Intracellular angiotensin II elicits Ca2+ increases in A7r5 vascular smooth muscle cells.

Recent studies show that angiotensin II can act within the cell, possibly via intracellular receptors pharmacologically different from typical plasma membrane angiotensin II receptors. The signal transduction of intracellular angiotensin II is unclear. Therefore, we investigated the effects of intracellular angiotensin II in cells devoid of physiological responses to extracellular angiotensin II (A7r5 vascular smooth muscle cells). Intracellular delivery of angiotensin II was obtained by using liposomes or cell permeabilisation. Intracellular angiotensin II stimulated Ca2+ influx, as measured by 45Ca2+ uptake and single-cell fluorimetry. This effect was insensitive to extracellular or intracellular addition of losartan (angiotensin AT(1) receptor antagonist) or PD123319 ((s)-1-(4-[dimethylamino]-3-methylphenyl)methyl-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylate) (angiotensin AT2 receptor antagonist). Intracellular angiotensin II stimulated inositol-1,4,5-trisphosphate (Ins(1,4,5,)P3) production and increased the size of the Ins(1,4,5,)P3 releasable 45Ca2+ pool in permeabilised cells, independent of losartan and PD123319. Small G-proteins did not participate in this process, as assessed by using GDPbetaS. Intracellular delivery of angiotensin I was unable to elicit any of the effects elicited by intracellular angiotensin II. We conclude from our intracellular angiotensin application experiments that angiotensin II modulates Ca2+ homeostasis even in the absence of extracellular actions. Pharmacological properties suggest the involvement of putative angiotensin non-AT1-/non-AT2 receptors.

Intracellular Angiotensin II and cell growth of vascular smooth muscle cells.

1. We recently demonstrated that intracellular application of Angiotensin II (Angiotensin II(intr)) induces rat aorta contraction independent of plasma membrane Angiotensin II receptors. In this study we investigated the effects of Angiotensin II(intr) on cell growth in A7r5 smooth muscle cells. 2. DNA-synthesis was increased dose-dependently by liposomes filled with Angiotensin II as measured by [(3)H]-thymidine incorporation at high (EC(50)=27+/-6 pM) and low (EC(50)=14+/-5 nM) affinity binding sites with increases in E(max) of 58+/-4 and 37+/-4% above quiescent cells, respectively. Cell growth was corroborated by an increase in cell number. 3. Extracellular Angiotensin II (10 pM - 10 microM) did not modify [(3)H]-thymidine incorporation. 4. Growth effects of Angiotensin II(intr) mediated via high affinity sites were inhibited by liposomes filled with 1 microM of the non-peptidergic antagonists losartan (AT(1)-receptor) or PD123319 (AT(2)-receptor) or with the peptidergic agonist CGP42112A (AT(2)-receptor). E(max) values were decreased to 30+/-3, 29+/-4 and 4+/-2%, respectively, without changes in EC(50). The Angiotensin II(intr) effect via low affinity sites was only antagonized by CGP42112A (E(max)=11+/-3%), while losartan and PD123319 increased E(max) to 69+/-4%. Intracellular applications were ineffective in the absence of Angiotensin II(intr). 5. Neither intracellular nor extracellular Angiotensin I (1 microM) were effective. 6. The Angiotensin II(intr) induced growth response was blocked by selective inhibition of phosphatidyl inositol 3-kinase (PI-3K) by wortmannin (1 microM) and of the mitogen-activated protein kinase (MAPK/ERK) pathway by PD98059 (1 microM) to 61+/-14 and 4+/-8% of control, respectively. 7. These data demonstrate that Angiotensin II(intr) induces cell growth through atypical AT-receptors via a PI-3K and MAPK/ERK -sensitive pathway.

Intracellular angiotensin II inhibits heterologous receptor stimulated Ca2+ entry.

