Phospholipase C-dependent control of cardiac calcium homeostasis involves a TRPC3-NCX1 signaling complex.

OBJECTIVE: Members of the classical transient receptor potential protein (TRPC) family are considered as key components of phospholipase C (PLC)-dependent Ca2+ signaling. Previous results obtained in the HEK 293 expression system suggested a physical and functional coupling of TRPC3 to the cardiac-type Na+/Ca2+ exchanger, NCX1 (sodium calcium exchanger 1). This study was designed to test for expression of TRPC3 (transient receptor potential channel 3) and for the existence of a native TRPC3/NCX1 signaling complex in rat cardiac myocytes. METHODS: Protein expression and cellular distribution were determined by Western blot and immunocytochemistry. Protein-protein interactions were investigated by reciprocal co-immunoprecipitation and glutathione S-transferase (GST)-pulldown experiments. Recruitment of protein complexes into the plasma membrane was assayed by surface biotinylation. The functional role of TRPC3 was investigated by fluorimetric recording of angiotensin II-induced calcium signals employing a dominant negative knockdown strategy. RESULTS: TRPC3 immunoreactivity was observed in surface plasma membrane regions and in an intracellular membrane system. Co-immunolabeling of TRPC3 and NCX1 indicated significant co-localization of the two proteins. Both co-immunoprecipitation and GST-pulldown experiments demonstrated association of TRPC3 with NCX1. PLC stimulation was found to trigger NCX-mediated Ca2+ entry, which was dependent on TRPC3-mediated Na+ loading of myocytes. This NCX-mediated Ca2+ signaling was significantly suppressed by expression of a dominant negative fragment of TRPC3. PLC stimulation was associated with increased membrane presentation of both TRPC3 and NCX1. CONCLUSION: These results suggest a PLC-dependent recruitment of a TRPC3-NCX1 complex into the plasma membrane as a pivotal mechanism for the control of cardiac Ca2+ homeostasis.

TRPC3: a multifunctional, pore-forming signalling molecule.

TRPC3 represents one of the first identified mammalian relatives of the Drosophila trp gene product. Despite intensive biochemical and biophysical characterization as well as numerous attempts to uncover its physiological role in native cell systems, this channel protein still represents one of the most enigmatic members of the transient receptor potential (TRP) superfamily. TRPC3 is significantly expressed in brain and heart and likely to play a role in both non-excitable as well as excitable cells, being potentially involved in a wide spectrum of Ca2+ signalling mechanisms. Its ability to associate with a variety of partner proteins apparently enables TRPC3 to form different cation channels in native cells. TRPC3 cation channels display unique gating and regulatory properties that allow for recognition and integration of multiple input stimuli including lipid mediators and cellular Ca2+ gradients as well as redox signals. The physiological/pathophysiological functions of this highly versatile cation channel protein are as yet barely delineated. Here we summarize current knowledge on properties and possible signalling functions of TRPC3 and discuss the potential biological relevance of this signalling molecule.

Activation of a small-conductance Ca(2+)-dependent K+ channel contributes to bradykinin-induced stimulation of nitric oxide synthesis in pig aortic endothelial cells.

Bradykinin-induced K+ currents, membrane hyperpolarization, as well as rises in cytoplasmic Ca2+ and cGMP levels were studied in endothelial cells cultured from pig aorta. Exposure of endothelial cells to 1 microM bradykinin induced a whole-cell K+ current and activated a small-conductance (approximately 9 pS) K+ channel in on-cell patches. This K+ channel lacked voltage sensitivity, was activated by increasing the Ca2+ concentration at the cytoplasmic face of inside-out patches and blocked by extracellular tetrabutylammonium (TBA). Bradykinin concomitantly increased membrane potential and cytoplasmic Ca2+ of endothelial cells. In high (140 mM) extracellular K+ solution, as well as in the presence of the K(+)-channel blocker TBA (10 mM), bradykinin-induced membrane hyperpolarization was abolished and increases in cytoplasmic Ca2+ were reduced to a slight transient response. Bradykinin-induced rises in intracellular cGMP levels which reflect Ca(2+)-dependent formation of EDRF(NO) were clearly attenuated in the presence of TBA (10 mM). Our results suggest that bradykinin hyperpolarizes pig aortic endothelial cells by activation of small-conductance Ca(2+)-activated K+ channels. Opening of these K+ channels results in membrane hyperpolarization which promotes Ca2+ entry, and consequently, NO synthesis.

