Activation of adenosine A1 and bradykinin receptors increases protein kinase C and phospholipase D activity in smooth muscle cells

Since adenosine A₁ receptors activate phospholipase C (PLC) in DDT₁ MF-2 smooth muscle cells we have examined whether phospholipase D (PLD) and protein kinase C (PKC) activities are also increased. The formation of diacylglycerol was also measured. PKC activity was determined by measuring the phosphorylation of two peptide substrates after rapidly permeabilizing the cells. PLD activity was determined by measuring the formation of phosphatidylethanol.N⁶-cyclopentyladenosine, a selective adenosine A₁ receptor agonist (100 nM) and bradykinin (1 M) both stimulated the formation of diacylglycerol. The activation was biphasic with a rapid, transient increase (within 1 min) followed by a second increase. N⁶-cyclopentyl-adenosine increased the activity of PKC (EC₅₀ 5.6 nM) and PLD (EC₅₀ 18.7 nM). This was blocked by treatment of cells with pertussis toxin or the adenosine A₁ receptor selective antagonist, 8-cyclopentyl-1,3-dipropylxanthine. K_i values (3 nM for PKC; 0.1 nM for PLD) were consistent with responses mediated via adenosine A₁ receptors. Bradykinin (1 M) also increased PKC and PLD activity, but these responses were insensitive to pertussis toxin treatment. The activation of PKC by N⁶-cyclopentyladenosine or bradykinin was transient, reaching a maximum at 1–2 min, and was preceded by increases in the formation of diacylglycerol. When adenosine A₁ and bradykinin receptors were activated simultaneously, a synergistic activation of PKC was seen. There was no synergistic effect on PLD activity.In summary, the present study shows that activation of adenosine receptors of the At subtype increases PKC and PLD activity. Simultaneous activation of adenosine A₁ and bradykinin receptors causes a synergistic increase in PKC.     

Involvement of reactive oxygen species, protein kinase C, and tyrosine kinase in prostaglandin E2 production in Balb/c 3T3 mouse fibroblast cells by quinolone phototoxicity

We examined the effect of an antioxidant and protein kinase inhibitors on prostaglandin E₂ (PGE₂) release from Balb/c 3T3 mouse fibroblast cells induced by quinolone phototoxicity. Simultaneous administration of sparfloxacin (SPFX) or lomefloxacin (LFLX) at 12.5 to 100 μ M and ultraviolet-A (UVA) irradiation for 10 min markedly elevated PGE₂ concentration in the incubation medium, whereas levofloxacin (LVFX) at concentrations up to 100 μ M and UVA irradiation did not increase PGE₂ concentration. Pretreatment with 100 μ M pyrrolidine dithiocarbamate (PDTC), an antioxidant, or 1 μ M calphostin C, a selective inhibitor of protein kinase C (PKC), completely inhibited the elevation of PGE₂ in the 24-h incubation medium; pre-treatment with 10 μ M H7, a cyclic nucleotide-dependent protein kinase, and PKC or 1 μ M herbimycin A, a tyrosine kinase inhibitor, inhibited the PGE₂ elevation by 29 to 39%. Conversely, 25 nM staurosporine significantly augmented the PGE₂ elevation by quinolones plus UVA. Interleukin-1β (IL-1β ) and tumor necrosis factor α (TNFα ) were not detected in the incubation medium of 3T3 cells after quinolone plus UVA, corresponding to the lack of effect of antibodies against IL-1α , IL-1β , and TNFα on PGE₂ release from 3T3 cells. These results suggest that PGE₂ production in 3T3 cells by quinolone phototoxicity is modulated by reactive oxygen species, PKC, and tyrosine kinase, but not by IL-1 or TNFα . Received: 23 September 1997 / Accepted: 1 December 1997     

Ca(2+)-dependent and -independent mechanism of cyclic-AMP reduction: mediation by bradykinin B2 receptors.

