beta-dependent pathways, whereas the responses to secretin and VIP are mediated by PLC beta-independent pathways.     

Effects of adrenomedullin and calcitonin gene-related peptide on contractions of the rat aorta and porcine coronary artery

1. Effects of adrenomedullin and alpha-calcitonin gene-related peptide (CGRP) on the contractions and cytosolic Ca2+ concentrations ([Ca2+]i) of the rat aorta and porcine coronary artery were investigated. Characteristics of the receptors mediating the effects of adrenomedullin and alpha-CGRP were also investigated. 2. Adrenomedullin and alpha-CGRP caused a concentration-dependent relaxation in the rat aorta contracted with noradrenaline. The IC50 values for adrenomedullin and alpha-CGRP were 2.4 nM and 4.0 nM, respectively. The relaxant effects of these peptides were abolished by removal of the endothelium and significantly attenuated by an inhibitor of nitric oxide synthase, NG-monomethyl-L-arginine (L-NMMA, 100 microM), but not by a cyclo-oxygenase inhibitor, indomethacin (10 microM). 3. Adrenomedullin and alpha-CGRP increased the endothelial [Ca2+]i in the rat aorta with endothelium, whereas they did not change [Ca2+]i in the smooth muscle. 4. An antagonist of the CGRP1 receptor, CGRP (8-37), antagonized the relaxant effects of alpha-CGRP and the beta-isoform of CGRP (beta-CGRP) but not those of adrenomedullin in the rat aorta. 5. In the porcine coronary artery contracted with U46619, adrenomedullin and alpha-CGRP caused a concentration-dependent relaxation with an IC50 of 27.6 and 4.1 nM, respectively. Removal of the endothelium altered neither the IC50 values nor the maximal relaxations induced by adrenomedullin or alpha-CGRP. When the artery was contracted with high K+ solution (72.7 mM), these peptides caused a small relaxation. 6. Adrenomedullin and alpha-CGRP increased cyclic AMP content and decreased the smooth muscle [Ca2+]i in the porcine coronary artery. 7. CGRP (8-37) significantly antagonized the relaxant effects of adrenomedullin and alpha-CGRP in the porcine coronary artery. However, it had little effect on the relaxations induced by the beta-isoform of CGRP (beta-CGRP). 8. These results suggest that in the rat aorta, adrenomedullin and alpha-CGRP increase the endothelial [Ca2+]i, activate nitric oxide synthase and release nitric oxide, without a direct inhibitory action on smooth muscle. In the porcine coronary artery, in contrast, adrenomedullin and alpha-CGRP directly act on smooth muscle, increase cyclic AMP content, decrease the smooth muscle [Ca2+]i and inhibit contraction. The rat aortic endothelium seems to express the CGRP receptor which is sensitive to alpha-CGRP, beta-CGRP and CGRP (8-37) and the adrenomedullin specific receptor. The porcine coronary smooth muscle, in contrast, seems to express two types of CGRP receptor; one of which is sensitive to alpha-CGRP, CGRP (8-37) and adrenomedullin and the other is sensitive only to beta-CGRP.     

Opposite modulation of noradrenaline and ATP release in guinea-pig vas deferens through prejunctional beta-adrenoceptors: evidence for the beta 2 subtype

Activation of prejunctional beta-adrenoceptors has been suggested to increase the release of noradrenaline but to decrease the neural release of ATP in the guinea-pig vas deferens. Experiments were carried out to determine the subtype of beta-adrenoceptor involved. In [3H]-noradrenaline-preincubated tissues superfused with medium containing prazosin and suramin, isoprenaline (1-100 nM), salbutamol (0.01-1 microM) and terbutaline (0.1-10 microM) increased the overflow of tritium but reduced the overflow of ATP elicited by electrical stimulation (210 pulses/7 Hz). The effects of isoprenaline were blocked by the beta 2-selective antagonist 1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3- [(1-methylethyl)amino]-2-butanol (ICI 118,551; 100 nM). In prazosin- and suramin-free medium, isoprenaline (100 nM) did not change the overflow of ATP elicited by exogenous noradrenaline (10 microM). Isoprenaline (1-100 nM), salbutamol (0.01-1 microM) and terbutaline (0.1-10 microM) reduced the initial twitch contraction elicited by electrical stimulation (210 pulses/7 Hz) in prazosin- and suramin-free medium as well as the isolated purinergic neurogenic contraction obtained by exposure to prazosin. They increased or tended to increase the secondary sustained concentration elicited by electrical stimulation in prazosin- and suramin-free medium as well as the isolated adrenergic neurogenic contraction obtained in the presence of suramin. The inhibition by isoprenaline of the isolated purinergic contraction was attenuated by ICI 118,551 (100 nM) but not by the beta 1-selective antagonist 1-[2-((3-carbamoyl- 4-hydroxy)phenoxy)ethylamino]-3-[4-(1-methyl-4- trifluoromethyl-2-imidazolyl)phenoxy]-2-propanol (CGP 20712A; 100 nM). The results confirm the opposite beta-adrenoceptor-mediated modulation of noradrenaline and neural ATP release in the guinea-pig vas deferens. They show that the prejunctional beta-adrenoceptor is of the beta 2-subtype.     