Intracellular Ca2+ inhibits smooth muscle L-type Ca2+ channels by activation of protein phosphatase type 2B and by direct interaction with the channel.

Modulation of L-type Ca2+ channels by tonic elevation of cytoplasmic Ca2+ was investigated in intact cells and inside-out patches from human umbilical vein smooth muscle. Ba2+ was used as charge carrier, and run down of Ca2+ channel activity in inside-out patches was prevented with calpastatin plus ATP. Increasing cytoplasmic Ca2+ in intact cells by elevation of extracellular Ca2+ in the presence of the ionophore A23187 inhibited the activity of L-type Ca2+ channels in cell-attached patches. Measurement of the actual level of intracellular free Ca2+ with fura-2 revealed a 50% inhibitory concentration (IC50) of 260 nM and a Hill coefficient close to 4 for Ca2+- dependent inhibition. Ca2+-induced inhibition of Ca2+ channel activity in intact cells was due to a reduction of channel open probability and availability. Ca2+-induced inhibition was not affected by the protein kinase inhibitor H-7 (10 microM) or the cytoskeleton disruptive agent cytochalasin B (20 microM), but prevented by cyclosporin A (1 microg/ ml), an inhibitor of protein phosphatase 2B (calcineurin). Elevation of Ca2+ at the cytoplasmic side of inside-out patches inhibited Ca2+ channels with an IC50 of 2 microM and a Hill coefficient close to unity. Direct Ca2+-dependent inhibition in cell-free patches was due to a reduction of open probability, whereas availability was barely affected. Application of purified protein phosphatase 2B (12 U/ml) to the cytoplasmic side of inside-out patches at a free Ca2+ concentration of 1 microM inhibited Ca2+ channel open probability and availability. Elevation of cytoplasmic Ca2+ in the presence of PP2B, suppressed channel activity in inside-out patches with an IC50 of approximately 380 nM and a Hill coefficient of approximately 3; i.e., characteristics reminiscent of the Ca2+ sensitivity of Ca2+ channels in intact cells. Our results suggest that L-type Ca2+ channels of smooth muscle are controlled by two Ca2+-dependent negative feedback mechanisms. These mechanisms are based on (a) a protein phosphatase 2B-mediated dephosphorylation process, and (b) the interaction of intracellular Ca2+ with a single membrane-associated site that may reside on the channel protein itself.

Evidence for a role of Trp proteins in the oxidative stress-induced membrane conductances of porcine aortic endothelial cells.

OBJECTIVE: Expression of homologues of the Drosophila transient receptor potential (Trp) protein has recently been demonstrated for vascular endothelium. Some Trp isoforms such as Trp3, are known to constitute cation conductances with biophysical properties similar to those of the endothelial oxidant-activated cation conductance. Therefore we tested whether Trp proteins provide the molecular basis of the oxidant-induced membrane currents in porcine aortic endothelial cells (ECAP). METHODS: Expression of the Trp3 isoform in ECAP was tested by RT-PCR and subsequent southern blot analysis. In order to knock-out the function of endogenous Trp channels, ECAP were transiently transfected to express NTRP3, a dominant negative fragment of Trp3. Oxidative-stress was introduced by exposure of cells to tert-butylhydroperoxide (tBHP; 400 microM), and membrane current as well as membrane potential were recorded using the conventional whole cell patch-clamp technique. RESULTS: RT-PCR experiments demonstrated the expression of a Trp3 isoform in ECAP. The oxidant tert.-butylhydroperoxide (tBHP) completely depolarized endothelial cells by activation of a cation conductance which allowed significant Na+ inward currents at negative potentials (mean inward current 462 pA at -80 mV). The tBHP-induced currents resembled Trp-related currents in terms of cation selectivity, La3+ sensitivity and lack of voltage dependence. Expression of the N-terminal fragment of hTrp3 (NTRP3), but not of a C-terminal fragment of hTrp3 (CTRP3), abolished the oxidant-induced cation current and reduced membrane depolarization. CONCLUSION: Our results strongly suggest Trp proteins as the molecular basis of endothelial oxidant-activated cation channels. It is concluded that Trp proteins play an important role in the redox sensitivity of the vascular endothelium.

