Plasminogen-stimulated airway smooth muscle cell proliferation is mediated by urokinase and annexin A2, involving plasmin-activated cell signalling

BACKGROUND AND PURPOSE

The conversion of plasminogen into plasmin by interstitial urokinase plasminogen activator (uPA) is potentially important in asthma pathophysiology. In this study, the effect of uPA-mediated plasminogen activation on airway smooth muscle (ASM) cell proliferation was investigated.

EXPERIMENTAL APPROACH

Human ASM cells were incubated with plasminogen (0.5–50 μg·mL⁻¹) or plasmin (0.5–50 mU·mL⁻¹) in the presence of pharmacological inhibitors, including UK122, an inhibitor of uPA. Proliferation was assessed by increases in cell number or MTT reduction after 48 h incubation with plasmin(ogen), and by earlier increases in [³H]-thymidine incorporation and cyclin D1 expression.

KEY RESULTS

Plasminogen (5 μg·mL⁻¹)-stimulated increases in cell proliferation were attenuated by UK122 (10 μM) or by transfection with uPA gene-specific siRNA. Exogenous plasmin (5 mU·mL⁻¹) also stimulated increases in cell proliferation. Inhibition of plasmin-stimulated ERK1/2 or PI3K/Akt signalling attenuated plasmin-stimulated increases in ASM proliferation. Furthermore, pharmacological inhibition of cell signalling mediated by the EGF receptor, a receptor trans-activated by plasmin, also reduced plasmin(ogen)-stimulated cell proliferation. Knock down of annexin A2, which has dual roles in both plasminogen activation and plasmin-signal transduction, also attenuated ASM cell proliferation following incubation with either plasminogen or plasmin.

CONCLUSIONS AND IMPLICATIONS

Plasminogen stimulates ASM cell proliferation in a manner mediated by uPA and involving multiple signalling pathways downstream of plasmin. Targeting mediators of plasminogen-evoked ASM responses, such as uPA or annexin A2, may be useful in the treatment of asthma.     

Protein kinase A and the exchange protein directly activated by cAMP (Epac) modulate phenotype plasticity in human airway smooth muscle

BACKGROUND AND PURPOSE

Platelet-derived growth factor (PDGF) modulates the airway smooth muscle (ASM) ‘contractile’ phenotype to a more ‘proliferative’ phenotype, resulting in increased proliferation and reduced contractility. Such phenotypic modulation may contribute to airway remodelling in asthma. We have previously shown that the cAMP effector molecules, protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac) inhibited PDGF-induced phenotypic modulation in bovine ASM. Here, we investigated these mechanisms in human ASM strips and cells.

EXPERIMENTAL APPROACH

ASM strips were incubated with PDGF in the absence or presence of the activators of Epac (8-pCPT-2′-O-Me-cAMP) or of PKA (6-Bnz-cAMP) for 4 days. Strips were mounted for isometric contraction experiments or analysed for the expression of contractile markers. Cell proliferation was measured and proliferative markers were analysed under similar conditions.

KEY RESULTS

Activation of Epac and PKA prevented PDGF-induced ASM strip hypocontractility, and restored the expression of smooth muscle actin, myosin and calponin, which had been markedly diminished by PDGF. Epac and PKA activation inhibited the PDGF-induced ASM cell proliferation and G₁/S phase transition and the expression and phosphorylation of cell cycle regulators.

CONCLUSIONS AND IMPLICATIONS

Epac and PKA maintain a normally contractile ASM phenotype in a mitogenic environment, suggesting that specific activators of Epac and PKA may be beneficial in the treatment of airway remodelling in asthma.     

Agonist-specific patterns of β2-adrenoceptor responses in human airway cells during prolonged exposure

Background and purpose:

β₂-Adrenoceptor agonists (β₂-agonists) are important bronchodilators used in the treatment of asthma and chronic obstructive pulmonary disease. At the molecular level, β₂-adrenergic agonist stimulation induces desensitization of the β₂-adrenoceptor. In this study, we have examined the relationships between initial effect and subsequent reduction of responsiveness to restimulation for a panel of β₂-agonists in cellular and in vitro tissue models.