Prejunctionally mediated inhibition of neurotransmission by isoprenaline in rat vas deferens

Effects of isoprenaline on monophasic contractions evoked by electric field stimulation were studied in rat isolated prostatic vas deferens. Isoprenaline reduced electrically evoked contractions (EC50: 0.27 +/- 0.05 microM), and propranolol concentration-dependently antagonized the effect of isoprenaline. In contrast, isoprenaline (0.3-3 microM) did not affect the contractile response induced by exogenous noradrenaline or ATP, while forskolin (100 nM) attenuated agonist-induced contraction. In some tissues, adrenergic and purinergic components of the electrically evoked contraction were isolated by exposure to alpha,beta-methylene ATP (3 microM) and prazosin (3 microM), respectively. Isoprenaline induced a greater inhibition of purinergic than adrenergic component of the electrically evoked contraction. Iberiotoxin (50 nM), glibenclamide (3 microM), 4-aminopyridine (0.3 mM) and tetraethylammonium ions (1 mM) attenuated the effect of isoprenaline. These results indicate that isoprenaline-induced inhibition of the electrically evoked (both purinergic and adrenergic) contraction was mediated primarily through activation of prejunctional beta-adrenoceptors, which probably inhibited release of contractile transmitters from sympathetic nerves supplying vas deferens. Lack of effect of isoprenaline on agonist-induced contraction does not favour a functional role of beta-adrenoceptors in vas smooth muscle.     

Inosine binds to A3 adenosine receptors and stimulates mast cell degranulation

We investigated the mechanism by which inosine, a metabolite of adenosine that accumulates to > 1 mM levels in ischemic tissues, triggers mast cell degranulation. Inosine was found to do the following: (a) compete for [125I]N6-aminobenzyladenosine binding to recombinant rat A3 adenosine receptors (A3AR) with an IC50 of 25+/-6 microM; (b) not bind to A1 or A2A ARs; (c) bind to newly identified A3ARs in guinea pig lung (IC50 = 15+/-4 microM); (d) lower cyclic AMP in HEK-293 cells expressing rat A3ARs (ED50 = 12+/-5 microM); (e) stimulate RBL-2H3 rat mast-like cell degranulation (ED50 = 2.3+/-0.9 microM); and (f) cause mast cell-dependent constriction of hamster cheek pouch arterioles that is attenuated by A3AR blockade. Inosine differs from adenosine in not activating A2AARs that dilate vascular smooth muscle and inhibit mast cell degranulation. The A3 selectivity of inosine may explain why it elicits a monophasic arteriolar constrictor response distinct from the multiphasic dilator/constrictor response to adenosine. Nucleoside accumulation and an increase in the ratio of inosine to adenosine may provide a physiologic stimulus for mast cell degranulation in ischemic or inflamed tissues.     

The role of histamine H1, H2 and H3 receptors on enteric ascending synaptic transmission in the guinea pig ileum