Protein kinase-C mediates dual modulation of L-type Ca2+ channels in human vascular smooth muscle.

The role of protein kinase C (PKC) in cellular regulation of L-type Ca2+ channels was investigated in human umbilical vein smooth muscle. Activation of PKC, by low concentrations (< 30 nM) of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) caused inhibition of Ca2+ channels, while higher concentrations of TPA (> 100 nM) elicited a transient rise, followed by sustained inhibition of Ca2+ channel activity in cell-attached patches. Low TPA concentrations predominantly reduced channel availability, while high concentrations of TPA (100 nM) transiently increased channel availability and, in addition, prolonged mean open time. The inactive 4-alpha-phorbol-12,13- didecanoate failed to affect channel activity, and pretreatment of the cells with PKC inhibitors (H-7, chelerythrine) antagonized inhibitory and stimulatory effects of TPA. Our results provide evidence for two distinct PKC-dependent mechanisms of L-type Ca2+ channel regulation in smooth muscle.

Basal dephosphorylation controls slow gating of L-type Ca2+ channels in human vascular smooth muscle.

The role of cellular phosphatase activity in regulation of smooth muscle L-type Ca2+ channels was investigated using tautomycin, a potent and specific inhibitor of serin/threonin phosphatases type 1 and 2A. Tautomycin (1-100 nM) inhibited Ca2+ channel activity in smooth muscle cells isolated from human umbilical vein. Tautomycin-induced inhibition of Ca2+ channel activity was due to a reduction of channel availability which originated mainly from prolongation of the lifetime of unavailable states of the channel. Pretreatment of smooth muscle cells with the protein kinase inhibitor H-7 (10 microM) prevented the inhibitory effect of tautomycin. Our results suggest modulation of slow gating between available and unavailable states as a mechanism of phosphorylation-dependent down-regulation of Ca2+ channels in vascular smooth muscle.

The cardiac acetylcholine-activated, inwardly rectifying K+-channel subunit GIRK1 gives rise to an inward current induced by free oxygen radicals.

Reactive oxygen species (ROS) play a crucial role in pathophysiology of the cardiovascular system. The present study was designed to analyze the redox sensitivity of G-protein-activated inward rectifier K+ (GIRK) channels, which control cardiac contractility and excitability. GIRK1 subunits were heterologously expressed in Xenopus laevis oocytes and the resulting K+ currents were measured with the two-electrode voltage clamp technique. Oxygen free radicals generated by the hypoxanthine/xanthine oxidase system led to a marked increase in the current through GIRK channels, termed superoxide-induced current (I(SO)). Furthermore, I(SO) did not depend on G-protein-dependent activation of GIRK currents by coexpressed muscarinic m2-receptors, but could also be observed when no agonist was present in the bathing solution. Niflumic acid at a concentration of 0.5 mmol/l did not abolish I(SO), whereas 100 micromol/l Ba2+ attenuated I(SO) completely. Catalase (10(6) i.u./l) failed to suppress I(SO), whereas H2O2 concentration was kept close to zero, as measured by chemiluminescence. Hence, we conclude that O2*- or a closely related species is responsible for I(SO) induction. Our results demonstrate a significant redox sensitivity of GIRK1 channels and suggest redox-activation of G-protein-activated inward rectifier K+ channels as a key mechanism in oxidative stress-associated cardiac dysfunction.

Trp proteins form store-operated cation channels in human vascular endothelial cells.

Members of the Trp protein family have been suggested as the structural basis of store-operated cation conductances. With this study, we provide evidence for the expression of three isoforms of Trp (hTrp1, 3 and 4) in human umbilical vein endothelial cells (HUVEC). The role of Trp proteins in store regulation of endothelial membrane conductances was tested by expression of an N-terminal fragment of hTrp3 (N-TRP) which exerts a dominant negative effect on Trp channel function presumably due to suppression of channel assembly. Depletion of intracellular Ca2+ stores with IP3 (100 microM) or thapsigargin (100 nM) induced a substantial cation conductance in sham-transfected HUVEC as well as in HUVEC transfected with hTrp3. In contrast, HUVEC transfected with N-TRP failed to exhibit store-operated currents. Our results suggest the involvement of Trp related proteins in the store-operated cation conductance of human vascular endothelial cells.