Recent studies show that angiotensin II (AngII) can act from within the cell, possibly via intracellular receptors pharmacologically different from typical plasma membrane AngII receptors. The role of this intracellular AngII (AngIIi) is unclear. Besides direct effects of AngIIi on cellular processes one could hypothesise a possible role of AngIIi in modulation of cellular responses induced after heterologous receptor stimulation. We therefore examined if AngIIi influences [Ca+]i in A7r5 smooth muscle cells after serotonin (5HT) or UTP receptor stimulation. Application of AngIIi using liposomes, markedly inhibited 45Ca2+ influx after receptor stimulation with 5HT or UTP. This inhibition was reversible by intracellular administration of the AT1-antagonist losartan and not influenced by the AT2-antagonist PD123319. Similar results were obtained in single cell [Ca2+]i measurements, showing that AngIIi predominantly influences Ca2+ influx and not Ca2+ release via AT1-like receptors. It is concluded that AngIIi modulates signal transduction activated by heterologous receptor stimulation.

Regulation of [Ca(2+)](i) homeostasis in MRP1 overexpressing cells.

Regulation of capacitative Ca(2+) entry was studied in two different multidrug resistance (MDR) protein (MRP1) overexpressing cell lines, HT29(col) and GLC4/ADR. MRP1 overexpression was accompanied by a decreased response to thapsigargin. Moreover, inhibition of capacitative Ca(2+) entry by D, L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) was abolished in MRP1 overexpressing cells. Both PDMP and the MRP1 inhibitor MK571 greatly reduced InsP(3)-mediated (45)Ca(2+) release from intracellular stores in HT29 cells. Again, these effects were virtually abolished in HT29(col) cells. Our results point to a modulatory role of MRP1 on intracellular calcium concentration ([Ca(2+)](i)) homeostasis which may contribute to the MDR phenotype.

Differential expression of sphingolipids in MRP1 overexpressing HT29 cells.

We have obtained a novel multidrug resistant cell line, derived from HT29 G(+) human colon carcinoma cells, by selection with gradually increasing concentrations of the anti-mitotic, microtubule-disrupting agent colchicine. This HT29(col) cell line displayed a 25-fold increase in colchicine resistance and exhibited cross-resistance to doxorubicin, VP16, vincristine and taxol. Immunoblotting, combined with RT-PCR showed that the multidrug resistance phenotype was conferred by specific overexpression of the multidrug resistance protein 1. Confocal scanning laser microscopy revealed that multidrug resistance protein 1 specifically localized in the plasma membrane of HT29(col) cells. In a functional assay, using the fluorescent multidrug resistance protein 1 substrate 5-carboxyfluorescein, an increased efflux activity of HT29(col) cells was measured, as compared to the wild-type HT29 G(+) cells. MK571, a specific inhibitor of multidrug resistance protein 1, blocked the 5-carboxyfluorescein efflux, but only partially reversed resistance to colchicine, indicating that additional multidrug resistance mechanisms operate in HT29(col) cells. In conclusion, these results show for the first time overexpression of a functional multidrug resistance protein 1 under colchicine pressure, indicating that colchicine is not a P-glycoprotein-specific substrate. Colchicine-induced overexpression of multidrug resistance protein 1 is accompanied by a changed sphingolipid composition, i.e., enhanced levels of glucosylceramide and galactosylceramide. In addition, ceramide, a lipid messenger molecule involved in apoptosis-related signal transduction processes, was much more abundant in HT29(col) cells, which is indicative of a stress response.

P2Y receptors contribute to ATP-induced increases in intracellular calcium in differentiated but not undifferentiated PC12 cells.

ATP-induced Ca2+ transients were examined in individual PC12 cells of a well defined clone, before and after treatment with nerve growth factor (NGF) to induce a neurone-like phenotype. Using reverse transcriptase PCR these cells were found to express mRNA for several P2 receptors. In undifferentiated cells the ATP-induced Ca2+ response was entirely dependent on Ca2+ influx, could not be mimicked by UTP, alpha,beta-methylene ATP or dibenzoyl ATP or be blocked by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS). ATP had no significant effect on levels of cyclic AMP or inositol 1,4,5-trisphosphate (InsP3). These results suggest that in undifferentiated PC12 cells ATP mainly acts on a P2X receptor, possibly the P2X4 subtype. After treatment with NGF for 7 days the ATP response was increased and partially sensitive to PPADS. A component of the ATP-induced Ca2+ increase was due to mobilisation of intracellular Ca2+ stores and another to capacitative Ca2+ entry. UTP caused an increase in intracellular Ca2+, and InsP3 formation could be stimulated by ATP and UTP. ATP also caused a small increase in cyclic AMP, but this was abolished in the presence of indomethacin. Thus, after NGF treatment ATP acts partially via a P2Y receptor, possibly the P2Y2 subtype.