1. Bradykinin caused a transient reduction of about 25% in the cyclic AMP level in forskolin prestimulated DDT1 MF-2 smooth muscle cells (IC50: 36.4 +/- 4.9 nM) and a pronounced, sustained inhibition (40%) of the isoprenaline-stimulated cyclic AMP level (IC50: 37.5 +/- 1.1 nM). 2. The Ca2+ ionophore, ionomycin, mimicked both the bradykinin-induced transient reduction in the forskolin-stimulated cyclic AMP level and the sustained reduction in the isoprenaline-stimulated cyclic AMP level. 3. The Ca(2+)-dependent effect on cyclic AMP induced by bradykinin was mediated solely by Ca2+ release from internal stores, since inhibition of Ca2+ entry with LaCl3 did not reduce the response to bradykinin. 4. The involvement of calmodulin-dependent enzyme activities, protein kinase C or an inhibitory GTP binding protein in the bradykinin-induced responses was excluded since a calmodulin inhibitor, calmidazolium, a PKC inhibitor, staurosporine and pertussis toxin, respectively did not affect the decline in the cyclic AMP level. 5. Bradykinin enhanced the rate of cyclic AMP breakdown in intact cells, which effect was not mimicked by ionomycin. This suggested a Ca(2+)-independent activation of phosphodiesterase activity by bradykinin in DDT1 MF-2 cells. 6. The bradykinin B1 receptor agonist, desArg9-bradykinin, did not affect cyclic AMP formation in isoprenaline prestimulated cells, while the bradykinin B2 receptor antagonists, Hoe 140 (D-Arg[Hyp3, Thi5, D-Tic7, Oic8]-BK) and D-Arg[Hyp3, Thi5,8, D-Phe7]-BK completely abolished the bradykinin response in both forskolin and isoprenaline prestimulated cells. 7. Bradykinin caused an increase in intracellular Ca2+, which was antagonized by the bradykinin B2 receptor antagonists, Hoe 140 and D-Arg[Hyp3, Thi5,8, D-Phe7]-BK.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of bradykinin B2 receptors on human IMR-90 lung fibroblasts: stimulation of 45Ca2+ efflux by D-Phe substituted bradykinin analogues.

[3H]Bradykinin binds to intact human IMR-90 fetal lung fibroblasts in a time and dose-dependent manner. Binding equilibrium was attained by 120 minutes at 4 degrees C. [3H]Bradykinin binding was saturable; Scatchard analysis of saturation binding data demonstrated a single binding site having a KD = 1.8 +/- 0.2 nM and a receptor concentration of 17.4 +/- 4.0 fmol/10(5) cells. The calculated value for KD(k-1/k1) from the association (k1 = 4.71 x 10(6) mol-1 min-1) and dissociation (k-1 = 1.13 x 10(-2) min-1) rate constants was 2.4 nM. The rank order of potency observed for bradykinin peptide agonists, bradykinin > Lys-bradykinin > Met,Lys-bradykinin > Ile,Ser-bradykinin > des-Arg9-bradykinin, is consistent with that of a bradykinin B2 receptor. Bradykinin stimulated efflux of 45Ca2+ from IMR-90 cells dose dependently with an EC50 = 331 +/- 50 pM. 45Ca2+ efflux was also demonstrated with Lys-bradykinin and Met-Lys-bradykinin but not by des-Arg10-kallidin (100 nM) or NKA (1 microM). Hoe-140 inhibited bradykinin-induced 45Ca2+ efflux (IC50 = 3 +/- 2 nM). D-Phe7-substituted bradykinin analogues stimulated 45Ca2+ efflux dose dependently and this stimulation of 45Ca2+ efflux was inhibited by Hoe-140. These results suggest that D-Phe7 substituted bradykinin analogues are agonists at the bradykinin B2 receptor in IMR-90 cells.     

Enhancement by captopril of bradykinin-induced calcium transients in cultured endothelial cells of the bovine aorta

Using microscopic fluorometry and fura-2-loaded cultured bovine aortic endothelial cells, we determined the effects of captopril, an angiotensin converting enzyme (ACE) inhibitor, on bradykinin-induced Ca²⁺ transients in endothelial cells. In the presence of extracellular Ca²⁺, 10⁻⁹ M bradykinin induced an early rise in the transients followed by sustained elevations of cytosolic calcium concentration ([Ca²⁺]_i). Bradykinin concentration-dependently increased [Ca²⁺]_i (EC₅₀ 6.7 × 10⁻⁹ M). Captopril, 10⁻⁵ M, enhanced and prolonged the bradykinin-induced Ca²⁺ transients and shifted the concentration-response curve to the left (EC₅₀ 8.5 × 10⁻¹⁰ M). In porcine coronary aterial strips with intact endothelium, cumulative applications of bradykinin induced an endothelium-dependent relaxation during prostaglandin F_2α-induced contraction (EC₅₀ = 2.0 × 10⁻⁹ M). Treatment with 10⁻⁵ M captopril enhanced the bradykinin-induced relaxation and shifted the concentration-response curve to the left (EC₅₀ = 7.6 × 10⁻¹⁰ M). Thus, captopril enhances the bradykinin-induced relaxation by mechanisms mainly dependent on the endothelium, namely the inhibition of ACE.     