Essential role of the beta subunit in modulation of C-class L-type Ca2+ channels by intracellular pH.

Elevation of intracellular pH (pHi) enhances the activity of native L-type Ca2+ channels in cardiac and smooth muscle. We studied the modulation by pHi of expressed L-type Ca2+ channels comprised of either the alpha1c subunits alone or of alpha1c plus beta2a subunits. Ca2+ channels were expressed in human embryonic kidney cells (HEK 293) and pHi was increased from a basal level of 7.3 to 8.3 by exposure of cells to NH4Cl (20 mM) or by elevation of extracellular pH to 8.5. Elevation of pHi enhanced the activity of Ca2+ channels derived by coexpression of alpah1c and beta2a subunits. This alkalosis-induced stimulation of channel activity was mainly due to an increase in channel availability. Channels derived by expression of alpha1c alone were not affected by intracellular alkalosis. Our results demonstrate that the pHi sensitivity of L-type Ca2+ channels is conferred by the beta subunit of the channel complex.

A sequence in the carboxy-terminus of the alpha(1C) subunit important for targeting, conductance and open probability of L-type Ca(2+) channels.

The role of the 80-amino acid motif 1572-1651 in the C-terminal tail of alpha(1C) Ca(2+) channel subunits was studied by comparing properties of the conventional alpha(1C,77) channel expressed in HEK-tsA201 cells to three isoforms carrying alterations in this motif. Replacement of amino acids 1572-1651 in alpha(1C,77) with 81 non-identical residues leading to alpha(1C,86) impaired membrane targeting and cluster formation of the channel. Similar to alpha(1C, 86), substitution of its 1572-1598 (alpha(1C,77L)) or 1595-1652 (alpha(1C,77K)) segments into the alpha(1C,77) channel yielded single-channel Ba(2+) currents with increased inactivation, reduced open probability and unitary conductance, when compared to the alpha(1C,77) channel. Thus, the C-terminal sequence 1572-1651 of the alpha(1C) subunit is important for membrane targeting, permeation and open probability of L-type Ca(2+) channels.

ATP inhibits smooth muscle Ca2(+)-activated K+ channels.

There has been much recent interest in the roles played by smooth-muscle K+ channels in protecting cells against ischemic and anoxic insults and in therapeutic vaso- and bronchodilation (Buckingham 1990; Longmore & Weston 1990). A K+ channel, which is uniquely sensitive to cytoplasmic ATP (KATP), has been identified as a likely candidate for mediating these important functions (Standen et al. 1989). We now show, by using electrophysiological techniques in three different types of smooth muscle, that a large-conductance voltage and Ca2(+)-sensitive channel, otherwise indistinguishable from the the large-conductance Ca2(+)-activated K+ channel (BK channel), is also sensitive to cytoplasmic ATP and cromakalim. ATP, in a dose-dependent manner, decreased the probability of channel opening (Po) of rabbit aortic, rabbit tracheal and pig coronary artery BK channels with a Ki of 0.2-0.6 mM. Cromakalim, 10 microM, partially reversed the ATP induced inhibition and increased Po. Our observations raise the possibility that the ubiquitous BK channel may play a role during pathophysiological events.

The effects of the stereoisomers of propafenone and diprafenone in guinea-pig heart.