Experimental approach:

β₂-Adrenoceptor-induced responses and subsequent loss of receptor responsiveness were studied in primary human airway smooth muscle cells and bronchial epithelial cells by measuring cAMP production. Receptor responsiveness was compared at equi-effective concentrations, either after continuous incubation for 24 h or after a 1 h pulse exposure followed by a 23 h washout. Key findings were confirmed in guinea pig tracheal preparations in vitro.

Key results:

There were differences in the reduction of receptor responsiveness in human airway cells and in vitro guinea pig trachea by a panel of β₂-agonists. When restimulation occurred immediately after continuous incubation, loss of responsiveness correlated with initial effect for all agonists. After the 1 h pulse exposure, differences between agonists emerged, for example isoprenaline and formoterol induced the least reduction of responsiveness. High lipophilicity was, to some extent, predictive of loss of responsiveness, but other factors appeared to be involved in determining the relationships between effect and subsequent loss of responsiveness for individual agonists.

Conclusions and implications:

There were clear differences in the ability of different β₂ agonists to induce loss of receptor responsiveness at equi-effective concentrations.     

ATP release and contraction mediated by different P2-receptor subtypes in guinea-pig ileal smooth muscle

1. The present study was addressed to clarify the subtypes of P2-purinoceptor involved in ATP release and contraction evoked by α,β-methylene ATP (α,β-mATP) and other P2-agonists in guinea-pig ileum.
2. α,β-mATP 100 μM produced a transient and steep contraction followed by ATP release from tissue segments. These maximum responses appeared with different time-courses and their ED₅₀ values were 5 and 25 μM, respectively. The maximum release of ATP by α,β-mATP was markedly reduced by 250 μM suramin, 30 μM pyridoxal-phosphate-6-azophenyl-2′,5′-disulphonic acid (PPADS) and 30 μM reactive blue 2 (RB-2), P2-receptor antagonists. However, the contractile response was inhibited by suramin, tetrodotoxin and atropine, but not by PPADS and RB-2.
3. Although the contraction caused by α,β-mATP was strongly diminished by Ca²⁺-removal and nifedipine, and also by tetrodotoxin and atropine at 0.3 μM, the release of ATP was virtually unaffected by these procedures.
4. UTP, β,γ-methylene ATP (β,γ-mATP) and ADP at 100 μM elicited a moderate release of ATP. The release caused by UTP was virtually unaffected by RB-2. However, these P2-agonists failed to elicit a contraction of the segment.
5. The potency order of all the agonists tested for the release of ATP was α,β-mATP>UTP>β,γ-mATP>ADP.
6. In superfusion experiments with cultured smooth muscle cells from the ileum, α,β-mATP (100 μM) enhanced the release of ATP 5 fold above the basal value. This evoked release was inhibited by RB-2.
7. These findings suggest that ATP release and contraction induced by P2-agonists such as α,β-mATP in the guinea-pig ileum result mainly from stimulation of different P2-purinoceptors, P2Y-like purinoceptors on the smooth muscles and, probably, P2X-purinoceptors on cholinergic nerve terminals, respectively. However, the ATP release may also be mediated, in part, by P2U-receptors, because UTP caused RB-2-insensitive ATP release.

     

cAMP inhibits modulation of airway smooth muscle phenotype via the exchange protein activated by cAMP (Epac) and protein kinase A

BACKGROUND AND PURPOSE

Changes in airway smooth muscle (ASM) phenotype may contribute to the pathogenesis of airway disease. Platelet-derived growth factor (PDGF) switches ASM from a contractile to a proliferative, hypo-contractile phenotype, a process requiring activation of extracellular signal-regulated kinase (ERK) and p70^S6 Kinase (p70^S6K). The effects of cAMP-elevating agents on these processes is unknown. Here, we investigated the effects of cAMP elevation by prostaglandin E₂ (PGE₂) and the activation of the cAMP effectors, protein kinase A (PKA) and exchange protein activated by cAMP (Epac) on PDGF-induced phenotype switching in bovine tracheal smooth muscle (BTSM).

EXPERIMENTAL APPROACH

Effects of long-term treatment with the PGE₂ analogue 16,16-dimethyl-PGE₂, the selective Epac activator, 8-pCPT-2′-O-Me-cAMP and the selective PKA activator, 6-Bnz-cAMP were assessed on the induction of a hypo-contractile, proliferative BTSM phenotype and on activation of ERK and p70^S6K, both induced by PDGF.

