8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8) acts as a muscarinic receptor antagonist in the epithelial cell line HT29

8-(N, N-diethyl amino) octyl-3,4,5-trimethoxybenzoate (TMB-8) is a widely used pharmacological tool to investigate the involvement of intracellular Ca²⁺ stores in cellular responses. In this study we investigate the effect of TMB-8 as a putative inhibitor of Ca²⁺ signalling in single fura-2 loaded HT₂₉ coIonic epithelial cells stimulated by ATP, carbachol (CCH) and neurotensin (NT). TMB-8 effectively inhibited the CCH-induced (100 mol/l intracellular Ca²⁺ ([Ca²⁺]_i) transient with an IC₅₀ of 20 mol/l. However, [Ca²⁺]_i transients induced by other phospholipase C coupled agonists ATP (10 mol/l, n = 4) and NT (10 nmol/l, n = 4) remained unaffected by TMB-8 (50 mol/l). The agonist-induced [Ca²⁺]_i transients remained equally unaffected by 100 mol/l TMB-8 when the stimulatory concentration was reduced to 0.5 mol/I for ATP (n = 4) or 1 nmol/l for NT (n = 4). The competitive nature of the TMB-8-induced inhibition of the CCH-induced [Ca²⁺]_i transient was demonstrated by examining the agonist at various concentrations in absence and presence of the antagonist. High TMB-8 concentrations (100 mol/l) alone induced a small [Ca²⁺]_i increase ([Ca²⁺]_i: 40 ± 5 nmol/l, n = 7). We assume that this increase is a consequence of a TMB-8 induced intracellular alkalinization ( pH: 0.1 ± 0.02, n = 7) occurring simultaneously with the increase in [Ca ⁺]_i. From these results we draw the following conclusions: (1) In sharp contrast to a large number of other studies, but in agreement with studies in other types of cells, these results substantially challenge the value of the tool TMB-8 as an intracellular Ca²⁺ antagonist; (2) TMB-8 acts a muscarinic receptor antagonist at the M₃ receptor; (3) TMB-8 does not influence the release of Ca²⁺ from intracellular stores when IP₃ signal transduction is activated by ATP or NT; (4) TMB-8 as a weak organic base alkalinizes the cytosol at high concentrations; and (5) TMB-8 induces small [Ca²⁺]_i transients at higher concentrations.     

Evidence for the existence of P2Y<Subscript>1,2,4</Subscript> receptor subtypes in HEK-293 cells: reactivation of P2Y<Subscript>1</Subscript> receptors after repetitive agonist application

ATP, ADPS and UTP induced a comparable rise in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in HEK-293 cells using fura-2 microfluorimetry. The responses persisted in Ca²⁺-free medium, but were abolished following depletion of intracellular Ca²⁺ stores by cyclopiazonic acid. Cross-desensitisation experiments demonstrated that exposure to ADPS has no marked effect on UTP-induced [Ca²⁺]_i transients and vice versa. Whereas the P2Y₁ receptor-selective antagonist 2-deoxy-N⁶-methyladenosine 3,5-diphosphate (MRS 2179) abolished the responses to ADPS, it decreased and did not alter the responses to ATP and UTP respectively. Although the P2Y₁/P2Y₄ receptor-preferential antagonist pyridoxalphosphate-6-azophenyl-2,4-disulphonic acid (PPADS) abolished the responses to ADPS, and decreased those to ATP, it also depressed the UTP-induced [Ca²⁺]_i transients. Suramin, an antagonist with preference for P2Y₂ receptors decreased both the ATP- and UTP-induced [Ca²⁺]_i reactions. After numerous splittings, HEK-293 cells failed to react to ADPS; however, repeated superfusion with this P2Y₁ receptor agonist restored the [Ca²⁺]_i signals. In agreement with the functional data, real-time polymerase chain reaction and immunocytochemical studies indicated the presence of P2Y₁, P2Y₂ and P2Y₄ receptors. Our findings raise doubt with respect to the reliability of HEK-293 cells as expression systems for recombinant P2X receptors, because of a possible functional interaction with endogenous P2Y receptors.     