1. Optically pure enantiomers of propafenone and diprafenone were prepared from their racemic mixtures and tested for their ability to block beta-adrenoceptors and to prolong functional refractory period in the guinea-pig heart. beta-Adrenoceptor affinity of the enantiomers was determined by the radioligand binding technique and in functional experiments. 2. Propafenone and diprafenone inhibited specific binding of the beta-adrenoceptor antagonist (-)-[3H]-CGP-12177 to guinea-pig myocardial membranes. beta-Adrenoceptor affinities of diprafenone enantiomers exceeded those of corresponding propafenone enantiomers by one order of magnitude. Displacement of (-)-[3H]-CGP-12177 by both antiarrhythmics was highly stereoselective, in that the (S)-enantiomers were 40-60 fold, i.e. 1.6-1.8 log units more potent than the (R)-enantiomers. 3. Propafenone and diprafenone antagonized the positive inotropic action of isoprenaline in isolated atria. beta-Adrenoceptor antagonist potencies of diprafenone enantiomers were about one order of magnitude higher than those of corresponding propafenone enantiomers. For both drugs the (S)-enantiomer was found to be considerably more potent (14-40 fold) than the (R)-enantiomer. 4. Propafenone and diprafenone prolonged functional refractory period of isolated auricles with equal potency and no difference in the antiarrhythmic activity of purified enantiomers was found. 5. It is concluded that the enantiomers of propafenone and diprafenone exert comparable antiarrhythmic activity, whereas only (S)-enantiomers block cardiac beta-adrenoceptors with high affinity, which explains the beta-adrenoceptor antagonist effects of the racemic drugs.

The role of myoendothelial cell contact in non-nitric oxide-, non-prostanoid-mediated endothelium-dependent relaxation of porcine coronary artery.

1. Experiments were designed to analyse the requirement of myoendothelial junctions by bradykinin-induced endothelium-dependent relaxations resistant to NG-nitro-L-arginine (L-NOARG) and indomethacin porcine coronary arteries. 2. Rings of porcine coronary arteries were contracted with the thromboxane receptor agonist, U46619 and relaxations to bradykinin recorded isometrically. All experiments were performed in the presence of indomethacin. Nitric oxide (NO)-mediated effects were blocked by the NO synthase inhibitor L-NOARG (250 microM) and myoendothelial contacts inhibited by treatment with hypertonic solution containing D-mannitol or sucrose (each 180 mM) or the gap junctional uncoupling agent 1-heptanol (2 mM). High [K+] solutions (40 mM) were used to probe a possible contribution of endothelium-derived hyperpolarizing factor (EDHF). 3. In the presence of endothelium, bradykinin induced concentration-dependent relaxations with a mean EC50 of 3.2 nM and a maximum response of 95 +/- 1% of papaverine-induced relaxation (control curve). 4. In the absence of endothelium, bradykinin failed to induce relaxations. Addition of cultured porcine aortic endothelial cells to the organ bath resulted in some relaxation and restored in part the relaxant effect of bradykinin. This endothelial cell-mediated relaxant effect was completely abolished in the presence of 250 microM L-NOARG. 5. Bradykinin-induced relaxations in endothelium-preserved rings were only slightly suppressed by L-NOARG (86% of control). In vessels partially depolarized by high extracellular [K+] (40 mM) relaxation was reduced to 72% of control. In the presence of L-NOARG, bradykinin failed to relax partially depolarized vessels. 6. In the presence of 2 mM -heptanol, 180 mM mannitol or 180 mM sucrose maximum relaxation to bradykinin was reduced to ~70%, i.e. to the same extent as in the presence of high [K+]. The remaining relaxation was sensitive to blockade by L-NOARG.7. Tissue cyclic GMP content which reflects NO activity, was increased about 4 fold by bradykinin(300 nM). This increase was unaffected by high [K+], heptanol or sucrose but blocked by L-NOARG.8 Our results suggest that non-nitric oxide- and non-prostanoid-mediated endothelium-dependent relaxation of porcine coronary artery requires functionally intact myoendothelial junctions.

Inhibition of a store-operated Ca2+ entry pathway in human endothelial cells by the isoquinoline derivative LOE 908.