Relaxant effect of prostaglandin D(2)--receptor DP agonist on liver myofibroblast contraction

Increased intrahepatic resistance causes portal hypertension in cirrhosis. Liver myofibroblasts (MFs) are now regarded as the principle cells involved in sinusoidal blood flow regulation. Many other prostaglandin-receptor agonists have been reported to regulate liver MF contraction, but the role of the prostaglandin D(2)-receptor DP is unknown. In this study, we investigated the effect of a synthetic agonist of prostanoid DP receptor, BW245C, on contractile properties of primary rat liver MFs. Collagen gel contraction assay revealed that BW245C alone (1 and 10 μM) did not induce contraction but induced cell relaxation. Pretreatment with BW245C (10 μM, 30 min) attenuated bradykinin (100 nM)-induced liver MF contraction. Elevation of [Ca(2+)](i) induced by bradykinin (100 nM) was partially suppressed by BW245C pretreatment (10 μM, 3 min). BW245C (1 and 10 μM) significantly increased intracellular cAMP level in a dose-dependent manner. Pretreatment with forskolin (30 - 300 nM, 30 min) and dibutyryl-cAMP (3 - 30 μM, 30 min) significantly reduced bradykinin-induced contraction. Furthermore, a protein kinase A (PKA) inhibitor KT5720 (10 nM to 1 μM, 30 min) blocked the relaxant effect of BW245C. These results suggest that prostanoid DP receptor agonism inhibits bradykinin-induced [Ca(2+)](i) elevation and contraction through cAMP-PKA signal activation in rat liver MFs.     

Differential sensitivity of macrophages to bradykinin

In this report, we investigated the responsiveness of subpopulations of elicited peritoneal macrophages between each other compared to resident tissue macrophages of alveoli of guinea pig to the action of bradykinin. Bradykinin stimulated the secretion of superoxide radical, arachidonic acid and prostaglandin E₂ (PGE₂) via the bradykinin B₂ receptor subtype in peritoneal macrophages, indicated by complete inhibitory effect of the bradykinin B₂ receptor antagonist HOE 140. The extent of the secretion, however, varied substantially between macrophages of different size. The highest level of the secretion was observed in the fraction containing the intermediate-size macrophages, while progressively lower level of the secretion was observed with decreasing size. In contrast, large macrophages obviously lost their secretory ability. Additionally, the bradykinin-stimulated release of cyclooxygenase products exerted an inhibitory action on NADPH-oxidase activity depending on size and stage of maturation/activation of macrophages, as judged by an increase in superoxide radical generation by indomethacin (100 μM) preincubation of cells. 2 receptor subtype. However, the receptor activity measured by bradykinin-induced increase in intracellular free calcium [Ca²⁺]_i was very low compared to elicited peritoneal macrophages. These findings indicate that the stage of differentiation/maturation and activation of macrophages may be important for the ability of bradykinin to stimulate these cells to inflammatory responses in vivo. Received: 27 June / Accepted: 17 October 1997     

Modulating effects of opioids,purine compounds, 5-hydroxytryptamine and prostaglandin E2 on cholinergic neurotransmission in a guinea-pig oesophagus preparation

The submucous plexus-longitudinal muscularis mucosae preparation of the guinea-pig oesophagus was used to study the actions of morphine, opioid peptides, purine compounds, 5-hydroxytryptamine (5-HT) and prostaglandin E₂ (PGE₂) on electrically-induced twitch contractions which are probably mediated by cholinergic nerve stimulation. The twitch contractions were inhibited by morphine (1–100 μM), methionine-enkephalin (1–100 μM) and β-endorphin (0.1–1 μM), but increased by adenosine (1–30 μM), adenosine 5′-triphosphate (1–30 μM), 5-HT (0.01–3 μM) and PGE² (1–10 nM). The submaximal contraction induced by acetylcholine (12 or 20 nM) which is nearly equivalent to the twitch contractions was unaffected by morphine, methionine-enkephalin, β-endorphin and 5-HT, but augmented by purine compounds and PGE₂. It is concluded that cholinergic neurotransmission in the submucous plexus-longitudinal muscularis mucosae of the guinea-pig oesophagus is inhibited by morphine and opioid peptides acting at prejunctional opiate receptors, and facilitated by 5-HT, purine compounds and PGE₂ via prejunctional or postjunctional mechanisms.     