K+ channel openers,cromakalim and Ki4032, inhibit agonist-induced Ca2+ release in canine coronary artery

Summary The effects of K⁺ channel openers, cromakalim and an acetoxyl derivative of KRN 2391 (Ki 4032), were studied on force of contraction, increases in intracellular calcium concentration ([Ca²⁺]_i) measured by fura-2 and inositol 1,4,5-trisphosphate (IP₃) production induced by the thromboxane A₂ analogue, U46619, in canine coronary arteries. Upon single dose applications of U46619 at 300 nmol/l, phasic and tonic increases in [Ca²⁺]_i and force were seen, which were almost abolished by cromakalim (10 mol/l) and Ki4032 (100 mol/l).In the absence of extracellular Ca²⁺, U46619 induced a transient increase in [Ca²⁺]_i with a contraction. Cromakalim (0.01–10 mol/l) and Ki4032 (0.1–100 mol/l) concentration-dependently inhibited the increases in [Ca²⁺]_i and contraction. The inhibitory effects of cromakalim and Ki4032 were blocked by the K⁺ channel blocker tetrabutylammonium (TBA) and counteracted by 20 mmol/l KCl-induced depolarization. Cromakalim and Ki4032 did not affect caffeine-induced Ca²⁺ release. Cromakalim reduced U46619-induced IP₃ production significantly and TBA blocked this inhibitory effect. These results suggest that the hyperpolarization of the plasma membrane by K⁺ channel openers inhibits the production of IP₃ and Ca ²⁺ release from intracellular stores related to stimulation of the thromboxane A₂ receptor.Correspondence to T. Yanagisawa at the above address     

Independent [Ca2+]i increases and cell proliferation induced by the carcinogen safrole in human oral cancer cells

The effect of the carcinogen safrole on intracellular Ca²⁺ movement and cell proliferation has not been explored previously. The present study examined whether safrole could alter Ca²⁺ handling and growth in human oral cancer OC2 cells. Cytosolic free Ca²⁺ levels ([Ca²⁺]_i) in populations of cells were measured using fura-2 as a fluorescent Ca²⁺ probe. Safrole at a concentration of 325 M started to increase [Ca²⁺]_i in a concentration-dependent manner. The Ca²⁺ signal was reduced by 40% by removing extracellular Ca²⁺, and was decreased by 39% by nifedipine but not by verapamil or diltiazem. In Ca²⁺-free medium, after pretreatment with 650 M safrole, 1 M thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor) barely induced a [Ca²⁺]_i rise; in contrast, addition of safrole after thapsigargin treatment induced a small [Ca²⁺]_i rise. Neither inhibition of phospholipase C with 2 M U73122 nor modulation of protein kinase C activity affected safrole-induced Ca²⁺ release. Overnight incubation with 1 M safrole did not alter cell proliferation, but incubation with 10–1000 M safrole increased cell proliferation by 60±10%. This increase was not reversed by pre-chelating Ca²⁺ with 10 M of the Ca²⁺ chelator BAPTA. Collectively, the data suggest that in human oral cancer cells, safrole induced a [Ca²⁺]_i rise by causing release of stored Ca²⁺ from the endoplasmic reticulum in a phospholipase C- and protein kinase C-independent fashion and by inducing Ca²⁺ influx via nifedipine-sensitive Ca²⁺ entry. Furthermore, safrole can enhance cell growth in a Ca²⁺-independent manner.     

Levcromakalim-induced modulation of membrane potassium currents,intracellular calcium and mechanical activity in rat mesenteric artery

In freshly-dispersed cells from rat mesenteric artery, levcromakalim (1 and 10 M) induced a non-inactivating potassium current (I_KCO), an event which was associated with increased current noise. I_KCO was fully inhibited in the presence of 10 M glibenclamide. Stationary fluctuation analysis of the current noise associated with I_KCO induced by levcromakalim at a holding potential of –10 mV indicated that the unitary conductance of the underlying K-channels was 10.2 pS at 0 mV under the quasi-physiological conditions of the experiment.In isolated arterioles both levcromakalim (10 nM - 10 M) and nifedipine (10 nM - 10 M) each elicted full, concentration-dependent, parallel reductions of the increases in [Ca²⁺]_i (assessed using fura-2) and tension induced by 10 M noradrenaline. However, the effects of both drugs on KCl-induced increases in tension and in [Ca²⁺]_i, did not follow a simple relationship. Levcromakalim relaxed KCl- and noradrenaline-induced sustained contractions with a similar potency. This was in contrast to nifedipine which was approximately 20 times more potent against KCl-induced contractions.It is concluded that levcromakalim relaxes rat mesenteric arterioles primarily by the opening of a small conductance, glibenclamide-sensitive K-channel. An additional action of levcromakalim is suggested by its relative inability to suppress the increase in [Ca²⁺]_i produced by 30 mM K⁺-PSS. Correspondence to: A. H. Weston at the above address     

The mechanism of honokiol-induced and magnolol-induced inhibition on muscle contraction and Ca2+ mobilization in rat uterus