The role of histamine H1-, H2- and H3-receptors was studied on neural transmission in ascending excitatory pathways of the guinea pig ileum. A two-compartment (oral and anal compartments) bath was used: ascending neural pathways were activated by electrical stimulation in the anal compartment and the resulting contraction of the circular muscle in the oral compartment was recorded. Drugs were applied in the anal compartment and each agonist was evaluated in the presence of the antagonists of the other two receptors. In the presence of cimetidine (10 microM) and thioperamide (1 microM), histamine (0.03-3 microM) depressed the nerve-mediated contractions (5-70% inhibition, P <.05-.01). The inhibitory effect of histamine was antagonized by mepyramine. At the higher concentrations (10 and 30 microM), histamine elicited contractions of the circular muscle in the oral compartment, and these were abolished by mepyramine (1 microM) and tetrodotoxin (0.6 microM). The H2 agonists dimaprit (30 and 100 microM) and amphamine (0.1-300 microM) produced small contractions of the circular muscle in the oral compartment. These contractile responses were abolished by tetrodotoxin (0.6 microM) and cimetidine (10 microM). The H3 agonist R-alpha-methylhistamine (0.001-1 microM) inhibited (2-58%, P <.05) the nerve-mediated contractions. This inhibitory effect was antagonized by the H3 antagonist thioperamide. These results indicate that 1) histamine, acting at H1 receptors, at lower concentrations depresses synaptic transmission, although at higher concentrations activates the enteric excitatory ascending pathway; 2) activation of H2 receptors by H2 agonists stimulates the enteric excitatory ascending pathways and 3) activation of H3 receptors inhibits synaptic transmission.     

Negative chronotropic and inotropic effects exerted by diadenosine hexaphosphate (AP6A) via A1-adenosine receptors

1. Diadenosine hexaphosphate (AP6A) exerts vasoconstrictive effects. The purpose of this study was to investigate whether AP6A has any effect on cardiac function. 2. The effects of AP6A (0.1-100 microM) on cardiac contractility and frequency were studied in guinea-pig and human isolated cardiac preparations. Furthermore, the effects of AP6A on the amplitude of the L-type calcium current, on the adenosine 3':5'-cyclic monophosphate (cyclic AMP) content and on the phosphorylation of regulatory phosphoproteins, i.e. phospholamban and troponin inhibitor, were investigated in guinea-pig isolated ventricular myocytes. 3. In isolated spontaneously beating right atria of the guinea-pig AP6A exerted a negative chronotropic effect and reduced the rate of contraction maximally by 35% (IC20 = 35 microM). 4. In isolated electrically driven left atria of the guinea-pig AP6A exerted a negative inotropic effect and reduced force of contraction maximally by 23% (IC20 = 70 microM). 5. In isolated electrically driven papillary muscles of the guinea-pig AP6A alone was ineffective, but attenuated isoprenaline-stimulated force of contraction maximally by 23% (IC20 = 60 microM). Furthermore, AP6A attenuated the relaxant effect of isoprenaline. 6. In human isolated electrically driven ventricular preparations AP6A alone was ineffective, but attenuated isoprenaline-stimulated force of contraction by maximally 42% (IC20 = 18 microM). Moreover, AP6A attenuated the relaxant effect of isoprenaline. 7. All these effects of AP6A were abolished by the selective A1-adenosine receptor antagonist 1,3-dipropyl-cyclopentyl-xanthine (DPCPX, 0.3 microM), whereas the M-cholinoceptor antagonist atropine (10 microM) and the P2-purinoceptor antagonist suramin (300 microM) failed to abolish the effects of AP6A. 8. AP6A 100 microM had no effect on the amplitude of the L-type calcium current, but attenuated isoprenaline-stimulated L-type calcium current. The maximum of the current-voltage relationship (I-V curve) was shifted to the left by isoprenaline and additional application of AP6A shifted the I-V curve back to the right to the control value. The phosphorylation state of phospholamban and the troponin inhibitor was unchanged by AP6A alone, but was markedly attenuated by AP6A in the presence of isoprenaline. Cyclic AMP levels remained unchanged by AP6A, even after stimulation with isoprenaline. 9. In summary, AP6A exerts negative chronotropic and inotropic effects in guinea-pig and human cardiac preparations. These effects are mediated via A1-adenosine receptors as all effects were sensitive to the selective A1-adenosine receptor antagonist DPCPX. Furthermore, the effects of AP6A on cyclic AMP levels, protein phosphorylation and the L-type calcium current are in accordance with stimulation of A1-adenosine receptors.     

Co-ordinated changes in cAMP, phosphorylated phospholamban, Ca2+ and contraction following beta-adrenergic stimulation of rat heart

