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Purpose: γ-rays (IR) cause an increase in intracellular calcium [Ca(2+)], alters contractility and triggers apoptosis via the activation of protein kinase C in intestinal guinea pig smooth muscle cells. The present study investigated the role of the mitochondria in these processes and characterized proteins involved in IR-induced apoptosis., Materials and Methods: Intestinal smooth muscle cells were exposed to 10-50 Gy from a (60)Co γ-source. Reactive oxygen species (ROS) levels were measured by colourimetry with a fluorescente probe. Protein expression was analyzed by immunoblotting and immunofluorescence., Results: Apoptosis was inhibited by glutathione, possible by inhibiting the generation or scavenging ROS. Apoptosis was mediated by the mitochondria releasing cytochrome c leading to caspase 3 activation. IR increased the expression of the cyclins A, B2 and E and led to unbalanced cellular growth in an absorption dose-dependent manner. However, radiation did not induce alterations in the mitochondrial ultrastructure or in transmembrane electric potential. In contrast, IR increased the nuclear expression of cytoplasmic proteins and cyclins A and E., Conclusion: Smooth muscle cells subjected to IR undergo mitochondrial-mediated apoptosis that involves oncoproteins activation and preserves mitochondrial structure. IR also cause alterations in the expression and localization of both pro- and anti-apoptotic proteins.

Background and Purpose: Adding spironolactone to standard therapy in heart failure reduces morbidity and mortality, but the underlying mechanisms are not fully understood. We analysed the effect of canrenone, the major active metabolite of spironolactone, on myocardial contractility and intracellular calcium homeostasis., Experimental Approach: Left ventricular papillary muscles and cardiomyocytes were isolated from male Wistar rats. Contractility of papillary muscles was assessed with force transducers, Ca(2+) transients by fluorescence and Ca(2+) fluxes by electrophysiological techniques., Key Results: Canrenone (300-600 micromol L(-1)) reduced developed tension, maximum rate of tension increase and maximum rate of tension decay of papillary muscles. In cardiomyocytes, canrenone (50 micromol L(-1)) reduced cell shortening and L-type Ca(2+) channel current, whereas steady-state activation and inactivation, and reactivation curves were unchanged. Canrenone also decreased the Ca(2+) content of the sarcoplasmic reticulum, intracellular Ca(2+) transient amplitude and intracellular diastolic Ca(2+) concentration. However, the time course of [Ca(2+)](i) decline during transients evoked by caffeine was not affected by canrenone., Conclusion and Implications: Canrenone reduced L-type Ca(2+) channel current, amplitude of intracellular Ca(2+) transients and Ca(2+) content of sarcoplasmic reticulum in cardiomyocytes. These changes are likely to underlie the negative inotropic effect of canrenone.

The role of intracellular Ca2+ (Ca2+i) on hematopoiesis was investigated in long term bone marrow cultures using cytokines and agonists of P2 receptors. Cytokines interleukin 3 and granulocyte/macrophage colony stimulator factor promoted a modest increase in Ca2+i concentration ([Ca2+]i) with activation of phospholipase Cgamma, MEK1/2, and Ca2+/calmodulin kinase II. Involvement of protein kinase C was restricted to stimulation with interleukin 3. In addition, these cytokines promoted proliferation (20 times) and an increase in the Gr-1(-)Mac-1+ population with participation of gap junctions (GJ). Nevertheless ATP, ADP, and UTP promoted a large increase in [Ca2+]i, moderate proliferation (6 times), a reduction in the primitive Gr-1(-)Mac-1(-)c-Kit+ population, and differentiation into macrophages without participation of GJ. It is likely that Ca2+i participates as a regulator of hematopoietic signaling: moderate increases in [Ca2+]i would be related to cytokine-dependent proliferation with participation of GJ, whereas high increases in [Ca2+]i would be related to macrophage differentiation without maintenance of the primitive population.