The effects of honokiol and magnolol extracted from the Magnolia officinalis on muscular contractile responses and intracellular Ca²⁺ mobilization were investigated in the non-pregnant rat uterus. Honokiol and magnolol (1–100 mol/l) were observed to inhibit spontaneous and uterotonic agonists (carbachol, PGF₂, and oxytocin)-, high K⁺-, and Ca²⁺ channel activator (Bay K 8644)-induced uterine contractions in a concentration-dependent manner. The inhibition rate of honokiol on spontaneous contractions appeared to be slower than that of magnolol-induced response. The time periods that were required for honokiol and magnolol, at 100 mol/l, to abolish 50% spontaneous contractions were approximately 6 min. Furthermore, honokiol and magnolol at 10 mol/l also blocked the Ca²⁺-dependent oscillatory contractions. Consistently, the increases in intracellular Ca²⁺ concentrations ([Ca²⁺]_i) induced by PGF₂ and high K⁺ were suppressed by both honokiol and magnolol at 10 mol/l. After washout of these treatments, the rise in [Ca²⁺]_i induced by PGF₂ and high K⁺ was still partially abolished. In conclusion, the inhibitory effects of honokiol and magnolol on uterine contraction may be mediated by blockade of external Ca²⁺ influx, leading to a decrease in [Ca²⁺]_i. Honokiol and magnolol may be considered as putative Ca²⁺ channel blockers and be of potential value in the treatment of gynecological dysfunctions associated with uterine muscular spasm and dysmenorrhea.     

Sulfhydryl modification by 4,4'-dithiodipyridine induces calcium mobilization in human osteoblast-like cells

The effect of oxidants on Ca²⁺ movement in osteoblasts is unclear. In this study, we show that 4,4-dithiodipyridine (4,4-DTDP), a reactive disulphide that mobilizes Ca²⁺ in muscle, induces an increase in cytoplasmic free-Ca²⁺ concentrations ([Ca²⁺]_i) in MG63 human osteosarcoma cells loaded with the Ca²⁺-sensitive dye fura-2. 4,4-DTDP acted in a concentration-dependent manner with an EC₅₀ of 10 M. Removing extracellular Ca²⁺ reduced the Ca²⁺ signal by 35%. In Ca²⁺-free medium, the 4,4-DTDP-induced [Ca²⁺]_i increase was not changed by depleting store Ca²⁺ with 50 M brefeldin A (a Golgi apparatus permeabilizer), by 2 M carbonylcyanide m-chlorophenylhydrazone (CCCP, a mitochondrial uncoupler), by 1 M thapsigargin (an inhibitor of the endoplasmic reticulum Ca²⁺ pump) or by 5 M ryanodine. Ca²⁺ signals induced by 4,4-DTDP in Ca²⁺-containing medium were not affected by modulation of protein kinase C activity or suppression of phospholipase C activity. However, 4,4-DTDP-induced Ca²⁺ release was inhibited by a thiol-selective reducing reagent, dithiothreitol (0.05-2.5 mM), in a concentration-dependent manner. Collectively, this study shows that 4,4-DTDP induced [Ca²⁺]_i increases in human osteosarcoma cells via releasing store Ca²⁺ from multiple stores in a manner independent of protein kinase C or phospholipase C activity. The store Ca²⁺ release induced by 4,4-DTDP appears to be associated with thiol oxidation. Furthermore, overnight incubation with 4,4-DTDP inhibited cell activity in a concentration-dependent manner.     

Effect of K+ channel blockers on the α2-adrenoceptor-coupled regulation of electrically evoked noradrenaline release in hippocampus

Summary The question whether presynaptic ₂-adrenoceptors regulating noradrenaline release in hippocampus directly couple to tetraethylammonium chloride (TEA) or -dendrotoxin (-DTX)-sensitive K⁺ channels was investigated. Hippocampal slices, prelabelled with [³H] noradrenaline, were superfused in the presence of (+)-oxaprotiline and electrically stimulated with 4 pulses delivered at 100 Hz, in order to avoid autoinhibition due to released noradrenaline.TEA enhanced the evoked [³H]noradrenaline release in rabbit hippocampus in a concentration-dependent manner, yielding an approximately 4-fold increase at 30 mmol/l, whereas the spontaneous outflow of tritium was only slightly affected at this concentration. The ₂-adrenoceptor agonist clonidine, at 10–100 nmol/l inhibited the evoked [³H]noradrenaline release between 77% and 96%. The inhibitory effect of the ₂-agonist was distinctly diminished in the presence of 30 mmol/l TEA but was restored in low Ca²⁺/high Mg²⁺ buffer. Therefore, the diminution of the ₂-agonist effect by TEA observed in experiments with normal Ca²⁺ can be explained by an increase of the Ca²⁺ availability for the release process due to the prolongation of action potentials. In rabbit hippocampus -DTX (10–200 nmol/l) did neither affect the evoked release of [³H]noradrenaline nor its ₂-agonist-induced modulation. However, in rat hippocampus -DTX significantly increased the evoked transmitter release and diminished the effect of clonidine.Taken together, the present data for the rabbit hippocampus exclude the possibility that activation of presynaptic ₂-adrenoceptors inhibits depolarization-evoked [³H]noradrenaline release by inducing an outward K⁺ current through TEA- or -DTX-sensitive K⁺ channels. However, there are species differences between the rabbit and the rat so that in the rat the ₂-adrenoceptors could actually be coupled to K⁺ channels — provided that the release-enhancing properties of -DTX do not account for the ₂-antagonism observed.Correspondence to C. Allgaier at the above address     