Concentration-dependent changes in cyclic AMP (cAMP), site-specific phosphorylation of phospholamban, the intracellular calcium ([Ca2+]i) transient and contraction were measured in isolated rat ventricular myocytes exposed to the beta-adrenoceptor agonist isoprenaline. Cyclic AMP was measured by [125I]-cAMP scintillation proximity assay, phosphorylation of phospholamban at Ser16 and Thr17 was assessed using a pair of site-specific polyclonal antibodies, and [Ca2+]i was monitored with the fluorescent dye fura 2. Cyclic AMP rose to twice basal levels in the presence of 10(-6) M isoprenaline. The maximum increase in phosphorylation at Ser16 and Thr17 of phospholamban was seen at 10(-7) M isoprenaline. At this stage Ser16 phosphorylation was six times higher, and Thr17 phosphorylation was three times higher than that recorded in the absence of isoprenaline. Phosphorylation at Ser16 correlated more closely with changes in the [Ca2+]i transient and contraction than did phosphorylation at Thr17. This is the first study of its kind to measure simultaneous changes in cAMP, the phosphorylation of phospholamban, the [Ca2+]i transient and contraction over a range of concentrations of beta-agonist. The results suggest that phosphorylation of phospholamban at Thr17 is of lesser physiological relevance to the effects of beta-adrenergic stimulation on the heart than phosphorylation at Ser16.     

Video-microscopic assessment of the role of tissue angiotensin-converting enzyme in the control of the renal microcirculation

In the present study, we assessed the role of tissue angiotensin-converting enzyme as a determinant of intrarenal hemodynamics by using the angiotensin-converting enzyme inhibitor trandolaprilat and the angiotensin II receptor antagonist losartan. Afferent and efferent arteriolar diameters were measured with computer-assisted vessel imaging in isolated perfused hydronephrotic rat kidneys. In response to the addition of 1.0 nM angiotensin I, afferent arterioles constricted by 27.3 +/- 2.4% and efferent arterioles by 20.9 +/- 2.4%. These constrictions were similar to those observed after the administration of 0.3 nM angiotensin 11 (33.7 +/- 2.3% and 20.9 +/- 2.4% in afferent and efferent arterioles, respectively). Pretreatment with the angiotensin-converting enzyme inhibitor trandolaprilat (0.1-10 microM) blunted the angiotensin I-induced constriction of afferent arterioles (12.7 +/- 1.4%) and completely abolished the angiotensin I-induced constriction of efferent arterioles. Subsequent addition of angiotensin II to the perfusate resulted in a marked decrease of afferent (39.9 +/- 1.8%) and efferent (27.8 +/- 3.3%) arteriolar diameters. Pretreatment with the angiotensin II receptor antagonist losartan completely blocked the angiotensin I-induced constriction of both afferent and efferent arterioles. Collectively, these data suggest that angiotensin I affects renal microvessels through its conversion to angiotensin II, mediated by locally available tissue angiotensin-converting enzyme, which subserves the local control of the renal microcirculation.     

Effects of dopamine on L-type Ca2+ current in single atrial and ventricular myocytes of the rat

1. The effects of dopamine on the L-type Ca2+ current (ICa,L) of both atrial and ventricular single myocytes and on the force of contraction of atrial trabeculae in rat heart were investigated. 2. Dopamine increased atrial ICa,L at concentrations higher than 1 microM, but had little or no effect on ICa,L at lower concentrations. The increase in ICa,L at high concentrations was reversed by propranolol and acetylcholine, but not by phentolamine. Activation and inactivation kinetics of ICa,L were not altered by dopamine. 3. In rat ventricular myocytes in which the D4 receptor mRNA does not express, dopamine (20-100 microM) also increased the ICa,L amplitude and propranolol reversed this effect. 4. Clozapine, a potent D4 receptor antagonist, blocked the augmenting effect of dopamine on ICa,L. However, this effect could be explained by beta-antagonism, since clozapine also inhibited the isoprenaline effect. 5. In the atrial trabeculae, the increase in contraction by dopamine (1 to 30 microM) was reversed by 1 microM propranolol, but not by 2 microM phentolamine. Low doses of dopamine (0.01 to 0.3 microM) did not affect the contraction in the controls or during a modest stimulation of the beta-adrenoceptor with 0.01 microM isoprenaline. 6. These results indicate that the positive inotropic action of dopamine is mediated through direct stimulation of the beta-adrenoceptor in both atrial and ventricular myocytes. Involvement of D4 receptor appears unlikely in the regulation of the atrial contraction.     