KEY RESULTS

Treatment with 16,16-dimethyl-PGE₂ inhibited PDGF-induced proliferation of BTSM cells and maintained BTSM strip contractility and contractile protein expression in the presence of PDGF. Activation of both Epac and PKA similarly prevented PDGF-induced phenotype switching and PDGF-induced activation of ERK. Interestingly, only PKA activation resulted in inhibition of PDGF-induced phosphorylation of p70^S6K.

CONCLUSIONS AND IMPLICATIONS

Our data indicate for the first time that both Epac and PKA regulated switching of ASM phenotype via differential inhibition of ERK and p70^S6K pathways. These findings suggest that cAMP elevation may be beneficial in the treatment of long-term changes in airway disease.     

The long-acting β-adrenoceptor agonist,indacaterol, inhibits IgE-dependent responses of human lung mast cells

Background and purpose:

The long-acting β₂-adrenoceptor agonist, indacaterol, has been developed as a bronchodilator for the therapeutic management of respiratory diseases. The aim of the present study was to determine whether indacaterol has any anti-inflammatory activity. To this end, the effects of indacaterol on human lung mast cell responses were investigated.

Experimental approach:

The effects of indacaterol, and the alternative long-acting β-agonists formoterol and salmeterol, were investigated on the IgE-dependent release and generation of histamine, cysteinyl-leukotrienes and prostaglandin D₂ from human lung mast cells. Moreover, the extent to which long-term (24–72 h) incubation of mast cells with long-acting β-agonists impaired the subsequent ability of β-agonists to inhibit mast cell responses was assessed.

Key results:

Indacaterol was as potent and as efficacious as the full agonist, isoprenaline (EC₅₀, ∼4 nmol·L⁻¹), at inhibiting the IgE-dependent release of histamine from mast cells. Formoterol was a full agonist whereas salmeterol was a partial agonist as inhibitors of histamine release. All three long-acting β-agonists were effective inhibitors of the IgE-dependent generation of cysteinyl-leukotrienes and prostaglandin D₂. Long-term incubation of mast cells with long-acting β-agonists led to a reduction in the subsequent ability of β-agonists to stabilize mast cell responses. This tendency to induce functional desensitization was least evident for indacaterol.

Conclusions and implications:

Indacaterol is an effective inhibitor of the release of mediators from human lung mast cells. This suggests that, as well as bronchodilation, mast cell stabilization may constitute an additional therapeutic benefit of indacaterol.     

Analgesic synergy between opioid and α2-adrenoceptors

Opioid and α₂-adrenoceptor agonists are potent analgesic drugs and their analgesic effects can synergize when co-administered. These supra-additive interactions are potentially beneficial clinically; by increasing efficacy and/or reducing the total drug required to produce sufficient pain relief, undesired side effects can be minimized. However, combination therapies of opioids and α₂-adrenoceptor agonists remain underutilized clinically, in spite of a large body of preclinical evidence describing their synergistic interaction. One possible obstacle to the translation of preclinical findings to clinical applications is a lack of understanding of the mechanisms underlying the synergistic interactions between these two drug classes. In this review, we provide a detailed overview of the interactions between different opioid and α₂-adrenoceptor agonist combinations in preclinical studies. These studies have identified the spinal cord as an important site of action of synergistic interactions, provided insights into which receptors mediate these interactions and explored downstream signalling events enabling synergy. It is now well documented that the activation of both μ and δ opioid receptors can produce synergy with α₂-adrenoceptor agonists and that α₂-adrenoceptor agonists can mediate synergy through either the α_2A or the α_2C adrenoceptor subtypes. Current hypotheses surrounding the cellular mechanisms mediating opioid–adrenoceptor synergy, including PKC signalling and receptor oligomerization, and the evidence supporting them are presented. Finally, the implications of these findings for clinical applications and drug discovery are discussed.