ATP priming of macrophage-derived chemokine responses in CHO cells expressing the CCR4 receptor

The mechanism by which ATP primes for subsequent macrophage-derived chemokine (MDC) mediated intracellular calcium (Ca²⁺_i) responses at the human CCR4 receptor stably expressed in Chinese hamster ovary (CHO) cells was investigated. MDC alone was unable to elicit a Ca²⁺_i response, but pre-stimulation of cells with ATP enabled a subsequent MDC-mediated Ca²⁺_i response with a pEC50 of 8.66±0.16. The maximal response elicited by MDC was dependent upon the concentration of ATP used to prime, but the pEC50 was stable at all ATP concentrations tested.Pertussis toxin pre-treatment did not effect the ATP response, but abolished that to MDC, demonstrating that priming with ATP did not alter G protein-coupling specificity of the CCR4 receptor. Ionomycin and thapsigargin both increased Ca²⁺_i concentrations (pEC50s of 7.59±0.57 and 6.81±0.31 respectively), but were unable to prime for MDC responses, suggesting the priming mechanism was not dependent upon increases in Ca²⁺_i concentrations. Priming of the MDC response was still observed when experiments were performed with low Ca²⁺_e (70 M), indicating that Ca²⁺ influx was not required for ATP to prime the CCR4 receptor.Neither Ro31–8220 nor wortmannin affected priming, suggesting that protein kinase C and phosphoinositol 3-kinase were not involved. In conclusion, pre-stimulation of endogenous P2Y receptors with ATP facilitates Ca²⁺ signalling at the recombinant CCR4 receptor in CHO cells, although the mechanism by which this occurs remains to be defined.Abbreviations ADP Adenosine 5-diphosphate - ATP Adenosine 5-triphosphate - BSA Bovine serum albumin - Ca²⁺_e Extracellular calcium - Ca²⁺_i Intracellular calcium - CCR4 CC chemokine receptor 4 - CHO Chinese hamster ovary cells - cpm Counts per minute - DMSO Dimethyl sulphoxide - FBS Foetal bovine serum - FLIPR Fluorometric imaging plate reader - HBSS Hanks balanced salt solution - MDC Macrophage-derived chemokine - PI3 K Phosphoinositol 3-kinase isoform - PKC Protein kinase C - PLC- Phospholipase C isoform - PTX Pertussis toxin - TARC Thymus and activation-regulated chemokine - UDP Uridine 5-diphosphate - UTP Uridine 5-triphosphate     

CGS 9343B and W7 (calmodulin antagonists) inhibit KCl-induced increase in cytosolic free calcium and insulin secretion of RINm5F cells

Summary CGS 9343B:1,3-Dihydro-l-[1-((4-methyl-4H,6H-pyrrolo[1,2-a]-[4,1]benzoxazepin-4-yl)methyl)-4-piperidinyl]-2 H-benzimidazol-2-one maleate and W7: N-(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide) are calmodulin antagonists with different specificities. The effects of CGS 9343B and W7 on cytosolic free calcium concentration ([Ca²⁺]_i) and insulin release were investigated in rat insulinoma cells (RINm5F). As measured with the Quin-2 technique, preincubation with CGS 9343B (0.3–10 M) and W7 (5–50 M) concentration dependently decreased KCl (25 mM)-mediated accumulation of cytosolic calcium. Both, CGS 9343B (10 M) and W7 (50–100 M) almost abolished the alanine- and KCl-induced increase in [Ca²⁺]_i and significantly inhibited KCl (25 mM)- and alanine (10 mM)-mediated insulin release. W5 (100 LM), the chlorine-deficient analogue of W7 with decreased affinity for calmodulin, did not inhibit the KCl-induced increase in [Ca²⁺]_i and enhanced basal and KCl-mediated insulin release by 56% and 189%, respectively. Our data suggest that CGS 9343B and W7 inhibit the depolarization-induced calcium uptake and subsequent increase in [Ca²⁺]_i. Send offprint requests to H. P. T. Ammon at the above address     

Modulation of cytosolic free calcium concentration by α1-adrenoceptors in rat atrial cells