Comparative study of potential for bisphosphonates to damage gastric mucosa of rats

1. The long-acting beta2-adrenoceptor agonist, salmeterol (10(-9)-10(-5) M), inhibited the IgE-mediated release of histamine from human lung mast cells (HLMC) in a dose-dependent fashion. Additional beta-adrenoceptor agonists were studied and the rank order of potency for the inhibition of histamine release from HLMC was isoprenaline > salmeterol > salbutamol. Approximate EC50 values for the inhibition of histamine release were 10 nM for isoprenaline and 100 nM for salbutamol. An EC50 value for salmeterol could not be calculated because maximal responses to salmeterol were not observed over the concentration range employed. 2. Both salmeterol and isoprenaline inhibited the generation of sulphopeptidoleukotrienes (sLT) more potently and more efficaciously than the release of histamine from immunologically-activated HLMC. Salmeterol (EC50 < 0.1 nM) was more potent than isoprenaline (EC50 0.4 nM) at attenuating sLT generation. 3. The beta-adrenoceptor antagonist, propranolol (1 microM), and the selective beta2-adrenoceptor antagonist, ICI 118,551 (0.1 microM), both caused rightward shifts in the dose-response curve for the inhibition of histamine release by isoprenaline. The antagonism of salmeterol effects by propranolol and ICI 118,551 was more complex. At lower concentrations (< 1 microM) of salmeterol, both antagonists shifted the dose-reponse curve to salmeterol rightward. At a higher concentration (10 microM) of salmeterol, neither ICI 118,551 nor propranolol was an effective antagonist of the salmeterol-mediated inhibition of histamine release. 4. Prolonged exposure (4 h) of HLMC to isoprenaline (1 microM) caused an approximately 50% reduction in the effectiveness of a second exposure to isoprenaline (10 microM) at inhibiting the release of histamine. whereas this pretreatment did not affect the salmeterol (10 microM) inhibition of histamine release. 5. Isoprenaline (10(-9)-10(-5) M) caused a dose-dependent increase in total cell cyclicAMP levels in purified HLMC which paralleled the inhibition of histamine release. Salmeterol (10(-9)-10(-5) M) was considerably less potent than isoprenaline at increasing HLMC cyclicAMP levels. 6. In summary, these data indicate that salmeterol is an effective inhibitor of the stimulated release of mediators from HLMC. The present data also suggest that salmeterol may act to inhibit mediator release from HLMC by beta-adrenoceptor-dependent and independent mechanisms.     

Electrophysiological basis for the enhanced cardiac arrhythmogenic effect of isoprenaline in aged spontaneously hypertensive rats

We used normotensive (WKY) and spontaneously hypertensive rats (SHR) of 2, 6 and 18 months of age to get insight into the mechanisms responsible for the increased incidence of ventricular arrhythmias in hypertensive heart disease. We studied: (1) isoprenaline-induced spontaneous activity in isolated papillary muscle; (2) action potential characteristics; (3) beta-adrenoceptor and A1-adenosine receptor number and affinity; and (4) intracellular cyclic AMP (cAMP) levels. The saturation binding assay was performed in enzymatically isolated ventricular myocytes using the hydrophilic antagonist 3H-CGP 12177 for beta-adrenoceptors and 3H-DPCPX for A1-adenosine receptors. cAMP was measured by a competitive binding assay in myocytes before and after exposure to isoprenaline. Intracellular action potential was recorded from papillary muscles by means of 3 M KCl-filled glass microelectrodes. The presence of isoprenaline-induced automaticity was assessed by interrupting the electrical stimulation periodically. In isolated papillary muscles, isoprenaline (10 nM) induced spontaneous activity more frequently in 18-month-old SHR (90.0%) than in 6-(25.0%, P < 0.05) and 2-month-old SHR (0%, P < 0.001). No age-related statistically significant differences in isoprenaline-induced automaticity were observed in WKY. The incidence of isoprenaline-induced automaticity was higher in 18-month-old SHR than in age-matched WKY (22.2%, P < 0.05). KD and Bmax for beta-adrenoceptors and A1-receptors were not significantly modified in different groups. cAMP levels before and after isoprenaline stimulation were significantly lower in 18-month-old SHR than in age-matched WKY. Action potential duration (APD) was markedly prolonged in both normotensive and hypertensive rats during aging. APD was significantly prolonged in 18-month-old SHR compared to the WKY. A "diastolic depolarization", caused by the presence of delayed after-depolarizations, became apparent in the oldest animals. The slope of the delayed after-depolarization was increased by isoprenaline (1-10 nM) in 18-month-old SHR and this effect was significantly greater than that observed in 18-month-old WKY (P < 0.05). In conclusion, beta-adrenergic responsiveness is enhanced in hypertensive rats during aging, resulting in a greater incidence of abnormal automaticity. This effect does not appear to be related to changes in beta-adrenoceptor or A1-receptor density or affinity; it is likely that the electrophysiological alterations may play a role in this phenomenon.     