LINKED ARTICLES

This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2     

Two imidazoquinoxaline ligands for the benzodiazepine site sharing a second low affinity site on rat GABAA receptors but with the opposite functionality

1. Imidazoquinoxaline PNU-97775 and imidazoquinoline PNU-101017 are benzodiazepine site ligands with a second low affinity binding site on GABA_A receptors, the occupancy of which at high drug concentrations reverses their positive allosteric activity via the benzodiazepine site, and may potentially minimize abuse liability and physical dependence.
2. In this study we discovered, with two imidazoquinoxaline analogues, that the functionality of the second site was altered by the nitrogen substituent on the piperazine ring moiety: PNU-100076 with a hydrogen substituent on the position produced a negative allosteric effect via the second low affinity site, like the parent compounds, while PNU-100079 with a trifluoroethyl substituent produced a positive allosteric response.
3. These functional characteristics were monitored with Cl⁻ currents measurements in cloned rat αxβ2γ2 subtypes of GABA_A receptors expressed in human embryonic kidney 293 cells, and further confirmed in rat cerebrocortical membranes containing complex subtypes of GABA_A receptors with binding of [³⁵S]-TBPS, which is a high affinity ligand specific for GABA_A receptors with exquisite sensitivity to allosteric modulations.
4. This structure-functional relationship could be exploited to further our understanding of the second allosteric site of imidazoquinoxaline analogues, and to develop more effective benzodiazepine site ligands without typical side effects associated with those currently available on the market.

     

Low Doses of Allyphenyline and Cyclomethyline,Effective against Morphine Dependence,Elicit an Antidepressant-like Effect

相似文献   

Species differences in the distribution of β-adrenoceptor subtypes in bladder smooth muscle

1. The β-adrenoceptor (β-AR) subtypes mediating relaxation of the rabbit, rat and canine detrusors were subjected to functional investigation using selective β-AR agonists and antagonists.
2. In all three species, isoprenaline, noradrenaline and adrenaline each produced a concentration-dependent relaxation of the detrusor. The rank order for their relaxing potency was isoprenaline> adrenaline>noradrenaline in rabbits and rats, but isoprenaline>noradrenaline>adrenaline in dogs.
3. Dobutamine did not produce relaxation of the detrusors at concentrations that are selective for β₁-AR. The selective β₂-AR agonist, procaterol, had a more potent relaxing effect on rabbit and rat detrusors than on the canine detrusor. CGP-12177A, a selective β₃-AR agonist, was more effective in the rabbit than in the other two species. On the other hand, the relaxing effect of another β₃-AR agonist, {"type":"entrez-nucleotide","attrs":{"text":"CL316243","term_id":"44896132","term_text":"CL316243"}}CL316243, was more pronounced in dogs and rats than in rabbits.
4. CGP-20712A (10⁻⁹ to 10⁻⁷ M), a selective β₁-AR antagonist, caused a slight rightward shift of the concentration-relaxation response curve for isoprenaline in the canine detrusor (pA₂ 9.41), but not in the rabbit and rat detrusors. ICI-118,551, a selective β₂-AR antagonist, antagonized the isoprenaline-induced relaxation in rabbits (pA₂ 9.45) and rats (pA₂ 9.05), but not in dogs. Bupranolol, a non-selective β-AR antagonist, caused a rightward shift of the concentration-relaxation curve for isoprenaline in the rabbit (pA₂ 9.32) and rat (pA₂ 8.98). However, higher concentrations (3×10⁻⁸ to 10⁻⁵ M) were needed to induce a rightward shift of the curve for isoprenaline in the dog (pA₂ 8.19) than in the other two species.
5. We have confirmed that the distribution of β-AR subtypes in the detrusor muscle varies significantly from species to species and we provide here the first evidence of the presence of β₃-AR in the detrusor. It is suggested that the relaxation induced by adrenoceptor agonists in urinary bladder smooth muscle may be mediated mainly via β₂-AR in rabbits, via both β₂- and β₃-AR in rats, but mainly via β₃-AR in dogs.