Summary The effects of ₁-adrenoceptor stimulation by phenylephrine (PE) and -adrenoceptor stimulation by isoprenaline (ISO) on Ca²⁺ current (I_Ca) and free intracellular Ca²⁺ concentration ([Ca²⁺]_i) were studied in isolated atrial myocytes from rat hearts. PE did not significantly affect the magnitude of I_Ca, whereas large increases of peak I_Ca were observed in response to ISO. In electrically driven cells, PE evoked a concentration-dependent, gradual increase in diastolic [Ca²⁺]_i and, initially, an increase in the height of peak [Ca²⁺]_i transients. When the diastolic [Ca²⁺]_i was increased to a greater extent, the amplitude of [Ca²⁺]_i transients was decreased. Simultaneous measurements of [Ca²⁺]_i and membrane potential showed that the increase in diastolic [Ca²⁺]_i was associated with a depolarization of the membrane, and the greater amplitude of [Ca²⁺]_i transients with a prolongation of the action potential (AP). The PE-induced increase in diastolic [Ca²⁺]_i was eliminated when the cells were voltage-clamped at the original resting membrane potential (RP); under these conditions, an increase in [Ca²⁺]_i transients was observed in response to PE. ISO usually caused larger increases in the amplitude of [Ca²⁺]_i transients with only minor changes in diastolic [Ca²⁺]_i. These results suggest that PE and ISO increase the amplitude of [Ca²⁺]_i transients in rat atrium in different ways. The increase in [Ca²⁺]_i transients in response to -adrenoceptor stimulation is commonly thought to be mediated by a greater conductance of voltage-dependent Ca²⁺ channels causing a greater Ca²⁺ influx and a release of more Ca²⁺ from the sarcoplasmic reticulum during the AP. The increase in diastolic [Ca²⁺]_i in response to PE is probably a consequence of the depolarization of the membrane, possibly involving the voltage-dependent Na⁺-Ca²⁺ exchange mechanism. The increase in the amplitude of the [Ca²⁺]_i transients in response to PE may be ascribed both to the initial increase in diastolic [Ca²⁺]_i and the prolongation of the AP. Send offprint requests to H. Nawrath at the above address     

An approach to differentiate between noradrenaline-elicited contractile processes in the rat isolated aorta

Summary The aim of the present study was to assess the different processes contributing to the contraction induced by noradrenaline (NA, 1 gmol/l) in the rat isolated aorta. Pretreatment with maximally effective concentrations of nifedipine or cromakalim reduced the NA-induced contraction to 80 ± 3.5% or 63 ± 2.0%, respectively, without alteration of the shape of the response. After pretreatment with Mn²⁺, NA caused a transient phasic contraction followed by a sustained tonic component, comparable to the response obtained in Ca²⁺-free medium. Ryanodine — in the presence of extracellular Ca²⁺ — caused a slight increase of resting tension, but did not modify the NA-induced contraction. In Ca²⁺-free medium the contraction elicited by NA consisted of a transient phasic and a sustained tonic component. The amplitude of the phasic contraction decreased exponentially with the time of exposure to Ca²⁺-free medium. The phasic component was identified as elicited by Ca²⁺ released from the sarcoplasmic reticulum (SR) by means of ryanodine. If Ca²⁺ depleted tissues (80 min in Ca²⁺-free solution) were exposed to Ca²⁺ in the presence of Mn²⁺ or cromakalim, the NA-induced phasic response was inhibited, suggesting that Mn²⁺ and cromakalim blocked the refilling of the store. It can be concluded that activation of ₁-adrenoceptors in the rat aorta by NA elicits Ca²⁺-entry processes which have a different sensitivity to nifedipine, cromakalim and Mn²⁺. The Ca²⁺ released from SR contributes about 20% to the overall contractile response. Our data suggest that the depleted SR can be refilled from the extracellular space via a direct cromakalim- and Mn²⁺-sensitive pathway. Send offprint requests to: B. Wilffert at the above address     

Functional characterisation of P2 purinoceptors in PC12 cells by measurement of radiolabelled calcium influx