1. The novel cation channel blocker, LOE 908, was tested for its effects on Ca2+ entry and membrane currents activated by depletion of intracellular Ca2+ stores in human endothelial cells. 2. LOE 908 inhibited store-operated Ca2+ entry induced by direct depletion of Ca2+ stores with 100 nM thapsigargin or 100 nM ionomycin with an EC50 of 2 microM and 4 microM, respectively. 3. LOE 908 did not affect thapsigargin- or ionomycin-induced Ca2+ release from intracellular stores up to concentrations of 3 microM. 4. LOE 908 reversibly suppressed thapsigargin- as well as ionomycin-induced whole-cell membrane currents. 5. The LOE 908-sensitive membrane conductance corresponded to a cation permeability of 5.5 and 6.9 fold selectivity for Ca2+ over K+ in the presence of thapsigargin and ionomycin, respectively. 6. Our results suggest that the isoquinoline, LOE 908 is a novel, potent inhibitor of the store-operated (capacitive) Ca2+ entry pathway in endothelial cells.

Inhibitory effects of aclarubicin on nitric oxide production in aortic smooth muscle cells and macrophages.

The effects of aclaruhicin (ACR), an anthracycline antibiotic, on inducible nitric oxide (NO) synthesis was investigated in rat aortic smooth muscle cells (RASMCs) and RAW macrophages. ACR at concentrations as low as 0.1 microM significantly inhibited NO production induced by interleukin-1beta in RASMCs. About 5- to 10-fold higher concentrations of ACR were required for inhibition of interferon-gamma and lipopolipopolysaccharide-induced NO production in RAW cells. When ACR was subsequently administered to inducible NO synthase (iNOS) induction, the NO production was barely suppressed in RASMCs. Moreover, ACR (up to 10 microM) lacked direct inhibitory effects on iNOS activity in homogenates of these cells. ACR (0.1 microM) inhibited the expression of iNOS protein and mRNA in RASMCs without concomitant cytotoxic effects. ACR (>0.5 microM)-induced inhibition of NO production in RAW cells was associated with substantial cytotoxic effects as shown by measurement of lactate dehydrogenase release. These results suggest that ACR is a potent inhibitor of iNOS induction in vascular smooth muscle, but inhibits iNOS induction in macrophage only at high cytotoxic

Intracellular alkalinization augments alpha(1)-adrenoceptor-mediated vasoconstriction by promotion of Ca(2+) entry through the non-L-type Ca(2+) channels.

Modulation by intracellular pH of the vasoconstriction induced by alpha-adrenoceptor agonists was investigated in isolated guinea pig aorta. NH(4)Cl (15 mM) increased intracellular pH of aortic smooth muscle cells by about 0.2 pH unit and significantly augmented KCl-induced contraction of aortic strips, whereas simultaneous administration of NH(4)Cl (15 mM) plus Na(+) propionate (30 mM) failed to affect intracellular pH or contractility. NH(4)Cl (15 mM) potentiated contractions induced by alpha-adrenoceptor agonists, norepinephrine, phenylephrine and clonidine. Contraction induced by alpha(1)-selective adrenoceptor agonist, phenylephrine, but not that induced by norepinephrine or clonidine, was insensitive to inhibition by verapamil (1 microM). Phenylephrine-induced contraction was not affected by NH(4)Cl in Ca(2+)-free medium whereas extracellular Ca(2+)-induced contraction of phenylephrine-stimulated aorta was significantly augmented by NH(4)Cl. Consistently, 45Ca(2+)uptake into phenylephrine 1 microM)-stimulated aortic strips was increased by incubation with NH(4)Cl. The potentiating effects of NH(4)Cl on both phenylephrine-induced Ca(2+) entry and contraction were antagonized by Na(+) propionate. These results suggest that intracellular alkalinization facilitates alpha(1)-adrenoceptor-mediated vasoconstriction by facilitation of an agonist-induced Ca(2+) entry pathway that is independent of L-type Ca(2+) channels.

NH4Cl-induced contraction of porcine coronary artery involves activation of dihydropyridine-sensitive Ca2+ entry.