     

Inhibition by ATP of calcium oscillations in rat cultured hippocampal neurones

1. The effect of adenosine 5′-triphosphate (ATP) on glutamatergic synaptic transmission in hippocampus was examined by an indicator of intracellular Ca²⁺ oscillations. These oscillations were postsynaptic responses by glutamate released from presynaptic sites. ATP completely inhibited the oscillations in a concentration-dependent manner.
2. The ATP-induced inhibition was mediated via P2-purinoceptors since ATP exhibited the inhibitory action even in the presence of P1-purinoceptor antagonists. Also non-hydrolysable ATP analogues and uridine 5′-triphosphate (UTP) inhibited the oscillation.
3. The rank order of agonist potency of ATP analogues for inhibition of the Ca²⁺ oscillation was as follows: 2-methyl-thio-adenosine 5′-triphosphate⩾ATP>adenosine 5′-O-(3-thiotriphosphate)>UTP>α,β-methylene-adenosine 5′-triphosphate. These inhibitory effects were insensitive to suramin. Judging from this rank order of potency, the inhibitory P2-purinoceptor could be assigned to a subclass of GTP-binding protein coupled-type receptors.
4. The site of action of ATP was thought to be presynaptic since ATP did not affect the postsynaptic Ca²⁺ responses by glutamate. These results suggest the existence of a presynaptic inhibitory P2-receptor that inhibits glutamate release in the hippocampus.

     

Pharmacological characterization of the receptors involved in the β-adrenoceptor-mediated stimulation of the L-type Ca2+ current in frog ventricular myocytes

1. The whole-cell patch-clamp was used for studying the effects of various β₁- and β₂-adrenoceptor agonists and antagonists on the L-type Ca current (I_Ca) in frog ventricular myocytes.
2. Dose-response curves for the effects of isoprenaline (non selective β-agonist), salbutamol (β₂-agonist), dobutamine (β₁-agonist) on I_Ca were obtained in the absence and presence of various concentrations of ICI 118551 (β₂-antagonist), metoprolol (β₁-antagonist) and xamoterol (partial β₁-agonist) to derive EC₅₀ (i.e. the concentration of β-agonist at which the response was 50% of the maximum) and E_max (the maximal response) values by use of a Michaelis equation. Schild regression analysis was performed to examine whether the antagonists were competitive and to determine the equilibrium dissociation constant (K_B) for the antagonist-receptor complex.
3. Isoprenaline increased I_Ca with an EC₅₀ of 20.0 nM and an E_max of 597%. ICI 118551 and metoprolol competitively antagonized the effect of isoprenaline with a K_B of 3.80 nM and 207 nM, respectively.
4. Salbutamol increased I_Ca with an EC₅₀ of 290 nM and an E_max of 512%. ICI 118551 and metoprolol competitively antagonized the effect of salbutamol with a K_B of 1.77 nM and 456 nM, respectively.
5. Dobutamine increased I_Ca with an EC₅₀ of 2.40 μM and an E_max of 265%. ICI 118551 and metoprolol competitively antagonized the effect of dobutamine with a K_B of 2.84 nM and 609 nM, respectively.
6. Xamoterol had no stimulating effect on I_Ca. However, xamoterol competitively antagonized the stimulating effects of isoprenaline, salbutamol and dobutamine on I_Ca with a K_B of 58–64 nM.
7. We conclude that a single population of receptors is involved in the β-adrenoceptor-mediated regulation of I_Ca in frog ventricular myocytes. The pharmacological pattern of the response of I_Ca to the different β-adrenoceptor agonists and antagonists tested suggests that these receptors are of the β₂-subtype.

     

Similarities and differences in the autonomic control of airway and urinary bladder smooth muscle

The airways and the urinary bladder are both hollow organs serving very different functions, i.e. air flow and urine storage, respectively. While the autonomic nervous system seems to play only a minor if any role in the physiological regulation of airway tone during normal breathing, it is important in the physiological regulation of bladder smooth muscle contraction and relaxation. While both tissues share a greater expression of M2 than of M3 muscarinic receptors, smooth muscle contraction in both is largely mediated by the smaller M3 population apparently involving phospholipase C activation to only a minor if any extent. While smooth muscle in both tissues can be relaxed by beta-adrenoceptor stimulation, this primarily involves beta2-adrenoceptors in human airways and beta3-adrenoceptors in human bladder. Despite activation of adenylyl cyclase by either subtype, cyclic adenosine monophosphate plays only a minor role in bladder relaxation by beta-agonists; an important but not exclusive function is known in airway relaxation. While airway beta2-adrenoceptors are sensitive to agonist-induced desensitization, beta3-adrenoceptors are generally considered to exhibit much less if any sensitivity to desensitization. Gene polymorphisms exist in the genes of both beta2- and beta3-adrenoceptors. Despite being not fully conclusive, the available data suggest some role of beta2-adrenoceptor polymorphisms in airway function and its treatment by receptor agonists, whereas the available data on beta3-adrenoceptor polymorphisms and bladder function are too limited to allow robust interpretation. We conclude that the distinct functions of airways and urinary bladder are reflected in a differential regulation by the autonomic nervous system. Studying these differences may be informative for a better understanding of each tissue.     