The aim of this study was to determine whether ⁴⁵Ca²⁺ influx could be used as a quantitative measure of channel activation for functional characterisation of P2X purinoceptors in cell lines. In undifferentiated PC12 cells, grown in suspension, ATP (EC₅₀ = 45 M), ATPTS (EC₅₀ = 50 M) and 2-meSATP (EC₅₀ = 81 M) but not meATP (I mM) stimulated ⁴⁵Ca²⁺ influx 2–5 fold. This effect did not appear to be due to activation of P2U or P2Y purinoceptors since 1 mM UTP, ADP or ADPS did not produce any significant effect. Similarly, the effects of ATP were not apparently mediated through activation of P2Z purinoceptors since dibenzylATP behaved as a weak (EC₅₀ = 191 M) partial agonist (Maximal effect 29.5% of ATP maximum) and there was no detectable ATP-stimulated ethidium bromide uptake in the PC12 cells.ATP-stimulated ⁴⁵Ca²⁺ influx was not affected by nifedipine suggesting that it was not secondary to activation of L-type calcium channels and rather reflected influx through a P2X purinoceptor present in these cells. The ATP-stimulated ⁴⁵Ca²⁺ influx could be reduced by monovalent cations, presumably as a result of direct competition for influx through the cation channel, with the following rank order of potency :- guanidinium (EC₅₀ = 16 mM) > sodium > Tris > choline > N-methyl-D-glucamine = sucrose.A number of P2 purinoceptor antagonists inhibited ATP-stimulated ⁴⁵Ca²⁺ influx. Pyridoxalphosphate-6-azophenyl-2,4-disulphonic acid (3–300 M), pyridoxal 5phosphate (3–300 M) and d-tubocurarine (30–300 M) produced an insurmountable antagonism of responses to ATP, with no marked change in agonist EC₅₀. Suramin (100–300 M) and cibacron blue (30–300 M) produced a surmountable antagonism while DIDS (4,4-diisothiocyana-tostilbene-2,2disulfonic acid) only antagonised responses to ATP at concentrations in excess of 300 M. The general properties of the P2X purinoceptor population identified in these cells were consistent with them being P2X₂ purinoceptors. These findings suggest that ATP-stimulated ⁴⁵Ca²⁺ influx may be used as a reliable and quantitative functional assay for characterisation of P2X purinoceptor subtypes in cell lines.     

Alpha-naphthoflavone induces vasorelaxation through the induction of extracellular calcium influx and NO formation in endothelium

The effect of -naphthoflavone (-NF) on vascular function was studied in isolated ring segments of the rat thoracic aorta and in primary cultures of human umbilical vein endothelial cells (HUVECs). -NF induced concentration-dependent relaxation of the phenylephrine-precontracted aorta endothelium-dependently and -independently at lower and higher concentrations, respectively. The cGMP, but not cAMP, content was increased significantly in -NF-treated aorta. Pretreatment with N -nitro-l-arginine methyl ester (L-NAME) or methylene blue attenuated both -NF induced vasorelaxation and the increase of cGMP content significantly. The increase of cGMP content induced by -NF was also inhibited by chelating extracellular Ca²⁺ with EGTA. These results suggest that the endothelium-dependent vasorelaxation induced by -NF is mediated most probably through Ca²⁺-dependent activation of NO synthase and guanylyl cyclase. In HUVECs, -NF induced concentration-dependent formation of NO and Ca²⁺ influx. -NF-induced NO formation was abolished by removal of extracellular Ca²⁺ and by pretreatment with the Ca²⁺ channel blockers SKF 96365 and Ni²⁺, but not by the L-type Ca²⁺ channel blocker verapamil. The Ca²⁺ influx, as measured by ⁴⁵Ca²⁺ uptake, induced by -NF was also inhibited by SKF 96365 and Ni²⁺. Our data imply that -NF, at lower concentrations, induces endothelium-dependent vasorelaxation by promoting extracellular Ca²⁺ influx in endothelium and the activation of the NO-cGMP pathway.     

Effects of the calmodulin antagonists fendiline and calmidazolium on aggregation,secretion of ATP,and internal calcium in washed human platelets

Summary Ca²⁺-calmodulin dependent phosphorylation of myosin is essential for the induction of platelet shape change and subsequent reactions. Therefore, we studied the effects of the calmodulin antagonists fendiline and calmidazolium on the thrombin-induced aggregation, secretion of ATP, and increases in the intracellular free calcium concentration ([Ca²⁺]_i) in washed human platelets in the absence and presence of extracellular Ca²⁺. In Ca²⁺ free medium, fendiline (10–100 M) and calmidazolium (3–30 M) concentration-dependently inhibited aggregation. The effect of fendiline could be partly reversed by extracellular Ca²⁺ and higher thrombin concentrations. Furthermore, aggregations induced by the calcium ionophore ionomycin and by the protein kinase C-activator 4--phorbol 12-myristate 13-acetate were inhibited by fendiline, although to a smaller degree than the thrombin-induced aggregation. Thrombin-induced secretion of ATP was attenuated by low concentrations of fendiline (1–30 M) and calmidazolium (1 M) but enhanced by higher concentrations (10–30 and 3–10 M, respectively), independently of extracellular Ca²⁺. Fendiline (1–10 M) did not affect [Ca²⁺]_i in resting and thrombin-stimulated platelets. At higher concentrations (30–100 M), it induced increases in [Ca²⁺]_i in unstimulated platelets and attenuated the response to thrombin in Ca²⁺ free medium, whereas thrombin-induced Ca²⁺ influx was markedly enhanced. Similar results were obtained with calmidazolium (1–3 M). These stimulating effects on ATP secretion and on [Ca²⁺]_i of fendiline and calmidazolium may be attributed to interactions with platelet membranes by which the permeability of small cations is increased. In contrast to these actions that would be normally considered platelet-activating, our study demonstrates that the two calmodulin antagonists effectively antagonize [Ca²⁺]_i ^– mediated induction of platelet aggregation. Send offprint requests to A. Lückhoff at the above address     