Contractile effects by intracellular angiotensin II via receptors with a distinct pharmacological profile in rat aorta.

1. We studied the effect of intracellular angiotensin II (Ang II) and related peptides on rat aortic contraction, whether this effect is pharmacologically distinguishable from that induced by extracellular stimulation, and determined the Ca2+ source involved. 2. Compounds were delivered into the cytoplasm of de-endothelized aorta rings using multilamellar liposomes. Contractions were normalized to the maximum obtained with phenylephrine (10(-5) M). 3. Intracellular administration of Ang II (incorporation range: 0.01-300 nmol mg(-1)) resulted in a dose-dependent contraction, insensitive to extracellular administration (10(-6) M) of the AT1 receptor antagonist CV11947, the AT2 receptor antagonist PD 123319, or the non-selective AT receptor antagonist and partial agonist saralasin ([Sar1,Val5,Ala8]-Ang II (P<0.05). 4. Intracellular administration of CV11947 or PD 123319 right shifted the dose-response curve about 1000 fold or 20 fold, respectively. PD 123319 was only effective if less than 30 nmol mg(-1) Ang II was incorporated. 5. Contraction was partially desensitized to a second intracellular Ang II addition after 45 min (P<0.05). 6. Intracellular administration of Ang I and saralasin also induced contraction (P<0.05). Both responses were sensitive to intracellular CV11947 (P<0.05), but insensitive to PD 123319. The response to Ang I was independent of intracellular captopril. 7. Contraction induced by extracellular application of Ang II and of Ang I was abolished by extracellular pre-treatment with saralasin or CV11947 (P<0.05), but not with PD 123319. Extracellular saralasin induced no contraction. 8. Intracellular Ang II induced contraction was not affected by pre-treatment with heparin filled liposomes, but completely abolished in Ca2+-free external medium. 9. These results support the existence of an intracellular binding site for Ang II in rat aorta. Intracellular stimulation induces contraction dependent on Ca2+-influx but not on Ins(1,4,5)P3 mediated release from intracellular Ca2+-stores. Intracellular Ang I and saralasin induce contraction, possibly via the same binding site. Pharmacological properties of this putative intracellular receptor are clearly different from extracellular stimulated AT1 receptors or intracellular angiotensin receptors postulated in other tissue.

PDMP blocks brefeldin A-induced retrograde membrane transport from golgi to ER: evidence for involvement of calcium homeostasis and dissociation from sphingolipid metabolism.

In this study, we show that an inhibitor of sphingolipid biosynthesis, D,L-threo-1-phenyl-2- decanoylamino-3-morpholino-1-propanol (PDMP), inhibits brefeldin A (BFA)-induced retrograde membrane transport from Golgi to endoplasmic reticulum (ER). If BFA treatment was combined with or preceded by PDMP administration to cells, disappearance of discrete Golgi structures did not occur. However, when BFA was allowed to exert its effect before PDMP addition, PDMP could not "rescue" the Golgi compartment. Evidence is presented showing that this action of PDMP is indirect, which means that the direct target is not sphingolipid metabolism at the Golgi apparatus. A fluorescent analogue of PDMP, 6-(N-[7-nitro-2,1, 3-benzoxadiazol-4-yl]amino)hexanoyl-PDMP (C6-NBD-PDMP), did not localize in the Golgi apparatus. Moreover, the effect of PDMP on membrane flow did not correlate with impaired C6-NBD-sphingomyelin biosynthesis and was not mimicked by exogenous C6-ceramide addition or counteracted by exogenous C6-glucosylceramide addition. On the other hand, the PDMP effect was mimicked by the multidrug resistance protein inhibitor MK571. The effect of PDMP on membrane transport correlated with modulation of calcium homeostasis, which occurred in a similar concentration range. PDMP released calcium from at least two independent calcium stores and blocked calcium influx induced by either extracellular ATP or thapsigargin. Thus, the biological effects of PDMP revealed a relation between three important physiological processes of multidrug resistance, calcium homeostasis, and membrane flow in the ER/ Golgi system.