Evidence that different mechanisms underlie smooth muscle relaxation to nitric oxide and nitric oxide donors in the rabbit isolated carotid artery

1. The endothelium-dependent relaxants acetylcholine (ACh; 0.03-10 microM) and A23187 (0.03-10 microM), and nitric oxide (NO), applied either as authentic NO (0.01-10 microM) or as the NO donors 3-morpholino-sydnonimine (SIN-1; 0.1-10 microM) and S-nitroso-N-acetylpenicillamine (SNAP; 0.1-10 microM), each evoked concentration-dependent relaxation in phenylephrine stimulated (1-3 microM; mean contraction and depolarization, 45.8+/-5.3 mV and 31.5+/-3.3 mN; n=10) segments of rabbit isolated carotid artery. In each case, relaxation closely correlated with repolarization of the smooth muscle membrane potential and stimulated a maximal reversal of around 95% and 98% of the phenylephrine-induced depolarization and contraction, respectively. 2. In tissues stimulated with 30 mM KCl rather than phenylephrine, smooth muscle hyperpolarization and relaxation to ACh, A23187, authentic NO and the NO donors were dissociated. Whereas the hyperpolarization was reduced by 75-80% to around a total of 10 mV, relaxation was only inhibited by 35% (n=4-7 in each case; P<0.01). The responses which persisted to ACh and A23187 in the presence of 30 mM KCl were abolished by either the NO synthase inhibitor L-NG-nitroarginine methyl ester (L-NAME; 100 microM) or the inhibitor of soluble guanylyl cyclase 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 microM; 10 min; n=4 in each case; P<0.01). 3. Exposure to ODQ significantly attenuated both repolarization and relaxation to ACh, A23187 and authentic NO, reducing the maximum changes in both membrane potential and tension to each relaxant to around 60% of control values (n=4 in each case; P<0.01). In contrast, ODQ almost completely inhibited repolarization and relaxation to SIN-1 and SNAP, reducing the maximum responses to around 8% in each case (n=3-5; P<0.01). 4. The potassium channel blockers glibenclamide (10 microM), iberiotoxin (100 nM) and apamin (50 nM), alone or in combination, had no significant effect on relaxation to ACh, A23187, authentic NO, or the NO donors SIN-1 and SNAP (n=4 in each case; P>0.05). Charybdotoxin (ChTX; 50 nM) almost abolished repolarization to ACh (n=4; P<0.01) and inhibited the maximum relaxation to ACh, A23187 and authentic NO each by 30% (n=4-8; P<0.01). Application of ODQ (10 microM; 10 min) abolished the ChTX-insensitive responses to ACh, A23187 and authentic NO (n=4 in each case; P<0.01 5. When the concentration of phenylephrine was reduced (to 0.3-0.5 microM) to ensure the level of smooth muscle contraction was the same as in the absence of potassium channel blocker, ChTX had no effect on the subsequent relaxation to SIN-1 (n=4; P>0.05). However, in the presence of tone induced by 1-3 microM phenylephrine (51.2+/-3.3 mN; n=4), ChTX significantly reduced relaxation to SIN-1 by nearly 50% (maximum relaxation 53.2+/-6.3%, n=4; P<0.01). 6. These data indicate that NO-evoked relaxation of the rabbit isolated carotid artery can be mediated by three distinct mechanisms: (a) a cyclic GMP-dependent, voltage-independent pathway, (b) cyclic GMP-mediated smooth muscle repolarization and (c) cyclic GMP-independent, ChTX-sensitive smooth muscle repolarization. Relaxation and repolarization to both authentic and endothelium-derived NO in this large conduit artery appear to be mediated by parallel cyclic GMP-dependent and -independent pathways. In contrast, relaxation to the NO-donors SIN-1 and SNAP appears to be mediated entirely via cyclic GMP-dependent mechanisms.     