BAY K 8644 stimulates glucose-dependent rise of cytoplasmic Ca2+ in hyperpolarized pancreatic β-cells

Summary The effect of BAY K 8644 on the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) was studied in pancreatic -cells hyperpolarized by the K⁺ channel-activating agent diazoxide. After 50–60 min preexposure to 0–20 mM glucose in the presence of 400 M diazoxide [Ca²⁺]_i was close to the level in unstimulated -cells. The addition of 5 M BAY K 8644 then triggereed a rise of [Ca²⁺]_i dependent on Ca²⁺ influx. The magnitude of the BAY K 8644 effect increased with the glucose concentration and was almost 10-fold higher in 20 mM than in the absence of the sugar. It is concluded that glucose can modulate Ca²⁺ entry through the voltage-dependent channels by a mechanism additional to depolarization. This action may help to explain why previous exposure to the sugar results in an augmented insulin response to a second challenge.     

Evidence for an A2 adenosine receptor in guinea pig lung

Summary Adenosine receptors in guinea pig lung were characterized by measurement of cyclic AMP formation and radioligand binding. 5-N-Ethylcarboxamidoadenosine (NECA) increased cyclic AMP levels in lung slices about 4-fold over basal values with an EC₅₀ of 0.32 mol/l. N⁶-R-(–)-Phenylisopropyladenosine (R-PIA) was 5-fold less potent than NECA. 5-N-Methylcarboxamidoadenosine (MECA) and 2-chloroadenosine had EC₅₀-values of 0.29 and 2.6 mol/l, whereas adenosine and inosine had no effect. The adenosine receptors in guinea pig lung can therefore be classified as A₂ receptors. Several xanthine derivatives antagonized the NECA-induced increase in cyclic AMP levels. 1,3-Diethyl-8-phenylxanthine (DPX; K _i 0.14 mol/l) was the most potent analogue, followed by 8-phenyltheophylline (K _i 0.55 mol/l), 3-isobutyl-1-methylxanthine (IBMX; K _i 2.9 mol/l) and theophylline (K _i 8.1 mol/l). In contrast, enprofylline (1 mmol/l) enhanced basal and NECA-stimulated cyclic AMP formation. In addition, we attempted to characterize these receptors in binding studies with [³H]NECA. The K _D for [³H]NECA was 0.25 mol/l and the maximal number of binding sites was 12 pmol/mg protein. In competition experiments MECA (K _i 0.14 mol/l) was the most potent inhibitor of [³H]NECA binding, followed by NECA (K _i 0.19 mol/l) and 2-chloroadenosine (K _i 1.4 mol/l). These results correlate well with the EC₅₀-values for cyclic AMP formation in lung slices. However, the K _i-values of R-PIA and theophylline were 240 and 270 mol/l, and DPX and 8-phenyltheophylline did not compete for [³H]NECA binding sites. Therefore, a complete characterization of A₂ adenosine receptors by [³H]NECA binding was not achieved. In conclusion, our results show the presence of adenylate cyclase-coupled A₂ adenosine receptors in lung tissue which are antagonized by several xanthines.     

Effect of endothelium on basal and α-adrenoceptor stimulated calcium fluxes in rat aorta

Summary The rate of unstimulated influx of Ca²⁺ into rat aorta smooth muscle, measured as uptake of ⁴⁵Ca, was inhibited in the presence of endothelium as compared to influx in the absence of endothelium. Efflux of ⁴⁵Ca from unstimulated prelabelled tissues was also reduced in the presence of endothelium. In normal physiological solution the rate of influx and efflux of Ca²⁺ stimulated by B-HT 920 (1 and 10 M), but not that stimulated by phenylephrine (30 nM and 1 M), was also reduced in the presence of endothelium. In the presence of the calcium entry blocker flunarizine (3 M), phenylephrine (1 M) stimulated efflux of Ca²⁺ was inhibited by the presence of endothelium. A correlation between inhibition of Ca²⁺ influx and modulation of -adrenoceptor agonist-induced contractions by endothelium could not be demonstrated, and methylene blue, an antagonist of endothelium mediated inhibition of B-HT 920 contractions, did not affect Ca²⁺ influx stimulated by the agonist. The effects of endothelium on Ca²⁺ influx and efflux are unlikely to be due to alterations by endothelium of diffusion of ⁴⁵Ca or the agonists in the vessel. The results demonstrate that an endothelial derived factor or factors can reduce calcium influx into smooth muscle cells and also modulate the release of calcium from cells, perhaps by affecting intracellular calcium pumping mechanisms. A reduction of calcium influx cannot be the sole explanation for the modulatory effect of endothelium on -adrenoceptor agonist-induced contractions but an effect on intracellular calcium metabolism may be important.     