We investigated the effects of gamma-radiation on cells isolated from the longitudinal smooth muscle layer of the guinea pig ileum, a relatively radioresistant tissue. Single doses (up to 50 Gy) reduced the amount of sarcoplasmatic reticulum and condensed the myofibrils, as shown by electron microscopy 3 days post-irradiation. After that, contractility of smooth muscle strips was reduced. Ca(2+) handling was altered after irradiation, as shown in fura-2 loaded cells, with elevated basal intracellular Ca(2+), reduced amount of intrareticular Ca(2+), and reduced capacitive Ca(2+) entry. Radiation also induced apoptosis, judged from flow cytometry of cells loaded with proprium iodide. Electron microscopy showed that radiation caused condensation of chromatin in dense masses around the nuclear envelope, the presence of apoptotic bodies, fragmentation of the nucleus, detachment of cells from their neighbors, and reductions in cell volume. Radiation also caused activation of caspase 12. Apoptosis was reduced by the administration of the caspase inhibitor Z-Val-Ala-Asp-fluoromethyl-ketone methyl ester (Z-VAD-FMK) during the 3 day period after irradiation, and by the chelator of intracellular Ca(2+), 1,2-bis(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA), from 1 h before until 2 h after irradiation. BAPTA also reduced the effects of radiation on contractility, basal intracellular Ca(2+), amount of intrareticular Ca(2+), capacitative Ca(2+) entry, and apoptosis. In conclusion, the effects of gamma radiation on contractility, Ca(2+) handling, and apoptosis appear due to a toxic action of intracellular Ca(2+). Ca(2+)-induced damage to the sarcoplasmatic reticulum seems a key event in impaired Ca(2+) handling and apoptosis induced by gamma-radiation.

The use of gamma-radiation in treatment of pelvic cancer is associated with injury of healthy surrounding tissues and disorders of intestinal motility; however, the cellular mechanisms involved are unclear. We tested the hypothesis that exposure of visceral smooth muscle cells (SMCs) to gamma-radiation induces apoptosis via activation of specific protein kinase C (PKC) isoforms. Cultured SMCs and slices from guinea pig ileum smooth muscle longitudinal layer (GPISMLL) were exposed to 10 to 50 Gy. Flow cytometry in gamma-radiated SMCs showed increased percentage of cells in the sub-G(0)/G(1) phase, a hallmark of apoptosis. gamma-Radiation-induced reduction in cell survival was partially but significantly alleviated with the PKC inhibitors. Sections of gamma-irradiated GPISMLL showed DNA fragmentation and apoptotic bodies analyzed by the terminal deoxynucleotidyl transferase dUTP nick-end labeling method, whereas the plasma and nuclear membranes were preserved. Confocal microscopy in gamma-radiated SMCs labeled with annexin V-fluorescein showed an increase in apoptotic cells and phosphatidylserine externalization. Contraction of GPISMLL strips in response to KCl and acetylcholine was reduced in tissues exposed to 30 and 50 Gy. gamma-Radiation of GPISMLL caused an increase in PKC activity in the particulate fraction, a decrease in the cytosolic fraction, and increased particulate/cytosolic PKC activity ratio. Western blot analysis revealed significant amounts of alpha- and epsilon-PKC in the cytosolic fraction of control GPISMLL. gamma-Radiation caused an increase in the amount of alpha- and epsilon-PKC in the particulate fraction and a decrease in the cytosolic fraction. Data suggest that gamma-radiation induces apoptosis, growth arrest, and contractile dysfunction in visceral SMCs of GPISMLL via activation and translocation of alpha- and epsilon-PKC isoforms.

Objective: To determine the effect of arachnotoxin (ATx), a venom extracted from the Chilean spider Latrodectus mactans, on intracellular calcium ([Ca(2+)](i)) and pH (pH(i)) in capacitated human spermatozoa., Design: Spermatozoa were collected from fertile adult men (n = 8). Mobile spermatozoa were collected by the "swim up" technique and stimulated with the crude extract of ATx and with progesterone (P)., Setting: Hospital of the Federal University of São Paulo, São Paulo, Brazil., Main Outcome Measure(s): [Ca(2+)](i) was measured in fura2-AM-loaded spermatozoa, and pH(i) was measured in spermatozoa loaded with the pH-sensitive dye [(2',7')-bis (carboxymethyl)-(5,6)-carboxyfluorescein]-AM (BCECF)., Result(s): The ATx and P induced a biphasic change in [Ca(2+)](i) consisting of a peak followed by a small but sustained elevation. The response to ATx was greatly reduced by pretreatment with P. The ATx caused intracellular acidification, whereas P induced alkalinization. Blockade of the NA(+)/H(+) exchanger with ethylisopropylamiloride (EIPA) sharply increased ATx-induced acidification., Conclusion(s): Arachnotoxin increased [Ca(2+)](i) through the opening of calcium channels and release of calcium from intracellular stores. The ATx reduced pH(i) in human sperm, possibly by inhibiting the Na(+)/H(+) exchanger.