Characterization of action potential-triggered [Ca2+]i transients in single smooth muscle cells of guinea-pig ileum

1. To characterize increases in cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) associated with discharge of action potentials, membrane potential and [Ca²⁺]_i were simultaneously recorded from single smooth muscle cells of guinea-pig ileum by use of a combination of nystatin-perforated patch clamp and fura-2 fluorimetry techniques.
2. A single action potential in response to a depolarizing current pulse elicited a transient rise in [Ca²⁺]_i. When the duration of the current pulse was prolonged, action potentials were repeatedly discharged during the early period of the pulse duration with a progressive decrease in overshoot potential, upstroke rate and repolarization rate. However, such action potentials could each trigger [Ca²⁺]_i transients with an almost constant amplitude.
3. Nicardipine (1 μM) and La³⁺ (10 μM), blockers of voltage-dependent Ca²⁺ channels (VDCCs), abolished both the action potential discharge and the [Ca²⁺]_i transient.
4. Charybdotoxin (ChTX, 300 nM) and tetraethylammonium (TEA, 2 mM), blockers of large conductance Ca²⁺-activated K⁺ channels, decreased the rate of repolarization of action potentials but increased the amplitude of [Ca²⁺]_i transients.
5. Thapsigargin (1 μM), an inhibitor of SR Ca²⁺-ATPase, slowed the falling phase and somewhat increased the amplitude, of action potential-triggered [Ca²⁺]_i transients without affecting action potentials. In addition, in voltage-clamped cells, the drug had little effect on the voltage step-evoked Ca²⁺ current but exerted a similar effect on its concomitant rise in [Ca²⁺]_i to that on the action potential-triggered [Ca²⁺]_i transient.
6. Similar action potential-triggered [Ca²⁺]_i transients were induced by brief exposures to high-K⁺ solution. They were not decreased, but rather increased, after depletion of intracellular Ca²⁺ stores by a combination of ryanodine (30 μM) and caffeine (10 mM) through an open-lock of Ca²⁺-induced Ca²⁺ release (CICR)-related channels.
7. The results show that action potentials, discharged repeatedly during the early period of a long membrane depolarization, undergo a progressive change in configuration but can each trigger a constant rise in [Ca²⁺]_i. Intracellular Ca²⁺ stores have a role, especially in accelerating the falling phase of the action potential-triggered [Ca²⁺]_i transients by replenishing cytosolic Ca²⁺. No evidence was provided for the involvement of CICR in the action potential-triggered [Ca²⁺]_i transient.

     

The effects of nifedipine and other calcium antagonists on the glibenclamide-sensitive K+ currents in smooth muscle cells from pig urethra

1. The effects of nifedipine on both levcromakalim-induced membrane currents and unitary currents in pig proximal urethra were investigated by use of patch-clamp techniques (conventional whole-cell configuration and cell-attached patches).
2. Nifedipine had a voltage-dependent inhibitory effect on voltage-dependent Ba²⁺ currents at −50 mV (K_i=30.6 nM).
3. In current-clamp mode, subsequent application of higher concentrations of nifedipine (⩾30 μM) caused a significant depolarization even after the membrane potential had been hyperpolarized to approximately −82 mV by application of 100 μM levcromakalim.
4. The 100 μM levcromakalim-induced inward current (symmetrical 140 mM K⁺ conditions, −50 mV) was inhibited by additional application of three different types of Ca antagonists (nifedipine, verapamil and diltiazem, all at 100 μM). In contrast, Bay K 8644 (1 μM) possessed no activating effect on the amplitude of this glibenclamide-sensitive current.
5. When 100 μM nifedipine was included in the pipette solution during conventional whole-cell recording at −50 mV, application of levcromakalim (100 μM) caused a significant inward membrane current which was suppressed by 5 μM glibenclamide. On the other hand, inclusion of 5 μM glibenclamide in the pipette solution prevented levcromakalim from inducing an inward membrane current.
6. The levcromakalim-induced K⁺ channel openings in cell-attached configuration were suppressed by subsequent application of 5 μM glibenclamide but not of 100 μM nifedipine.
7. These results suggest that in pig proximal urethra, nifedipine inhibits the glibenclamide-sensitive 43 pS K⁺ channel activity mainly through extracellular blocking actions on the K⁺ channel itself.