Modulation of 5-HT<Subscript>1A</Subscript> receptor-mediated Ca<Superscript>2+</Superscript> responses by co-expression with various recombinant G<Subscript>α</Subscript> proteins in CHO-K1 cells

The ability of the human 5-HT_1A receptor to activate different recombinant G proteins was investigated in CHO-K1 cells by monitoring 5-HT ligand-mediated Ca²⁺ responses upon co-expression with either G_q, G₁₅ or chimeric G_q/i3 proteins. Each G protein yielded a typical 5-HT-dependent Ca²⁺ response with different kinetic parameters both for the onset-time of maximal Ca²⁺ response (21 to 30 s) and time-dependent attenuation (43 to 73% of residual activity at 1 min upon peak Ca²⁺ response). Pertussis toxin-treatment fully abolished the Ca²⁺ responses mediated by both the endogenous G_i/o and the chimeric-PTX-sensitive G_q/i3 proteins. In contrast, Ca²⁺ responses driven by recombinant G_q and G₁₅ proteins were decreased by PTX, respectively by 52% and 35%, corresponding to the level of endogenous G protein activation. The pharmacology of the 5-HT ligand-mediated Ca²⁺ responses was highly affected by both the presence and nature of the co-expressed G protein. This influence was more pronounced for the partial agonists L 694247, 8-OH-DPAT, flesinoxan and buspirone in contrast to ipsapirone. The G protein rank order for apparent increase of ligands' intrinsic activity was: G_q <G_q/i3 <G₁₅ protein. Each of the 5-HT-mediated Ca²⁺ responses could be antagonised by WAY 100635, buspirone and methiothepin regardless of the absence or presence of a G_q, G_q/i3 or G₁₅ protein. In conclusion, these data reinforce that depending on the presence and nature of the G protein environment, 5-HT_1A ligands may display a large spectrum of activities.Abbreviations AFU Arbitrary fluorescence unit - 5-CT 5-Carboxamidotryptamine - 5-HT 5-Hydroxytryptamine (serotonin) - 8-OH-DPAT 8-(Hydroxy-2-(di-n-propylamino)tetralin - CHO Chinese hamster ovary - PLC Phospholipase C - WAY 100635 N-[2-[4-(2-methoxyphenyl)1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexanecarboxamide - PTX Bordetella pertussis toxin - wt Wild-type     

Differential calcium signalling by m2 and m3 muscarinic acetylcholine receptors in a single cell type

We have compared muscarinic acetylcholine receptor (mAChR) coupling to phospholipase C (PLC) and increases in cytoplasmic Ca²⁺ concentration [Ca²⁺]_i in human embryonic kidney (HEK) cells, stably expressing either the human m3 or m2 receptor subtype. In m3 mAChR-expressing cells, carbachol stimulated inositol phosphate (InsP) formation and increased [Ca²⁺]_i with EC₅₀ values of about 2 M and 30 nM, respectively. Maximal inositol 1,4,5-trisphosphate (InsP₃) production (about fourfold) was rapid (15 s) and stable for 2 min. Maximal increases in [Ca²⁺]_i were 300–350 nM and mainly, almost 90%, due to influx of extracellular Ca²⁺. The efficacy of pilocarpine for stimulating InsP andCa²⁺ responses was not significantly different from that of carbachol. All m3 mAChR-mediated responses were pertussis toxin (PTX)-insensitive. In m2 mAChR-expressing cells, carbachol stimulated InsP formation and increased [Ca²⁺]_i with EC₅₀ values of about 20 M and 7 M, respectively. Maximal InsP formation was only 10–15% of that observed in m3 mAChR-expressing cells, whereas maximal elevations of [Ca²⁺]_i were similar in both cell types. Formation of InsP₃ was rapid (15 s to 2 min) and about twofold above basal. In contrast to m3 mAChR activation, [Ca²⁺]_i increases induced by m2 mAChR activation were exclusively due to Ca²⁺ mobilization from intracellular stores.The efficacy of pilocarpine for stimulating InsP and Ca²⁺ responses was 50% and 20% of the efficacy of carbachol, respectively. PTX treatment did not affect m2 mAChR-induced PLC stimulation, but reduced the m2 mAChR-mediated increases in [Ca²⁺]_i to 50%. In conclusion, m3 and m2 mAChRs stably expressed in HEK cells can induce similar cellular responses; however, they do so by activating apparently distinct signalling pathways. While coupling of m2 mAChR to PLC occurs in a PTX-insensitive manner, coupling to mobilization of Ca²⁺ from intracellular stores is partly PTX-sensitive and this may occur at least partly independent of PLC activation.