In the current study, the ability of ATP to promote apoptosis in myeloblasts at different ages was investigated. We have observed that high concentration of extracellular ATP (>1mM), which activates P2X(7) receptor, produced cell shrinkage an increase in the number of events in the sub-G(0)/G(1) region of the cellular cycle and annexin-V/propidium iodide label, which characterizes the apoptotic cell death. In addition, BzATP produced apoptosis, but not ADP and UTP. Gr-1(+) cells express the P2X(7) receptor and oxidized ATP, a specific P2X(7) inhibitor, blocked the ATP-dependent apoptosis. ATP-dependent apoptosis is decreased by aging in myeloblasts of 12 and 22-month-old mice. Furthermore, P2X(7) expression decrease was observed in older mice, explaining apoptosis decrease. This decrease in apoptosis by aging may be related to some diseases in the myelocyte lineage.

In this study, we describe the presence of P2 receptor subtypes and Ca2+ signaling in erythroblasts. ATP and ADP produced a biphasic increase of intracellular Ca2+ concentration ([Ca2+]i), with an initial transient phase followed by a sustained phase. Reverse transcription polymerase chain reaction (RT-PCR) showed the expression of P2Y1, P2Y2 and P2Y12. The selective P2Y1 receptor antagonist 2'-deoxy-N6-methyl-adenosine-3',5'-diphosphate (MRS2179) and the G(i) protein inhibitor pertussis toxin blocked Ca2+ increase. The initial transient [Ca2+]i increase phase was sensitive to the 1,4,5-inositol trisphosphate (IP3) receptor blocker 2-aminoethoxy-diphenylborate (2-APB), while the sustained phase was sensitive to the protein kinase C (PKC) inhibitor 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide (GF109203X) and calcium calmodulin kinase II (CaMKII) inhibitor 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62). In addition, the PKC activator phorbol-12,13-dibutyrate (PDBu) produced increase of [Ca2+]i. Flow cytometry analysis showed the expression of Ca2+-dependent PKC alpha, betaI, gamma and phospho-CaMKII. These results suggest that the activation of the P2Y1 receptor triggers two different [Ca2+]i increase pathways, one IP3-dependent and the other kinase-dependent.

In this study, we investigated the effect of aging on intracellular Ca2+ stores, as sarcoendoplasmic reticulum (SR) and mitochondria, and the influence of these compartments on contraction of rat colon smooth muscle [Bitar, K.N., 2003. Aging and neural control of the GI tract V. Aging and gastrointestinal smooth muscle: from signal transduction to contractile proteins. Am. J. Physiol. Gastrointest. Liver. Physiol. 284(1), G1-G7; Marijic, J., Li, Q.X., Song, M., Nishimaru, K., Stefani, E., Toro, L., 2001. Decreased expression of voltage-and Ca2+-activated K+ channels in coronary smooth muscle during aging. Circ. Res. 88, 210-234; Rubio, C., Moreno, A., Briones, A. Ivorra, M.D., D'Ocon, P., Vila, E., 2002. Alterations by age of calcium handling in rat resistance arteries. J. Cardiovasc. Pharmacol. 40(6), 832-840]. Calcium stores and contraction were evaluated by simultaneous measurements of fluorescence and tension in smooth muscle strips loaded with fura-2. Results showed that activation of muscarinic receptors by methylcholine (MCh, 10 microM), induced a greater contraction in aged rats than in adult animals. The inhibition of Ca2+ ATPase by thapsigargin (TG, 1 microM) did not prevent the refilling of SR either in adult or aged rats. MCh, in the presence of TG, induced an increase in transient fluorescence, indicating a release of Ca2+ from TG-insensitive compartment. The mitochondrial uncoupler, FCCP (5 microM), caused a greater increase in intracellular Ca2+ and tension in aged rats, indicating that mitochondria may accumulate more Ca2+ during aging. The present results show that changes in intracellular Ca2+ stores, such as mitochondria and SR, affect contraction and may cause dysfunctions during aging that could culminate in severe alterations of Ca2+ homeostasis and cell damage.