     

Blockade of calcium entry in smooth muscle cells by the antidepressant imipramine

The present study was designed to evaluate the effects of antidepressants on smooth muscle contractile activity. In rat aortic rings, the antidepressants imipramine, mianserin and sertraline provoked concentration-dependent inhibitions of the mechanical responses evoked by K+ (30 mM) depolarization. These myorelaxant effects were not modified by the presence of glibenclamide or 80 mM K+ in the bathing medium. Moreover, the vasodilator properties of imipramine were not affected by atropine, phentolamine and pyrilamine. Radioisotopic experiments indicated that imipramine failed to enhance 86Rb outflow from prelabelled and perifused aortic rings whilst counteracting the increase in 45Ca outflow provoked by a rise in the extracellular K+ concentration. Simultaneous measurements of contractile activity and fura-2 fluorescence revealed that, in aortic rings, imipramine reduced the mechanical and fluorimetric response to K+ challenge. In A7r5 smooth muscle cells, whole cell recordings further demonstrated that imipramine inhibited the inward Ca2+ current. Under different experimental conditions, the ionic and relaxation responses to the antidepressants were reminiscent of those mediated by the Ca2+ entry blocker verapamil. Lastly, it should be pointed out that imipramine exhibited a myorelaxant effect of similar amplitude on rat aorta and on rat distal colon. All together, these findings suggest that the myorelaxant properties of imipramine, and probably also setraline and mianserin, could result from their capacity to inhibit the voltage-sensitive Ca2+ channels.     

Action potential shortening through the putative β4-adrenoceptor in ferret ventricle: comparison with β1- and β2-adrenoceptor-mediated effects

The electrophysiological responses to (−)-CGP 12177 ((−)-4-(3-tertiarybutylamino-2-hydroxypropoxy) benzimidazol-2-one), an agonist for the putative β₄-adrenoceptor, were investigated on isolated perfused ferret hearts paced at 100 min⁻¹ and compared to those of (−)-noradrenaline and (−)-adrenaline, mediated through β₁- and β₂-adrenoceptors respectively. The three agonists decreased ventricular monophasic action potential duration but prolonged the action potential plateau; β₃-adrenoceptor-selective agonists had no effect. (−)-CGP 12177 was the most potent, but (−)-noradrenaline the most efficacious; both agonists caused ventricular extra-systoles. Because only (−)-noradrenaline but not (−)-CGP 12177 elicited shortening of the refractory period, the mechanism of arrhythmias mediated through β₁- and putative β₄-adrenoceptors may be different.     

Effects of inhaled beclomethasone dipropionate on beta2-receptor function in the airways and adrenal responsiveness in bronchial asthma

Summary Sixteen patients suffering from bronchial astham, with or without chronic bronchitits, sufficiently severe to be treated with inhaled corticosteroids, were studied in a single-blind trial (blind observer) of beclomethasone dipropionate (BDP) given in three randomized dosage regimens: 500, 1000 and 2000 µg per day, each for 4 weeks. The ₂-adrenergic agoinst response curve showed a dose-dependent increase in FEV₁ which was not affected by different doses of BDP. A small but significant reduction in basal cortisol levels was observed after BDP 500 µg/day. There was no significant difference between the various doses of BDP in reducting cortisol level and stimulation with tetracosactide remained unchanged. The study showed a gradual, dose-dependent improvement in lung function, statistically significant for morning peak expiratory flow rate at BDP 2000 µg/day. Dyspnoea score and ₂-agonist use decreased, reflecting the anti-asthmatic effects. An increase in total leukocyte count was observed, together with a decrease in the eosinophil count. Oral candidiasis was seen in 2 out of 16 patients. It is concluded that the clinical anti-asthmatic effects of corticosteroid treatment by inhalation are not due to modulation of ₂-receptor function in the airways.