Characterization of bradykinin receptors mediating catecholamine release in PC12 cells

The rat pheochromocytoma cell line PC12, which is a widely used model for analyzing stimulus-secretion coupling, was investigated for the effects of kinins on catecholamine release. Subtypes of kinin receptors were characterized using the B₁ agonist desArg⁹-bradykinin, the B₂ agonist bradykinin and the B₂ antagonists [Thi^5,8, D-Phe⁷]-bradykinin, D-Arg[Hyp³, D-Tic⁷, Oic⁸]-bradykinin (HOE 890307) and D-Arg-[Hyp³, Thi⁵, D-Tic⁷, Oic⁸]-bradykinin (HOE 140). The effectiveness of acute and chronic exposure to angiotensin I converting enzyme inhibitors as well as pretreatment of the cells with bacterial lipopolysaccharides in modulating B₁ or B₂ receptor systems was also tested.Bradykinin stimulated noradrenaline release from PC12 cells at low concentrations (EC₅₀ = 1 nM), maximally inducing a release of 43.7% of the cellular content within 15 min. In comparison with acetylcholine and K⁺-induced depolarization, bradykinin was the most effective stimulus. DesArg⁹-bradykinin was only effective at very high concentrations (> 30 M). Like in other neuronal cells, the B₂-specific partial antagonist [Thi^5,8, D-Phe⁷]-bradykinin acted as a low-affinity agonist without any antagonistic effects. The B₂ antagonists HOE 890307 and HOE 140 exerted no agonistic effects and concentration-dependently inhibited bradykinin-induced noradrenaline release, showing competitive antagonism with K_i values of 1.38 nM and 0.66 nM, respectively. Only at the highest concentration used (1 M), HOE 140 did depress the maximal response to bradykinin. HOE 890307 also abolished the effects of desArg⁹-bradykinin and [Thi^5,8, D-Phe⁷]-bradykinin. Acute or chronic inhibition of the angiotensin I converting enzyme or application of lipopolysaccharides, which all can lead to induction of the B₁ receptor subtype in vivo, did not alter the secretory response of PC12 cells to either bradykinin (0.1 and 30 nM) or desArg⁹-bradykinin (1 M).In conclusion, noradrenaline release from PC12 cells is stimulated via B₂, but not B₁, receptors. Despite the fact that the receptor system is highly susceptible to stimulation by low-affinity ligands, HOE 890307 and HOE 140 are pure antagonists, with only high concentrations of HOE 140 (> 1 M) showing a non-competitive type of inhibition. Induction of B₁ receptors which could stimulate noradrenaline release could not be demonstrated in this model. The possible role of bradykinin in modulating sympathetic neurotransmission during inhibition of angiotensin I converting enzyme is discussed.     

PARTICIPATION OF PROSTANOIDS IN CHEMICAL ACTIVATION OF THE PERICARDIAL PRESSOR REFLEX IN DOGS

1. Experiments were performed on anaesthetized, open-chest dogs to determine the reflex effects on systemic blood pressure and heart rate produced by stimulation of the parietal pericardium with bradykinin, prostacyclin, prostaglandin E₂ (PGE₂), prostaglandin D₂ (PGD₂) and arachidonic acid. 2. Pericardial application of bradykinin (1 μg) consistently elicited reflex increases in blood pressure and heart rate, whereas application of prostanoids or arachidonic acid in doses up to 10 μg failed to produce any cardiovascular responses. 3. Indomethacin, applied either directly to the parietal pericardium (1 μg/ml) or given intravenously (5 mg/kg) caused a long lasting reduction of the reflex responses to bradykinin. The reflex effects of bradykinin could be temporarily restored by treatment of the pericardium with either prostacyclin (0.1 μg/min) or PGE₂ (0.1 μg/min). PGD₂ (0.1-1 μg/min) did not influence the bradykinin induced pericardial reflex. 4. Superfusion of arachidonic acid (3 μg/min) over the pericardium amplified the reflex effects of bradykinin when given before, but not when given after indomethacin treatment. 5. The results indicate that locally formed prostanoids, specifically prostacyclin and PGE₂, can facilitate activation of the pericardial pressor reflex by bradykinin. The findings may be relevant to the changes in cardiovascular activity occurring during pericardial inflammation.     