Fura-2 is one of the most used fluorophore for measuring intracellular calcium concentration ([Ca2+]i). In mouse bone marrow cell suspensions ATP produces a biphasic effect: till 1 mM, ATP produces increases in [Ca2+]i; from 1 mM on an increase is observed, that is followed by the decrease in the 340/380 nm ratio (R340/380). At high ATP (4 mM) concentration fura-2 leaked from loaded bone marrow cell suspensions. We observed that ATP decreases fluorescence in the absorption and excitation spectra of fura-2, consequently the emitted one is decreased including the isobestic point (360 nm). ATP analogs: BzATP, ATPyS and UTP, but not alphabetaATP, ADP or AMP, promote decrease of fluorescence in the isobestic point of fura-2. The physical/chemical process that reduces the absorption and excitation of fura-2 by ATP is unknown. The P2X7 inhibitors, Mg2+ (5 mM), OxATP (300 microM) and Brilliant Blue (100 nM), blocked the efflux of fura-2 and ATP-induced R340/380 decrease. The J774 cell line and mononuclear cells with a higher expression of P2X7 receptors show the same decrease in R340/380 as that induced by ATP. In the HL-60 cell line, myeloid cells and erythroblasts extracted from bone marrow, such effect does not occur. It is concluded that the use of the fluorescent Ca2+ indicator fura-2 does not allow the correct measurement of [Ca2+]i in these cells in the presence of a higher concentration of ATP which activated the P2X7 receptor.

Heparin and heparan sulfate fragments, obtained by bacterial heparinase and heparitinases, bearing an unsaturation at C4-C5 of the uronic acid moiety, are able to produce up to 80% reduction of the cytosolic calcium of smooth muscle cell lines. Unsaturated disaccharides from chondroitin sulfate, dermatan sulfate, and hyaluronic acid are inactive, indicating that, besides the unsaturation of the uronic acid, a vicinal 1 --> 4 glycosidic linkage is needed. An inverse correlation between the molecular weight and activity is observed. Thus, the ED(50) of the N-acetylated disaccharide derived from heparan sulfate (430 Da) is 88 microm compared with 250 microm of the trisulfated disaccharide (650 Da) derived from heparin. Except for enoxaparin (which contains an unsaturation at the non-reducing end and 1 --> 4 glycosidic linkage), other low molecular weight heparins and native heparin are practically inactive in reducing the cytosolic calcium levels. Thapsigargin (sarcoplasmic reticulum Ca(2+)-ATPase inhibitor), vanadate (cytoplasmic membrane Ca(2+)-ATPase inhibitor), and nifedipine and verapamil (Ca(2+) channel antagonists) do not interfere with the effect of the trisulfated disaccharide upon the decrease of the intracellular calcium. A significant decrease of the activity of the trisulfated disaccharide is observed by reducing extracellular sodium, suggesting that the fragments might act upon the Na(+)/Ca(2+) exchanger promoting the extrusion of Ca(2+). This was further substantiated by binding experiments and circular dichroism analysis with the exchanger inhibitor peptide.