Effects of the immunosuppressant cyclosporin A on neurotransmitter release from peripheral non-adrenergic, non-cholinergic nerves

This study was undertaken to determine the effect of the immunosuppressant cyclosporin A on neurotransmitter release from non-adrenergic, non-cholinergic nerves (tachykininergic nerves) in the rabbit iris sphincter muscle. Cumulative application of cyclosporin A (0.1 to 10 microM) caused a slow onset of contraction in a concentration-dependent manner. Both FK888 (1 microM) and capsaicin (10 microM), a substance P receptor antagonist and a substance P-depleting agent, respectively, inhibited the contractile effect of cyclosporin A, whereas atropine (1 microM) had no effect. Both cyclosporin A and capsaicin (10 microM) stimulated the release of substance P-like immunoreactivity in the iris. Neither the sodium channel blocker tetrodotoxin (1 microM), the N-type voltage-dependent Ca2+ channel blocker omega-conotoxin GVIA (1 microM) nor the P-type channel blocker omega-agatoxin IVA (0.2 microM) affected cyclosporin A (1 microM)-induced contraction. In contrast, the L-type Ca2+ channel blocker nicardipine (10 microM) inhibited this contractile effect. These results suggest that cyclosporin A stimulates substance P-like tachykinin release by activating L-type voltage-dependent Ca2+ channels, resulting in contraction of the rabbit iris sphincter muscle.     

Characterization of VIP receptor-effector system antagonists in rat and mouse peritoneal macrophages

In the present study we show that the synthetic peptides [4-Cl-D-Phe6,Leu17]VIP and the growth hormone releasing factor (GRF) analog [Ac-Tyr1,D-Phe2]GRF-(1-29)-NH2 inhibit in a competitive manner the specific [125I]VIP binding to both rat and mouse peritoneal macrophages. In rat peritoneal macrophages, the order of potency of the different peptides, as expressed by the IC50 values was: VIP (IC50 = 1.90 +/- 0.16 nM) > [4-Cl-D-Phe6,Leu17]VIP (IC50 = 125.8 +/- 13.2 nM) > [Ac-Tyr1,D-Phe2]GRF-(1-29)-NH2 (IC50 = 354.8 +/- 21.2 nM). In mouse peritoneal macrophages a similar pattern of potency was observed: VIP (IC50 = 1.58 +/- 0.12 nM) > [4-Cl-D-Phe6,Leu17]VIP (IC50 = 110.8 +/- 10.7 nM) > [Ac-Tyr1,D-Phe2]GRF-(1-29)-NH2 (IC50 = 251 +/- 19.2 nM). The behavior as VIP receptor antagonists of both [4-Cl-D-Phe6,Leu17]VIP and [Ac-Tyr1,D-Phe2]GRF-(1-29)-NH2 in rat and mouse peritoneal macrophages was confirmed by: (a) the shift to the right of VIP dose-stimulated cyclic AMP production curves in the presence of the two antagonists; (b) the agreement between the order of efficacy of the two peptides in competition experiments with the corresponding inhibition of cyclic AMP production; (c) the inefficiency of the two antagonists on the stimulation of cyclic AMP production by the beta-adrenoceptor agonist isoproterenol, which indicates the specificity of the interaction; (d) the synergic effect of VIP on isoproterenol-stimulated cyclic AMP production was completely abolished by [4-Cl-D-Phe6,Leu17]VIP or [Ac-Tyr1,D-Phe2]GRF-(1-29)-NH2, suggesting that both antagonists acted via specific VIP receptors. Moreover, propranolol, a beta-adrenoceptor antagonist, did not affect the VIP-stimulated cyclic AMP production and the antagonist role of [4-Cl-D-Phe6,Leu17]VIP or [Ac-Tyr1,D-Phe2]GRF-(1-29)-NH2; (e) in cross-linking experiments, the intensity of the labeling of the [125I]VIP/receptor complexes was significantly lower with the antagonists than in the control experimental situation in both mouse and rat peritoneal macrophage membranes.     

Dependence of P2-nucleotide receptor agonist-mediated endothelium-independent relaxation on ectonucleotidase activity and A2A-receptors in rat portal vein

