B Hoiting

Modulation of agonist-induced phosphoinositide metabolism, Ca2+ signalling and contraction of airway smooth muscle by cyclic AMP-dependent mechanisms.

1 The effects of increased cellular cyclic AMP levels induced by isoprenaline, forskolin and 8‐bromoadenosine 3′:5′‐cyclic monophosphate (8‐Br‐cyclic AMP) on phosphoinositide metabolism and changes in intracellular Ca2+ elicited by methacholine and histamine were examined in bovine isolated tracheal smooth muscle (BTSM) cells. 2 Isoprenaline (pD2 (‐log10 EC50) = 6.32±0.24) and forskolin (pD2 = 5.6±0.05) enhanced cyclic AMP levels in a concentration‐dependent fashion in these cells, while methacholine (pD2 = 5.64±0.12) and histamine (pD2 = 4.90±0.04) caused a concentration‐related increase in [3H]‐inositol phosphates (IP) accumulation in the presence of 10 mM LiCl. 3 Preincubation of the cells (5 min, 37°C) with isoprenaline (1 μm), forskolin (10 μm) and 8‐Br‐cyclic AMP (1 mM) did not affect the IP accumulation induced by methacholine, but significantly reduced the maximal IP production by histamine (1 mM). However, the effect of isoprenaline was small (15.0±0.6% inhibition) and insignificant at histamine concentrations between 0.1 and 100 μm. 4 Both methacholine and histamine induced a fast (max. in 0.5‐2 s) and transient increase of intracellular Ca2+ concentration ([Ca2+]i) followed by a sustained phase lasting several minutes. EGTA (5 mM) attenuated the sustained phase, indicating that this phase depends on extracellular Ca2+. 5 Preincubation of the cells (5 min, 37°C) with isoprenaline (1 μm), forskolin (10 μm) and 8‐Br‐cyclic AMP (1 μm) significantly attenuated both the Ca2+‐transient and the sustained phase generated at equipotent IP producing concentrations of 1 μm methacholine and 100 μm histamine (approx. 40% of maximal methacholine‐induced IP response), but did not affect changes in [Ca2+]i induced by 100 μm methacholine (95.2±3.5% of maximal methacholine‐induced IP response). 6 Significant correlations were found between the isoprenaline‐induced inhibition of BTSM contraction and inhibition of Ca2+ mobilization or influx induced by methacholine and histamine, that were similar for each contractile agonist. 7 These data indicate that (a) cyclic AMP‐dependent inhibition of Ca2+ mobilization in BTSM cells is not primarily caused by attenuation of IP production, suggesting that cyclic AMP induced protein kinase A (PKA) activation is effective at a different level in the [Ca2+]i homeostasis, (b) that attenuation of intracellular Ca2+ concentration plays a major role in β‐adrenoceptor‐mediated relaxation of methacholine‐ and histamine‐induced airway smooth muscle contraction, and (c) that the relative resistance of the muscarinic agonist‐induced contraction to β‐adrenoceptor agonists, especially at (supra) maximal contractile concentrations is largely determined by its higher potency in inducing intracellular Ca2+ changes.

P2-purinoceptor-activated membrane currents and inositol tetrakisphosphate formation are blocked by suramin

The effect of suramin on the ATP-induced response in vas deferens DDT1 MF-2 smooth muscle cells was studied. Stimulation of P2-purinoceptors by ATP caused a change in membrane currents, measured by using the whole-cell patch-clamp configuration, and enhanced the formation of inositol phosphates, as analysed by high performance liquid chromatography. The ATP-induced membrane current consisted of a triphasic response, carried by a fast inward current, followed by a transient outward current and a sustained inward current. Inositol tetrakisphosphate (InsP4) formation increased in the presence of ATP. The formation of the isomers Ins(1,3,4,5)P4, Ins(1,3,4,6)P4 and Ins(3,4,5,6)P4 increased significantly after 5 min stimulation with ATP. Suramin inhibited the ATP-evoked membrane currents and the ATP-induced formation of inositol tetrakisphosphate isomers concentration dependently, but did not affect the basal inositol phosphate levels in the absence of ATP. These results indicate that suramin inhibits ATP-activated cellular processes in DDT1 MF-2 vas deferens cells, most likely by acting on P2-purinoceptors.