The role of voltage-dependent, dihydropyridine-sensitive Ca2+ channels in NH4Cl-induced vasoconstriction was investigated in isolated porcine coronary arteries by measuring in parallel isometric tone and 45Ca2+ uptake. NH4Cl (10-80 mM) concentration dependently induced tonic contractions which were preceded by a time lag of several minutes. Contractile responses to high (60 mM) as well as low (25 mM) concentrations of NH4Cl were markedly inhibited by 1 microM nifedipine or removal of extracellular Ca2+. The contractile effect of 25 mM NH4Cl was substantially enhanced by increasing extracellular K+ to 14.7 mM or by pretreatment of coronary arteries with either 5 mM tetraethylammonium chloride or 0.1 microM 1,4-dihydro- 2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)-phenyl]-3-pyridine carboxylic acid methyl ester (BAY K8644). NH4Cl (60 mM) significantly increased 45Ca2+ uptake with a lag time of more than 5 min. The increase in 45Ca2+ uptake induced by 60 mM NH4Cl was abolished in the presence of 1 microM nifedipine. Although NH4Cl (25 mM) did not detectably stimulate 45Ca2+ uptake in normal K+ solution, it significantly augmented 45Ca2+ uptake when extracellular K+ was increased to 14.7 mM. Furthermore, NH4Cl (20 mM) potentiated histamine-induced contraction of coronary arteries. This potentiating effect of NH4Cl was completely antagonized by nifedipine. Our results suggest an involvement of nifedipine-sensitive Ca2+ channels in NH4Cl-induced vasoconstriction of porcine coronary artery.

Characterization of muscarinic receptors mediating endothelium-dependent relaxation of bovine coronary artery.

In order to identify the receptor subtype responsible for acetylcholine (ACh)-induced relaxation of bovine coronary artery, we determined the affinity of six subtype-selective muscarinic antagonists and compared them with affinity estimates obtained for bovine left atria. At low concentrations, ACh potently relaxed circular strips of coronary artery with endothelium (EC50 0.15 microM), but contracted them at higher agonist concentrations with potencies that depended on the presence or absence of endothelium: EC50 1.8 microM (without endothelium); 4.6 microM (with endothelium). The pA2 values obtained for antagonism of relaxant responses to ACh were: pirenzepine (M1-selective) 7.38 +/- 0.12; AF-DX 116 (11-[2-(diethylamino-methyl)-1-piperidinyl-acetyl]-5,11- dihydro-6H-pyrido(2,3-b)1,4-benzodiazepine-6-one; M2-selective) 5.79 +/- 0.09; and 4-diphenylacetoxy-N-methyl-piperidine-methobromide (4-DAMP; M3/M1-selective) 9.07 +/- 0.12. The corresponding Schild slopes were 0.98 +/- 0.07 for pirenzepine, 1.17 +/- 0.09 for AF-DX 116 and 1.01 +/- 0.04 for 4-DAMP. For the following three antagonists, pKB values were determined at two different antagonist concentrations: dicyclomine (M1-selective) 7.49 +/- 0.10, cyclohexylphenyl-(2-piperidinoethyl)-silanol (CPPS; M3-selective) 8.0 +/- 0.10, and parafluoro-hexahydrosila-difenidol (pFHHSiD; M3-selective) 7.87 +/- 0.10. For comparison, the antagonism of methacholine-induced negative inotropy in left atria was determined for three antagonists, yielding the following pA2 values: pirenzepine 5.98 +/- 0.14; AF-DX 116 6.81 +/- 0.14 and 4-DAMP 7.99 +/- 0.14. The slopes of the corresponding Schild plots were 1.05 +/- 0.10, 1.14 +/- 0.12 and 1.08 +/- 0.08, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of DPI 201-106 with cardiac muscarinic receptors.

The effects of the new cardiotonic compound, DPI 201-106, on muscarinic responses and muscarinic receptor binding were studied in the guinea pig heart. DPI 201-106 exerted a pronounced anticholinergic action in isolated auricles and a moderate one in papillary muscles, which resembled the pattern of a functional antagonism. However, in competition binding experiments, DPI 201-106 inhibited binding of the specific muscarinic antagonist [3H]NMS with equal potency in atrial and ventricular homogenates (apparent KI = 0.7 mumol/l in atria and 1.2 mumol/l in ventricles). At higher concentrations (greater than 3 mumol/l), DPI 201-106 slowed the dissociation of [3H]NMS from cardiac muscarinic receptors, indicating that DPI 201-106 affects in addition a site allosteric to the muscarinic receptor. It is concluded that DPI 201-106 is able to inhibit cholinergic responses in the heart, not only by a functional antagonism but also by direct interaction with muscarinic receptors.