Extracellular and intracellular arachidonic acid-induced contractions in rat aorta.

Arachidonic acid induced contractions of de-endothelized rat aortic rings. A more potent effect was obtained after intracellular administration of arachidonic acid using liposomes. Contractions induced by extracellular arachidonic acid were inhibited similarly to phenylephrine-induced contractions by the L-type Ca2+ channel blocker, methoxyverapamil (D600), and the calmodulin inhibitor, calmidazolium. In contrast, contractions induced by arachidonic acid-filled liposomes were not affected by these compounds. Indomethacin did not affect the contractions induced by either extra- or intracellular arachidonic acid, whereas nordihydroguaiaretic acid relaxed contractions induced by extracellular arachidonic acid but not those induced by arachidonic acid-filled liposomes. Apart from a relaxing effect on contractions induced by extracellular arachidonic acid or by phenylephrine, protein kinase C inhibition with 1-(5-isoquinolinesulphonyl-2-methylpiperazine (H7)) had an even more prominent relaxing effect on contractions induced by arachidonic acid-filled liposomes. Therefore, arachidonic acid exerts a contractile effect on rat aorta, and this effect is regulated differently depending on the site of application.

D-myo-inositol derivatives alter liposomal membrane fluidity.

We investigated the effect on membrane fluidity induced by D-myo-inositol derivatives (IP3, IP4, IP5, IP6). Fluidity was determined as the anisotropy of fluorescence polarisation from liposome model membranes labelled with DPH (1,6-diphenyl-1,3,5 hexatriene). IP3 (10(-10) to 10(-5) M) increased the membrane fluidity with a maximum effect at 10(-5) M. For IP4, IP5 and IP6, at concentrations less than 10(-6) M these derivatives increased the membrane viscosity (i.e. reduced fluidity). This effect was enhanced when the derivatives were incorporated in the vesicles, rather than added to the vesicle suspension. In this case IP5 and IP6 increased viscosity over the reference values. We conclude that inositol derivatives directly modified membrane fluidity which could play a role in their effects in biological systems, beside the one mediated by binding to specific receptors.

Vasorelaxant properties of brefeldin A in rat aorta.

The effects of brefeldin A, a putative specific agent that disassembles the Golgi apparatus were assessed on the contractility of de-endothelised rat aorta. Brefeldin A inhibited, either as pre- or as post-treatment, the contractions elicited by K+ (75 mM) or phenylephrine (10 microM), being significantly more potent upon the latter. The thapsigargin (1 microM)-induced rat aorta contraction was less sensitive to brefeldin A inhibition. Pre-treatment with brefeldin A (30-100 microM) did not affect phenylephrine-induced transient contractions in Ca2+-free medium, but strongly inhibited the phenylephrine-induced sustained contractions upon re-admission of Ca2+ to the medium. Brefeldin A was unable to prevent Ca2+ stores refilling. We concluded that brefeldin A inhibits Ca2+ entry but not the pathways activated after Ca2+ stores depletion or the pathways responsible for replenishment of these stores in rat aorta, presumably by disassembling the Golgi apparatus network.

Delta9-tetrahydrocannabinol activates [Ca2+]i increases partly sensitive to capacitative store refilling.

Delta9-tetrahydrocannabinol induces [Ca2+]i increases in DDT1MF-2 smooth muscle cells. Both Ca2+ entry and release from intracellular Ca2+ stores were concentration dependently activated. The Ca2+ entry component contributed most to the increases in [Ca2+]i. Stimulation with delta9-tetrahydrocannabinol after functional downregulation of intracellular Ca2+ stores by longterm thapsigargin treatment, still induced a major Ca2+ entry and a minor Ca2+ release component. Thapsigargin sensitive influx and release were selectively inhibited by the cannabinoid CB1 receptor antagonist SR141716A. No effects on [Ca2+]i were obtained after stimulation with the CB2 receptor agonist palmitoylethanolamide. This study is the first demonstration of (1) Ca2+ release from thapsigargin sensitive intracellular stores and capacitative Ca2+ entry via CB1 receptor stimulation and of (2) an additional delta9-tetrahydrocannabinol induced thapsigargin insensitive component, mainly representing Ca2+ influx which is neither mediated by CB1 nor CB2 receptor stimulation.