Evidence for the involvement of multiple mechanisms in the excitatory action of bradykinin in the circular muscle of guinea-pig colon

We have investigated, by using the sucrose gap technique, the mechanisms of the excitatory action of bradykinin in the circular muscle of the guinea-pig proximal colon. In the presence of atropine (1μM) and S-ketoprofen (3μM), the application of bradykinin (1μM for 20s) produced complex changes in membrane potential and muscle tension. The prevailing response was a small hyperpolarization followed by a slowly developing depolarization and a tonic contraction. The selective B₂ receptor antagonist, HOE 140 (0.3μM) blocked the responses to bradykinin (1μM) while tetrodotoxin (0.3μM) had no affect. The selective B₁ receptor agonist, [des-Arg⁹]bradykinin (1μM) did not affect the electrical or mechanical activities of the circular muscle. Apamin (0.1μM) blocked the transient hyperpolarization and potentiated the bradykinin-induced depolarization and contraction. In the presence of apamin, nifedipine (1μM) blocked spikes (when present) and the phasic contraction while leaving the tonic contraction unaffected. The excitatory action of bradykinin was further investigated in the presence of atropine (1μM), S-ketoprofen (3μM), apamin (0.1μM) and nifedipine (1μM). The depolarization but not the contraction induced by bradykinin was reduced by about 30% in low-Na⁺ (25mM) but not in low Cl^– (9.7mM) Krebs solution. The depolarization and contraction evoked by bradykinin were reduced (by about 30 and 75%, respectively) in Ca²⁺-free (2min) Krebs solution. The blocker of the sarcoplasmic reticulum Ca²⁺ pump, cyclopiazonic acid (CPA, 10μM) reduced the nifedipine-resistant depolarization and contraction induced by bradykinin by about 40 and 60%, respectively. The inhibitor of receptor-operated cation channels, SKF 96365 (50μM) reduced the nifedipine-resistant bradykinin-induced depolarization and contraction by about 40 and 30%, respectively, whereas the inhibitor of Ca²⁺-dependent chloride channels, niflumic acid (100μM) was without effect. The inhibitory effect of SKF 96365 (50μM) and CPA (10μM) was additive: in the presence of both drugs the bradykinin-induced depolarization and contraction were reduced by about 70–80%. The proteine kinase C inhibitor, GF 109203x (10μM) did not affect the nifedipine-resistant bradykinin-induced depolarization and contraction. At a concentration of 30μM, GF 109203x reduced the bradykinin-induced contraction by about 50% while leaving the bradykinin-induced depolarization unaffected. The KCl (40mM)-induced contraction was significantly reduced (by about 30%) by GF 109203x (30μM). The present findings indicate that, in the presence of apamin and nifedipine, the bradykinin-induced contraction of circular muscle of the guinea-pig colon is due to the influx of extracellular Ca²⁺ via non-selective cation channels and, in part, to the release of Ca²⁺ from a loosely bound internal store. Intracellular Ca²⁺ facilitates the bradykinin-induced depolarization, a response which does not involve a protein kinase C-dependent mechanism. Received: 4 August 1997 / Accepted: 26 November 1997     

Characteristics of the bradykinin-induced changes in intracellular calcium ion concentration of single bovine tracheal smooth muscle cells.

1. Single bovine tracheal smooth muscle (BTSM) cells were cultured and used to measure bradykinin-induced changes in [Ca2+]i by dynamic video imaging. 2. Bradykinin (10 pM-10 microM)-induced an increase in [Ca2+]i over basal levels (69 +/- 2 nM; n = 353) which was concentration-dependent (log EC50 = -8.7 M) in the presence of extracellular calcium ions (2 mM). The bradykinin B2 receptor antagonist, D-Arg[Hyp3,Thi5,8,D-Phe7]- bradykinin, produced a parallel shift to the right of the bradykinin concentration-response curve (log EC50 = -7.1 M and -5.8 M in the presence of 1 microM and 10 microM antagonist respectively) yielding an apparent KD of 26 nM. 3. In the absence of extracellular calcium ions (with 0.1 mM EGTA), bradykinin (10 pM-10 microM) produced a uniform increase in [Ca2+]i from a basal level of 33 +/- 2 nM (n = 140) to approximately 180 nM in BTSM cells indicating an 'all-or-nothing' release of intracellular calcium ions. In the presence of 10 microM D-Arg[Hyp3,Thi5,8,D-Phe7]-bradykinin no responses could be induced by bradykinin at concentrations below 100 nM. However, at 100 nM and 1 microM bradykinin there was no change in the uniform increase in [Ca2+]i in these cells previously observed. 4. In both the absence or presence of D-Arg[Hyp3,Thi5,8,D-Phe7]-bradykinin, there was a concentration-dependent increase in the percentage of cells responding to bradykinin (frequency) under calcium-rich or calcium-free conditions. Individual cells also demonstrated a difference in the sensitivity to any particular concentration of bradykinin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes and new initiatives for the Journal