Regulation of histamine‐ and UTP‐induced increases in Ins(1,4,5)P3, Ins (1,3,4,5)P4 and Ca2+ by cyclic AMP in DDT1 MF‐2 cells

1 Stimulation of P2U‐purinoceptors with UTP or histamine H1‐receptors with histamine gave rise to the formation of inositol 1,4,5‐trisphosphate (Ins(1,4,5)P3) and inositol 1,3,4,5‐tetrakisphosphate (Ins(1,3,4,5)P4) in DDT1 MF‐2 smooth muscle cells. 2 Stimulation of P2U‐purinoceptors or histamine H1‐receptors caused an increase in cytoplasmic Ca2+, consisting of an initial peak, representing the release of Ca2+ from internal stores and a sustained phase representing Ca2+ influx. 3 The P2U‐purinoceptor‐mediated Ca2+‐entry mechanism was more sensitive to UTP than Ca2+‐mobilization (EC50: 3.3 μm ± 0.4 μm vs 55.1 μm ± 9.2 μm), in contrast to these processes activated by histamine H1‐receptors (EC50: 5.8 μm ± 0.6 μm VS 3.1 μm ± 0.5 μm). 4 Pre‐stimulation of cells with several adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) elevating agents, reduced the histamine H1‐receptor‐mediated formation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4. Forskolin completely inhibited Ins(1,4,5)P3 formation (IC50: 158 ± 24 nm) whereas Ins(1,3,4,5)P4 formation was inhibited by only 45% (IC50: 173 ± 16 nm). The P2U‐purinoceptor‐mediated production of these inositol phosphates was not affected by cyclic AMP. 5 Forskolin and isoprenaline reduced the histamine‐induced increase in cytoplasmic Ca2+, as measured in Ca2+ containing medium and in nominally Ca2+‐free medium but did not change the UTP‐induced increase in cytoplasmic Ca2+. 6 These results clearly demonstrate that cyclic AMP differentially regulates components of the histamine induced phospholipase C signal transduction pathway. Furthermore, cyclic AMP does not affect the phospholipase C pathway activated by stimulation of P2U‐purinoceptors in DDT1 MF‐2 cells.

CALCIUM RELEASE FROM SEPARATE RECEPTOR-SPECIFIC INTRACELLULAR STORES INDUCED BY HISTAMINE AND ATP IN A HAMSTER-CELL LINE

1. The specificity of intracellular Ca2+ stores to Ca(2+)‐mobilizing agonists was studied in DDT1 MF‐2 vas deferens cells of the Syrian hamster. 2. Application of histamine (100 microM) or ATP (100 microM) to the DDT1 MF‐2 cells caused an initial increase of intracellular Ca2+ followed by a lower phase as measured by using Indo‐1 as fluorescent probe at 22 degrees C. The basal Ca2+ level (146 nM) was enhanced to 309 nM by histamine and to 379 nM by ATP. 3. A transient rise in intracellular Ca2+ lasting for about 2 min was measured in the presence of histamine or ATP in the absence of extracellular Ca2+. The basal Ca2+ level (78 nM) was increased to 128 nM by histamine and to 145 nM by ATP. 4. A transient hyperpolarization was elicited in single cells as measured with microelectrodes by both agonists under Ca(2+)‐free conditions with a similar time course as the change in internal Ca2+. The hyperpolarization observed in the presence of histamine amounted to 23 mV and 31 mV with ATP. The histamine‐induced responses were abolished by the H1 histaminoceptor antagonist mepyramine (10 microM) and the responses evoked by ATP were blocked by the P2 purinoceptor antagonist suramin (300 microM). 5. A second internal Ca2+ response could only be evoked under Ca(2+)‐free conditions by applying a higher agonist concentration or after replenishing the intracellular stores with Ca2+ from the extracellular space. 6. A second addition of an optimal concentration (100 microM) of the agonist to the cells under Ca(2+)‐free conditions did not evoke mobilization of internal Ca2+ or hyperpolarization, but resulted in a rise of the cellular inositol (1,4,5)‐trisphosphate content (Ins(1,4,5)P3) as determined by a radioligand binding assay. 7. The cells responded to both agonists (100 microM) with a transient Ca2+ response if successively applied at a maximal effective concentration (100 microM) under Ca(2+)‐free conditions. 8. Simultaneous stimulation of H1 histaminoceptors and P2 purinoceptors resulted in the absence of external Ca2+ in an additional increase in internal Ca2+ represented by the amplitude and area of the response and in an increased response area of the hyperpolarization.(ABSTRACT TRUNCATED AT 400 WORDS)

P2 –Purinoceptor-mediated inositol phosphate formation in relation to cytoplasmic calcium in DDT1 MF-2 smooth muscle cells