TLC characterization of small unilamellar liposomes containing D-myo-inositol derivatives.

The thin-layer chromatographic (TLC) behaviour of small unilamellar liposomes containing inositol phosphates (IPs) was studied. The vesicles contained different concentrations of D-myo-inositol 1,4,5-triphosphate (IP3), D-myo-inositol 1,2,6-triphosphate (alpha-trinositol, PP 56, a novel Perstorp Pharma derivative), D-myo-inositol 1,3,4,5-tetraphosphate (IP4), D-myo-inositol 1,3,4,5,6-pentakisphosphate (IP5) and D-myo-inositol 1,2,3,4,5,6-hexakisphosphate (IP6). Migration of all liposome batches was compared to that of control liposomes (multilamellar and small unilamellar, both containing only triple-distilled water), and to that of free phosphatidylcholine (PC). The same amount of lipid was used in all situations. Thin-layer chromatography was performed with silica gel as adsorbent. The developing solvent was an n-buthanol:ethanol:water mixture in a 4:3:3 volume ratio. At doses higher than 10(-2) M liposomes containing alpha-trinositol and IP6 had a different migration than PC, MLV or SUV as well as all batches of liposomes. Physiological studies (using as model endothelized rat aorta rings) proved that in this situation they had no effects.

Effects of alpha-trinositol administered extra- and intracellularly (using liposomes) on rat aorta rings.

The effects of alpha-trinositol, a D-myo-inositol [1,2,6]trisphosphate derivative, were studied on de-endothelised rat aorta rings. The substance was applied extracellularly as well as intracellularly (by using liposomes as drug carriers). Upon extracellular administration, the drug reduced the level of contraction induced by 40 mM K+ or by phenylephrine (10(-5) M). No effects were observed on relaxed preparations. Liposomes containing alpha-trinositol induced a dose-dependent contraction of the preparations under resting tension with a threshold of 10(-5) M in the aqueous phase. These contractions were heparin-insensitive but were significantly blocked by D-600 (10(-5) M) (an L-type Ca2+ channel blocker) or in Ca(2+)-free medium. Our data suggest that alpha-trinositol has a plasmalemmal mechanism of action which could involve Ca2+ influx from the extracellular space.

TLC characterization of liposomes containing D-myo-inositol derivatives.

The thin-layer chromatographic (TLC) behaviour of liposomes containing inositol phosphates (IPs) was studied. The liposomes contained different concentrations of D-myo-inositol 1,4,5k-trisphosphate (IP3), D-myo-inositol 1,2,6-trisphosphate (alpha-trinositol, PP 56, a novel Perstorp Pharma derivative), D-myo-inositol 1,3,4,5-tetrakisphosphate (IP4), D-myo-inositol 1,3,4,5,6-pentakisphosphate (IP5) and D-myo-inositol 1,2,3,4,5,6-hexakisphosphate (IP6). Migration of all liposome batches was compared to that of control liposomes (containing only triple distilled water), and to that of free phosphatidylcholine (PC); the same amount of lipid was used in all situations. Thin-layer chromatography was performed on silica gel as adsorbent. As solvent we used an n-buthanol:ethanol:water mixture in a 4:3:3 volume ratio. Significant differences were found between PC and all liposome batches, as well as between control liposomes and the ones containing IP3, alpha-trinositol, IP4, or IP5, in various concentrations. Liposomes containing IP6 migrate completely differently compared not only to phosphatidylcholine and control liposomes, but also to the ones containing other IPs ( < 10(-3) M). Unlike the other IPs studied, liposome-entrapped IP6 elicits dose-dependent contractions of the isolated rat aorta. This suggests that liposomes loaded with IP6 undergo, during or after their preparation, physico-chemical alterations that eventually change their drug-delivery capacity.