Abstract: The synthesis and pharmacological evaluation of dimer derivatives of the C-terminal fragments of the potent bradykinin antagonist HOE-140, linked through their N-termini, were performed. The influence of peptide moiety length was studied using the succinyl moiety as a linker. Our attention focused on the dimer of the C-terminal tetrapeptide of HOE-140 (compound JMV 980 ), which displayed some inhibiting activity (IC₅₀= 247 nM) for bradykinin B₂ receptors. Unexpectedly, it was orally active in inhibiting bradykinin-induced hypotension in the rat. Based on this tetrapeptide dimer model, we synthesized pseudotetrapeptide dimer bradykinin antagonists 29 and 33 , which exhibited high affinity (K_i= 76 and 61 nM, respectively) for the human cloned B₂ receptor. In addition, compound 29 inhibited bradykinin-induced contraction of the human umbilical vein giving a pK_B value of 6.45. Compounds 29 and 33 were selective toward B₂, receptors because they did not bind to the cloned human B₁ receptor up to 10 μM.     

P-glycoprotein phosphorylation/dephosphorylation and cellular accumulation of L-DOPA in LLC-GA5 Col300 cells

1 The present work was aimed to study the effect of PKC activation and protein-serine/threonine phosphatase (PP1/PP2 A) inhibition on P-glycoprotein (P-gp) mediated transport of L-DOPA in LLC-GA5 Col300 cells, a renal cell line expressing the human P-glycoprotein in the apical membrane. 2 L-DOPA accumulation was a time-and concentration-dependent process with the following kinetic characteristics: k_in, 57.3 ± 1.2 pmol mg protein^?1 min^?1; k_out, 3.3 ± 0.1 pmol mg^?1 protein min^?1; A_max, 10.6 ± 0.8; K_m, 198 ± 64 μm ; V_max, 5.2 ± 0.7 nmol mg protein^?1. 3 Verapamil (25 μm ), a P-glycoprotein inhibitor, markedly increased (≈ 40% increase) the accumulation of a non-saturating concentration of L-DOPA (2.5 μm ) at both initial rate of uptake (IRU, 6 min incubation) and at steady-state (SS, 30 min incubation). 4 PKC activation with phorbol 12,13-dibutyrate (PDBu, 1, 3 and 10 n m ) produced a concentration-dependent decrease in L-DOPA accumulation at SS, but not at IRU. The inactive phorbol ester, 4α-phorbol 12,13-didecanoate (100 n m ), produced no change in L-DOPA accumulation. The effect of PDBu was completely reverted by staurosporine (100 n m ). The phosphatase inhibitor okadaic acid (100 n m ) reduced by 20% the accumulation of L-DOPA at IRU, but not at SS. 5 It is suggested that P-glycoprotein plays a role in regulation of intracellular availability of L-DOPA in renal epithelial cells, and phosphorylation/dephosphorylation of P-glycoprotein may be involved in the regulation of the transporter.     

Regulation of cyclic AMP levels by phorbol esters in bovine adrenal medullary cells

Cyclic AMP responses to phorbol esters were studied in cultured bovine adrenal medullary cells. Phorbol esters that activate protein kinase C (PKC: phorbol 12,13-dibutyrate, phorbol 12,13-didecanoate) increased cellular cyclic AMP levels by up to 100% over 5 min, and this was maintained for up to 3 h. The effect was mimicked by 1,2-dioctanoyl-sn-glycerol but not by inactive phorbol esters. The effect of active phorbol esters was concentration dependent over the range 50–500 nM, and was abolished by the PKC inhibitor, Ro 31–8220 (10μM). The response was enhanced by 3-isobutyl-1-methylxanthine (1 mM) and by forskolin (0.3 μM), was enhanced following pertussis toxin pretreatment (100 ng/ml, 7.5 h) and was unaffected by removing extracellular Ca²⁺. The phorbol ester cyclic AMP response was additive with that to K⁺ depolarisation, and synergised with those to prostaglandin E₁ and dimaprit. The results indicate PKC activation increases cyclic AMP formation in bovine adrenal medullary cells, probably by a direct action on adenylate cyclase or G_s.     

The character of the antagonism by polyphloretin phosphate of contractions to prostaglandins E1 and F2α in guinea-pig ileum

Polyphloretin phosphate (PPP) produced a dose-dependent decrease in the tone and reduction of the spontaneous phasic contractions of the longitudinal muscle of guinea-pig isolated ileum. PPP (100 μg ml^?1) after a 2 min contact with the ileum decreased the contractile effects of PGE₁ 0·1 μM by 40·6 ± 7·4%, of PGE₁ 0·01 μM by 86·7 ± 3·3% and of PGF_2α 0·1 μM by 62·2 ± 8·6%. After 10 min contact of PPP the contractile effect of PGE₁ 0·1 μM was decreased by 47·7 ± 4·7% and that of PGF_2α0·1 μM by 89·6 ± 1·7%. When the contact was longer, PPP showed a pronounced after-effect in respect to the effects of PGE₁ and particularly of PGF_2α. PPP significantly reduced contractions to 5-HT and BaCl₂, but not to acetylcholine, histamine or substance P. The type of antagonism of PGE₁ by PPP was examined using cumulative concentration-effect curves for PGE₁ in the presence of increasing concentrations of PPP. We conclude that on guinea-pig ileum PPP acts as a non-competitive antagonist of PGE₁ and PGF_2α.     