We combined fluorogenic substrates or internally quenched fluorescent peptides with specific inhibitors in the pH profile of proteolytic activity experiments in order to detect proteolytic activities in lysates of MDCK cells. Hydrolytic activities related to cathepsin B, L, and D were observed. Serine-proteinase was not detected; however, we clearly demonstrated the presence of a thiol-metallo-endo-oligopeptidase, also called thimet-oligopeptidase (TOP). This peptidase from MDCK cells has substrate and inhibitor specificities as well as an activation profile with mercaptoethanol that are indistinguishable from the recombinant rat testis TOP (EC 3. 4.24.15). In addition, polyclonal purified antibodies to this enzyme depleted the TOP activity of MDCK cells in whole homogenate. Although we present only preliminary data, TOP is secreted by MDCK cells. The presence of TOP in a phenotype polarized MDCK cells can have special significance in the cytoplasmic selection, transport, or clearance of short peptides due to restriction of the enzyme to sequences from 6 to 17 amino acids. Therefore, the MDCK cell could be a very useful cellular model with which to study some of the suggested TOP biological functions as processing of biological active peptides and antigen presentation., (Copyright 2000 Wiley-Liss, Inc.)

The effects of recombinant human erythropoietin (rHuEPO)-induced polycythemia on renal function and glomerular hemodynamics were evaluated in Munich-Wistar rats (MW+EPO) before and after infusion of indomethacin; the rHuEPO effects on total renal function were also evaluated in 5/6 nephrectomized (CRF) MW and spontaneously hypertensive rats (MW-CRF+EPO and SHR-CRF+EPO, respectively). In normal MW rats, rHuEPO (300 IU/kg BW, 3 x /week, during 2 weeks) induced elevation in MAP, with maintenance of GFR, paralleled by superficial vasodilatation and elevation in SNGFR, suggesting cortical blood redistribution. These hemodynamic alterations induced by rHuEPO were blunted by indomethacin, suggesting a participation of the vasodilator prostaglandins in the renal compensatory mechanism of polycythemia. Elevation in MAP and reduction in GFR occurred in the MW-CRF+EPO group compared with the group receiving vehicle. In contrast, the SHR-CRF+EPO presented a reduction in MAP and maintenance of GFR, suggesting different rHuEPO effects depending on previous renal function and/or hypertensive state.

Mammalian cell invasion assays, using metacyclic trypomastigotes of Trypanosoma cruzi G and CL strains, showed that the CL strain enters target cells in several-fold higher numbers as compared with the G strain. Analysis of expression of surface glycoproteins in metacyclic forms of the two strains by iodination, immunoprecipitation and FACS, revealed that gp90, undetectable in the CL strain, is one of the major surface molecules in the G strain, that expression of gp82 is comparable in both strains and that gp35/50 is expressed at lower levels in the CL strain. Purified gp90 and gp35/50 bound more efficiently than gp82 to cultured HeLa cells. However, the intensity of the Ca2+ response triggered in HeLa cells by gp82 was significantly higher than that induced by gp35/50 or gp90. Most of the Ca2+ signalling activity of the metacyclic extract towards HeLa cells was due to gp82 and was inhibitable by gp82-specific monoclonal antibody 3F6. Ca2+ mobilization was also triggered in metacyclic trypomastigotes by host-cell components; it was mainly gp82-mediated and more intense in the CL than in the G strain. We propose that expression of gp90 and gp35/50 at high levels impairs binding of metacyclic forms to host cells through productive gp82-mediated interaction, which leads to the target-cell and parasite Ca2+ mobilization required for invasion. Analysis of metacyclic forms of eight additional T. cruzi strains corroborated the inverse correlation between infectivity and expression of gp90 and gp35/50.

In rat stomach fundus, contractions induced by Ca2+ (1.8 mM) were strikingly potentiated by thapsigargin. This potentiation was partially inhibited by the blockers of Ca2+ release activated channels (CRACs), miconazole and SK&F96365 ([1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole, HCL]) and slightly blocked by the antagonist of calcium voltage-operated channels (VOCs), isradipine. In dissociated cells in a 0Ca solution, thapsigargin potentiated the increase in intracellular calcium after reintroduction of Ca2+. This potentiation was partially reduced by the CRAC blockers, but not by the VOC blockers. This data suggests that calcium influx increased due to the depletion of intracellular calcium by thapsigargin and that this influx occurs predominantly through CRACs.