The effect of P2 purinoceptor stimulation on inositol phosphate (InsP) formation in relation to the intracellular Ca2+ concentration was measured in vas deferens DDT1 MF-2 smooth muscle cells. The different [3H]myo-inositol-labelled InsP fractions were analyzed by high performance liquid chromatography and intracellular Ca2+ was determined by measuring fluorescence using Indo-1 as indicator. Stimulation with ATP (10(-4) M) resulted in an enhanced formation of inositol mono-, bis-, tris- and tetrakisphosphate (InsP1, InsP2, InsP3 and InsP4), but no changes occurred in the formation of inositol pentakis- and hexakisphosphate (InsP5 and InsP6). The putative second messenger Ins(1,3,4,5)P4 rapidly increased after addition of the agonist, reaching a maximum after about 2 min. The isomer Ins(1,4,5)P3 showed a delayed rise starting after about 2 min. The formation of Ins(1,3,4,5)P4 in the presence of ATP (2 min) was concentration-dependent, reaching a half maximal value at about 50 microM of the agonist. The intracellular Ca2+ concentration showed an initial increase after P2 purinoceptor stimulation, reaching a plateau after 2 min. Both the top of the initial phase and the plateau value of the response reached a half maximal value at an ATP concentration of about 7 microM. This Ca2+ response could be evoked repeatedly by ATP and was not affected by diltiazem (10(-5) M). In the absence of external Ca2+, the internal Ca2+ concentration increased transiently in the presence of ATP without showing the plateau phase. This response could be evoked only once under Ca2(+)-free conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

β‐Agonist‐induced constitutive β2‐adrenergic receptor activity in bovine tracheal smooth muscle

According to the two state receptor model, the β2‐adrenergic receptor (β2‐AR) isomerizes between an inactive state and a constitutively active state, which couples to the stimulatory G‐protein in the absence of agonist. In bovine tracheal smooth muscle (BTSM), we investigated the effect of short and long term β2‐AR activation by fenoterol on constitutive receptor activity. Preincubation of the BTSM strips for 5 min, 30 min and 18 h with 10 μM fenoterol, followed by extensive washout (3 h, 37°C), caused a rapid and time‐dependent inhibition of KCl‐induced contraction, reaching 68±10, 51±6 and 46±4% of control, respectively, at 40 mM KCl (P<0.05 all). At all time points, the EC50 values to KCl were significantly reduced as well. Preincubation of BTSM with 0.1, 1.0 and 10 μM fenoterol during 18 h caused a concentration‐dependent decrease of the 40 mM KCl response to 70±5, 47±12 and 43±9% of control, respectively (P<0.05 all). The reduced KCl contractions were reversed in the presence of 1 μM timolol. Moreover, the sensitivity to KCl in the presence of timolol was enhanced after fenoterol incubation. Inverse agonism was also found for other β‐blockers, with a rank order of efficacy of pindolol timolol=propranolol>alprenololsotalol>labetalol. At 25 mM KCl‐induced tone, the contraction induced by cumulative timolol administration was competitively antagonized by the less efficacious inverse agonist labetalol, indicating that the fenoterol‐induced effects cannot be explained by residual β‐agonist binding. In conclusion, fenoterol treatment of BTSM causes a time‐ and concentration‐dependent development of constitutive β2‐AR activity, which can be reversed by various inverse agonists. The β‐agonist‐induced changes could represent a novel regulation mechanism of β2‐AR activity.

α-adrenoceptor regulation of inositol phosphates, internal calcium and membrane current in DDT1 MF-2 smooth muscle cells

The effect of alpha 1-adrenoceptor stimulation on inositol phosphates (InsPs), intracellular Ca2+ and membrane current was measured in vas deferens DDT1 MF-2 cells. The InsPs were analyzed after labelling the cells with [3H]myo-inositol using high performance liquid chromatography and the internal Ca2+ concentration was determined by measuring fluorescence using Indo-1 as probe. Noradrenaline stimulated the formation of inositol mono-, bis-, tris- and tetrakisphosphate (InsP, InsP2, InsP3 and InsP1) concentration-dependently in the presence of LiCl, but no changes occurred in the formation of inositol pentakis- and hexakisphosphate (InsP5 and InsP6). Various isomers of InsP3 and InsP4 were detected after noradrenaline (10(-5) M) stimulation (without LiCl). Only the formation of the putative second messenger Ins(1,4,5)P3 formation was increased in the presence of noradrenaline. The internal Ca2+ concentration was enhanced both in the presence and absence of external Ca2+ upon addition of noradrenaline. The response in the presence of extracellular Ca2+ was not affected by diltiazem (10(-5) M). The increase in cytoplasmic Ca2+ could be repeatedly evoked, but could be elicited only once under Ca2(+)-free conditions. The relation between the noradrenaline concentration and the rise in internal Ca2+ was similar to that obtained for the InsP formation. Half-maximal effects were obtained at about 1 microM. The membrane current measured in these cells by using the whole-cell patch-clamp method was not changed by the agonist (10(-5) M). These results suggest that noradrenaline acts on DDT1 MF-2 smooth muscle cells, represented by the formation of InsP3 and enhancement of internal Ca2+ originating from internal structures, via the alpha 1B-adrenoceptor subtype.

Characteristics of the P2-purinoceptor- and H1-receptor mediated response in DDT1 MF-2 vas deferens smooth muscle cells

Original article can be found at: http://www.sciencedirect.com/science/journal/00142999 Copyright Elsevier B.V. [Full text of this article is not available in the UHRA]