Accumulation of adenosine 3′, 5′-monophosphate induced by prostaglandin E1 binding to mastocytoma P-815 cells

Addition of prostaglandin E₁ (PGE₁) to intact mouse mastocytoma P-815 cells stimulated adenosine 3', 5'-monophosphate (cAMP) accumulation and retarded cellular growth. To study the effects of prostaglandin (PG) binding on cAMP accumulation, specific [³H]PG binding to intact mastocytoma cells was examined. Intact mastocytoma cells have two types of binding sites for PGE₁ with high (K_d = 2.14 × 10^?9 M) and low (k_d = 1.05 × 10^?8 M) affinities and one type of binding site for PGF_2α. Mastocytoma cells, however, did not have a binding site for PGA₁ or PGD₂. The order of potencies of nonradioactive prostaglandins in competing with [³H]PGE₁ for binding sites was as follows: PGE₁ ? PGE₂ ? PGF_2α ? PGA₁ ? PGD₂ ? PGI₂. In contrast, the relative potencies of the prostaglandins in enhancing cAMP accumulation were PGI₂ ? PGE₁ ? PGE₂ ? PGA₁ ? PGF_2α = PGD₂, indicating that the receptors for E type and I type of PGs were different. Refractoriness of mastocytoma cells to PGE₁ stimulation of cAMP accumulation developed within 1 min of incubation of the cells at 37°, but disappeared immediately after decreasing the temperature to below 23°. A change in the number of PGE₁ receptors was not observed. cAMP concentrations were quickly restored by increasing temperatures from below 23° to 37°. This refractoriness did not develop in the presence of phosphodiesterase inhibitors. Furthermore, the activity of phosphodiesterase in mastocytoma cells was enhanced within 2 min by PGE₁ treatment.     

Pharmacology and mode of action of bradykinin on mouse-isolated trachea

In the present study, the effect of bradykinin on basal and precontracted mouse-isolated trachea was investigated. In basal conditions mouse-isolated tracheal rings do not respond to bradykinin. However, when the tracheal rings were precontracted with carbachol (10^–7M) a relaxation with bradykinin (3·10^–9–3·10^–7) was found. The maximal response amounted 69.7±4.1% (n=15) with a pD₂ value of 7.2±0.21. The selective bradykinin B₂ receptor antagonist HOE 140 (10^–10–10^–8M) antagonized the bradykinin-induced relaxation, while the bradykinin B₁ receptor antagonist des-Arg⁹-Leu⁸-bradykinin (10^–6M) had no influence. The selective bradykinin B₁ receptor agonist des-Arg⁹-bradykinin (10^–6M) caused a small relaxation (8.4±2.5%, n=6), which could be antagonized completely by the selective bradykinin B₁ receptor antagonist des-Arg⁹-Leu⁸-bradykinin (10^–6M) while addition of the selective bradykinin B₂ receptor antagonist HOE 140 (10^–8M) was without effect. In the presence of indomethacin (10^–6M) the relaxation of bradykinin was completely abolished. Pretreatment of the tracheal rings with capsaicin, or the presence of the selective NK₁ receptor antagonist RP 67851 (10^–6M) or the presence of the nitric oxide synthase inhibitor L-NAME (3·10^–4M) had no effect on the bradykinin-induced relaxation. In conclusion, these results demonstrate that the mouse-isolated tracheal is a preparation in which bradykinin exerts a relaxant response via stimulation of bradykinin B₂ receptors. This response is probably mediated by prostaglandines. Received: 14 November 1996 / Accepted: 5 March 1997     

Inhibition of prostanoid formation in intact cells by 2,5-di-(tert-butyl)-1,4-benzohydroquinone,a blocker of Ca(2+)-ATPases.