1. The mechanism of action of P2 nucleotide receptor agonists that produce endothelium-independent relaxation and the influence of ecto-ATPase activity on this relaxing effect have been investigated in rat portal vein smooth muscle. 2. At 25 degrees C, ATP, 2-methylthioATP (2-MeSATP) and 2-chloroATP (2-ClATP), dose-dependently inhibited spontaneous contractile activity of endothelium-denuded muscular strips from rat portal vein. The rank order of agonist potency defined from the half-inhibitory concentrations was 2-CIATP (2.7+/-0.5 microM, n=7) >ATP (12.9+/-1.1 microM, n=9) > or =2-MeSATP (21.9+/-4.8 M, n=4). In the presence of alphabeta-methylene ATP (alphabeta-MeATP, 200 microM) which itself produced a transient contractile effect, the relaxing action of ATP and 2-MeSATP was completely abolished and that of 2-ClATP strongly inhibited. 3. The non-selective P2-receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 100 microM) did not affect the relaxation induced by ATP, 2-MeSATP, and 2-ClATP. 4. The A2A-adenosine receptor antagonist ZM 241385 inhibited the ATP-induced relaxation in a concentration-dependent manner (1-100 nM). In the presence of 100 nM ZM 241385, the relaxing effects of 2-MeSATP and 2-ClATP were also inhibited. 5. ADP, AMP and adenosine also produced concentration-dependent inhibition of spontaneous contractions. The relaxing effects of AMP and adenosine were insensitive to alphabeta-MeATP (200 microM) but were inhibited by ZM 241385 (100 nM). 6. Simultaneous measurements of contraction and ecto-ATPase activity estimated by the degradation of [gamma-32P]-ATP showed that muscular strips rapidly (10-60 s) hydrolyzed ATP. This ecto-ATPase activity was abolished in the presence of EDTA and was inhibited by 57+/-11% (n=3) by 200 microM alphabeta-MeATP. 7. These results suggest that ATP and other P2-receptor agonists are relaxant in rat portal vein smooth muscle, because ectonucleotidase activity leads to the formation of adenosine which activates A2A-receptors.     

Alpha2-adrenoceptor-mediated contractions of the porcine isolated ear artery: evidence for a cyclic AMP-dependent and a cyclic AMP-independent mechanism

1. The aim of this study was to determine the conditions under which the alpha2-adrenoceptor agonist UK14304 produces vasoconstriction in the porcine isolated ear artery. 2. UK14304 (0.3 microM) produced a small contraction of porcine isolated ear arteries which was 7.8+/-3.3% of the response to 60 mM KCl. Similar sized contractions were obtained after precontraction with either 30 nM angiotensin II, or 0.1 microM U46619 (8.2+/-1.8% and 10.2+/-2.6% of 60 mM KCl response, respectively). However, an enhanced alpha2-adrenoceptor response was uncovered if the tissue was precontracted with U46619, and relaxed back to baseline with 1-2 microM forskolin before the addition of UK14304 (46.9+/-9.6% of 60 mM KCl response). 3. The enhanced responses to UK14304 in the presence of U46619 and forskolin were not inhibited by the alpha1-adrenoceptor antagonist prazosin (0.1 microM), but were inhibited by the alpha2-adrenoceptor antagonist rauwolscine (1 microM), indicating that the enhanced responses were mediated via postjunctional alpha2-adrenoceptors. 4. In the presence of 0.1 microM U46619 and 1 mM isobutylmethylxanthine (IBMX), 1 microM forskolin produced an increase in [3H]-cyclic AMP levels in porcine isolated ear arteries. Addition of 0.3 microM UK14304 prevented this increase. 5. The enhanced UK14304 response was dependent upon the agent used to relax the tissue. After relaxation of ear arteries precontracted with 10 nM U46619 and relaxed with forskolin the UK14304 response was 46.9+/-9.6% of the 60 mM KCl response, and after relaxation with sodium nitroprusside (SNP) the response was 24.8+3.3%. However, after relaxation of the tissue with levcromakalim the UK14304 response was only 8.2+/-1.7%, which was not different from the control response in the same tissues (12.2+/-5.6%). An enhanced contraction was also obtained after relaxation of the tissue with the cyclic AMP analogue dibutyryl cyclic AMP (23.2+/-1.3%) indicating that at least part of the enhanced response to UK14304 is independent of the ability of the agonist to inhibit cyclic AMP production. 6. Relaxation of U46619 contracted ear arteries with SNP could be inhibited by the NO-sensitive guanylyl-cyclase inhibitor 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) indicating that production of cyclic GMP is necessary for the relaxant effect of SNP. However, ODQ had no effect on the relaxation of tissue by forskolin, suggesting that this compound does not act via production of cyclic GMP. Biochemical studies showed that while forskolin increases the levels of cyclic AMP in the tissues, SNP had no effect on the levels of this cyclic nucleotide. 7. In conclusion, enhanced contractions to the alpha2-adrenoceptor agonist UK14304 can be uncovered in porcine isolated ear arteries by precontracting the tissue with U46619, followed by relaxation back to baseline with forskolin, SNP or dibutyryl cyclic AMP before addition of UK14304. There was a greater contractile response to UK14304 after relaxation with forskolin than with SNP or dibutyryl cyclic AMP, suggesting that cyclic AMP-dependent and- independent mechanisms are involved in the enhancement of the UK14304 response.