Multiple effects of tyrosine kinase inhibitors on vascular smooth muscle contraction.

The effects of three tyrosine kinase inhibitors: genistein, quercetin and psi-tectorigenin, were investigated on contractions evoked in de-endothelised rat aortic rings, either by phenylephrine or 70 mM K+. A dose-dependent inhibition of both contractions by all three compounds was observed, the phenylephrine-mediated contractions being more sensitive to genistein. No differences between genistein or quercetin effects in pre-treatment or post-treatment protocols were found. Ca2+ store refilling, expressed in terms of phenylephrine-induced tension in Ca(2+)-free medium, was dose-dependently blocked by quercetin and genistein. Sodium orthovanadate, an inhibitor of tyrosine phosphatase, contracted the rat aortic rings with an IC50 of 0.66 microM. Its presence during the refilling period after exposure to Ca(2+)-free medium completely prevented the subsequent response to phenylephrine. One can conclude that the use of the above-mentioned protein tyrosine kinase inhibitors in the rat aorta blocks a step involved in Ca2+ entry and Ca2+ store refilling. A definite conclusion regarding the vanadate effects is not possible due to the fact that this compound also affects Ca2+ ATP-ases.

Effects of liposome-entrapped platelet-activating factor in the isolated rat trachea.

The effects of platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine)-filled liposomes upon rat tracheal rings in vitro were examined. The capture of liposomes by the smooth muscle cells of the isolated tracheal rings as well as the release of their content into the cytoplasm was shown by using Evans blue (5 x 10(-4) M)-loaded liposomes. Administration of PAF (10(-3) M)-filled liposomes contracted the preparations, in contrast with extracellular administration of PAF and control liposomes, which had no effect. Administration during the plateau or pretreatment with liposomes containing BN 52021 (3-t-butylhexahydro-4,7b-trihydroxy-8-methyl-9H-1,7a-epoxymethano- 1H,6aH- cyclopenta[c]furo(2,3-b)furo[3',2':3,4]cyclopental [1,2-d]furan-5,9,12(4H)-trione) ((10(-3) M, a selective PAF receptor antagonist) or heparin (5 x 10(-5) M) blocked this contraction. BN 52021 and heparin, not entrapped in liposomes, had no such effect. Our data suggest an intervention of PAF in the mechanisms of contraction of tracheal smooth muscle, involving a direct or indirect intervention (intracellular receptors for PAF cannot be excluded). At the same time, the rat trachea contraction induced by PAF-loaded liposomes could be linked to the PtdIns(1,4,5)P3-dependent Ca2+ channels from the endoplasmic reticulum and/or to the interaction with G proteins, as shown by the blocking effects of heparin-containing liposomes.

TLC--a rapid method for liposome characterization.

One of the most important problems for the use of liposomes as a drug delivery system is the modification of the vesicle induced by the liquid medium in which they are introduced (blood plasma for in vivo studies and the saline buffer solution for in vitro studies). Using thin-layer chromatography (TLC) we compared the behaviour of phosphatidylcholine (used for liposomes preparation) to that of the following unfilled liposomes: multilamellar liposomes (MLV); small unilamellar vesicles (SUV); and reverse phase evaporation vesicles (REV), before and after storage for 15 min in Krebs-Henseleit solution (37 degrees C, pH 7.4, aerated continuously with 95% O2 + 5% CO2). All variants contained the same amount of phosphatidylcholine. Thin-layer chromatography was performed on silica gel 60 as adsorbent. Two types of solvents were tested: one based on chloroform/alcohol (n-butanol or n-propanol or methanol)/water mixture (in different ratios) and another based on alcohol/alcohol/water mixture (n-butanol/n-propanol/water in 4/3/3 volume ratio). In all variants of chloroform containing solvents no differences were found between phosphatidylcholine and all types of liposomes. When using as solvent n-butanol/n-propanol/water significant differences were found between all types of liposomes before and after storage in Krebs-Henseleit solution. Their presence, after TLC treatment, was shown in electron microscopy studies.