1 The blocker of endoplasmic reticulum Ca(2+)-ATPase, 2,5-di-(tert-butyl)-1,4-benzohydroquinone (BHQ) was shown to inhibit formation of prostaglandin E2 and prostacyclin in the osteoblast-like cell lines, MC3T3-E1 and ROS 17/2.8, respectively, in a dose-dependent manner with an IC50 of 0.5-1 microM. Inhibition was observed with various stimuli (arachidonic acid, bradykinin, melittin and calcium ionophore, A23187). 2 This effect was also observed in human platelets, where BHQ dose-dependently blocked thromboxane biosynthesis and formation of 12-hydroxy-heptadecatrienoic acid after stimulation with arachidonic acid, but not formation of 12-hydroxy-eicosatetraenoic acid. 3 Inhibition of prostaglandin E2 formation in MC3T3-E1 cells was not observed with thapsigargin after stimulation with arachidonic acid, A23187 or melittin, whereas bradykinin-induced prostaglandin E2 biosynthesis was blocked. 4 Taken together, the results suggest a direct inhibitory action of BHQ on the cyclo-oxygenase in these three cell systems.     

Independent coupling of the human tachykinin NK2 receptor to phospholipases C and A2 in transfected Chinese hamster ovary cells

The human tachykinin NK₂ receptor stably expressed in Chinese hamster ovary cells (CHO-hNK₂R cells) was characterized by studying the effect of neurokinin A (NKA), the preferred natural ligand, and that of other agonists and antagonists in both binding experiments and functional assays. Competition experiments using [¹²⁵I]NKA showed that CHO-hNK₂R cells express binding sites which have high affinity for NKA (K _i=3.4±0.9 nM), GR 64349 (K _i=12±3 nM) and [βAla⁸]NKA(4–10) (K _i=21±8 nM) and for the antagonists MEN 10627 (K _i=0.55±0.2 nM), and MEN 11420 (K _i=2.4±0.8 nM). In contrast, the tachykinin NK₁ and NK₃ receptor agonists [Sar⁹,Met(O₂)¹¹]SP and senktide, respectively, were recognized with low affinity (K _i>10 μM). NKA (EC₅₀=68±18 nM) induced a rapid and concentration-dependent increase in the intracellular level of inositoltrisphosphate (IP₃). The concentration-response curve to GR 64349 (EC₅₀=155±14 nM) was close to that of NKA, whereas [βAla⁸]NKA(4–10) (EC₅₀=445±78 nM) and SP (EC₅₀=3197±669 nM) were 7- and 50-fold less potent, respectively. In addition, NKA stimulated the release of arachidonic acid and the production of prostaglandin E₂ (PGE₂) in a concentration-dependent manner. Also in this assay, NKA was found to be more potent than the other agonists tested (the EC₅₀ values were 3±0.3, 9±3, 7.8±0.9 and 217±37 nM for NKA, GR 64349, [βAla⁸]NKA(4–10) and SP, respectively). MEN 10627 and MEN 11420 were potent and competitive antagonists in blocking NKA-induced IP₃ formation and PGE₂ release: MEN 10627 and MEN 11420 displayed comparable potencies in blocking the two functional responses initiated by occupancy of the NK₂ receptor by NKA. Pretreatment of the cells with pertussis toxin (500 ng/ml for 18 h) did not significantly modify the basal or stimulated phosphatidylinositol turnover but reduced the basal and NKA-induced PGE₂ release by about 35%. The phospholipase C inhibitor U-73122 (10 μM) prevented the NKA-induced formation of IP₃ but did not affect PGE₂ release. Conversely, the phospholipase A₂ inhibitor quinacrine (100 μM) blocked the release of arachidonic acid and PGE₂ without affecting the NKA-stimulated formation of IP₃. Chelation of extracellular calcium with 3 mM EGTA inhibited the NKA-induced PGE₂ release by 81% but was without effect on basal and NKA-stimulated IP₃ production. The calcium channel blockers verapamil (10 μM) and ω-conotoxin GVIA (0.1 μM) did not modify the basal PGE₂ production and had no significant effect on the response to tachykinins while the blocker of non-selective cation channels, SKF-96365 (10 μM), inhibited the response to NKA by about 74%. SKF-96365 did not affect the basal or the NKA-induced IP₃ formation. In conclusion, our data demonstrate that the human tachykinin NK₂ receptor expressed in CHO cells displays binding affinity and functional properties which are those of a native NK₂ receptor. No pharmacological evidence for heterogeneity of the human NK₂ receptor was obtained in this study. Our findings indicate that the human tachykinin NK₂ receptor is independently coupled to both PLC and PLA₂ signaling pathways. Activation of the PLA₂ pathway may be linked to the opening of a voltage-independent cation channel which activates a Ca²⁺-dependent PLA₂. Received: 7 April 1998 / Accepted: 17 July 1998