To evaluate functional alterations of mesangial cells induced by diabetes (DMC), we observed the changes of cytosolic calcium ([Ca]i) in response to the vasoconstrictor agonists angiotensin II (Ang II) and norepinephrine (NOR). DMC were obtained from rats with streptozotocin-induced diabetes, cultured in normal medium and identified as mesangial cells (MC) in the third subculture. [Ca]i was measured using fura-2 as a fluorophore. Basal calcium levels (60 to 80 nM) in DMC were not different from control mesangial cells (CMC). The high glucose (30 mM) medium concentration reduced the response of CMC and DMC to Ang II and NOR. This was not an osmotic effect since mannitol did not alter these responses. When DMC were stimulated with Ang II, a desensitized response was always observed, with a transient variation of [Ca]i (N = 6, P < 0.05). In contrast, a non-desensitized response with a sustained pattern of [Ca]i increases was obtained in NOR-stimulated DMC. Therefore, the present results suggest that DMC show a modified response to stimulation of the Ang II receptor, which is expressed phenotypically in culture by desensitization. Furthermore, these alterations induced by diabetes environment in MC in vivo were maintained in vitro despite a long period (approximately 5 months) in which the cells were grown in normal culture medium.

The 35/50 kDa mucin-like surface glycoprotein (gp35/50) of Trypanosoma cruzi metacyclic trypomastigotes has been implicated in mammalian cell invasion. In this study we investigated whether the sialyl residues of gp35/50 are required for interaction of parasites with target cells. After treatment with bacterial neuraminidase, the metacyclic forms (G strain) remained reactive with the monoclonal antibody (mAb) 10D8 but lost their reactivity with mAb 3C9, that recognizes sialic acid-containing epitopes on gp35/50, and entered HeLa cells in significantly higher numbers as compared to untreated controls. Resialylation of gp35/50, by incubation of parasites with T. cruzi trans-sialidase and sialyl lactose, restored the reactivity with mAb 3C9 as well as the affinity for sialic acid specific lectin. Accordingly, the rate of invasion of resialylated parasites was reduced to levels similar to those observed before desialylation. Purified G strain gp35/50, desialylated by neuraminidase treatment, bound to HeLa cells more than its sialylated counterpart. The Ca2+ signaling activity, which has been associated with cell invasion, was also determined by measuring the cytosolic Ca2+ concentration ([Ca2+]i), in HeLa cells upon interaction with sonicated extracts from untreated or neuraminidase-treated parasites, or with purified gp35/50 in its sialylated or desialylated form. Consistent with the results of cell invasion assay, the desialylated parasite preparations, as well as the sialic acid free gp35/50, induced an average elevation in [Ca2+]i significantly higher than that triggered by untreated controls. None of these effects, namely the increase in infectivity and Ca2+ signaling activity, was observed with neuraminidase-treated CL strain metacyclic trypomastigotes, which express a variant form of sialic acid gp35/50 molecule that is not recognized by mAb 10D8 and apparently is not involved in target cell invasion.

FK 506 is a new immunosuppressive drug that, like cyclosporine A (CsA), presents nephrotoxicity. Glomerular hemodynamic studies showed that acute FK 506 infusion (N = 9, 3 mg/kg body wt, i.v. in bolus) caused a 57% reduction in glomerular filtration rate (GFR) (0.74 +/- 0.03 to 0.32 +/- 0.02 ml/min, P < 0.05) and a 40% reduction in single nephron glomerular filtration rate (SNGFR; 43.0 +/- 5.2 to 26.0 +/- 2.5 nl/min, P < 0.05) due to a 25% reduction in glomerular plasma flow rate (QA) (133.4 +/- 19.8 to 99.8 +/- 12.0 nl/min) and a 22% reduction in glomerular ultrafiltration coefficient (Kf; 0.1009 +/- 0.0203 to 0.0790 +/- 0.0130 nl/sec. mm Hg). After 10 days of FK treatment (N = 8, 0.6 mg/kg body wt, i.p.), we observed a reduction of 23% in GFR (0.97 +/- 0.02 to 0.75 +/- 0.04 ml/min, P < 0.05) and of 23% in SNGFR (37.9 +/- 3.0 to 29.1 +/- 1.9 nl/min, P < 0.05) due to a 42% reduction in Kf (0.1486 +/- 0.0101 to 0.0870 +/- 0.0110 nl/sec.mm Hg, P < 0.05) and a 38% reduction in QA (117.6 +/- 10.2 to 73.5 +/- 6.1 nl/min, P < 0.05). The latter was consequent to the increment of 72% in total arteriolar resistance (RT) (3.1 +/- 0.2 to 5.2 +/- 0.5 +/- 0.5 10(10).dyn.sec.cm-5, P < 0.05). Thus, the pattern of FK 506 effect on glomerular hemodynamics was similar in both acute and chronic treatments.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of angiotensin II (ANG II) and atrial natriuretic peptide (ANP) on intracellular free calcium concentration [Ca2+]i was investigated in Mandin-Darby canine kidney (MDCK) cells in culture. Changes in [Ca2+]i were monitored fluorometrically with the Ca(2+)-sensitive probe fura-2/AM at 37 degrees C using a Perkin-Elmer LS-5 spectrofluorimeter (excitation 340/380 nm, slit 3 nm; emission 520 nm, slit 10 nm). MDCK cells exhibited a mean baseline [Ca2+]i of 98 +/- 10 nM. The addition of increasing concentrations of ANG II (1 pM to 1 microM) to the cell suspension led to a progressive increase in [Ca2+]i to 2-3 times basal levels. In contrast, addition of 1 microM ANP to the cell suspension led to a very rapid 60% decrease in [Ca2+]i. The addition of 1 pM to 1 microM ANG II immediately after 1 microM ANP caused an increase in [Ca2+]i which never exceeded the basal level in the absence of ANP. The data indicate that ANG II increases cell [Ca2+]i, as expected, and provide the new observation that ANP reduces [Ca2+]i in these cells. Furthermore, ANP reduces the increase in [Ca2+]i elicited by ANG II, thus modulating the effect of ANG II on [Ca2+]i.

Simultaneous recordings of the tension and intracellular Ca2+ concentration of guinea-pig ileum longitudinal smooth muscle strips, as well as 24Na+ and 45Ca2+ influx measurements in cultured myocytes from the same tissue, were used to investigate the mechanisms underlying angiotensin-induced desensitization and tachyphylaxis. Angiotensin II and [2-lysine]-angiotensin II (Lys2All), incubated for prolonged periods (10 min) with muscle strips, induced fading of the contractile response (desensitization) and reappearance of the intracellular Ca2+ concentration oscillations, which were inhibited during the initial increase in cytosolic Ca2+. The desensitization was paralleled, in cultured myocytes, by inhibition of the 45Ca2+ but not of the 24Na+ influxes which were initially stimulated by the peptides. On the other hand, repeated administrations of angiotensin II (but not of Lys2All) caused gradual reduction of the contractile response and of the 24Na+ influx stimulation evoked by the agonist (tachyphylaxis). Treatment with phorbol 12-13 dibutyrate accelerated the desensitization induced by both angiotensin II and by Lys2All and aggravated the tachyphylaxis to angiotensin II. The results support the hypothesis that activation of protein kinase C is responsible for the desensitization and that tachyphylaxis is due to the slow dissociation of angiotensin II from a postulated Na(+)-dependent regulatory site on the receptor.

The effects of angiotensin II (ANG) on Na+ and Ca++ fluxes in cultured intestinal smooth muscle cells from the guinea pig ileum were studied and correlated with the contraction and desensitization observed in whole muscles. The effects of ANG were compared with those of acetylcholine (ACh), an agonist that acts at muscarinic receptors in the intestinal smooth muscle and which does not induce desensitization. Both ANG and ACh stimulated 24Na+ influx upon addition to the cells, and this stimulation persisted for at least 30 min. Both agonists also stimulated 45Ca++ uptake but ANG's effect was transient, whereas that of ACh was persistent. Short-term (30 min) treatment with PMA (phorbol-12-myristate-13-acetate) caused a fade of the tonic response of the whole muscle to ANG, and also blocked this hormone's stimulating effect on 45Ca++, but not on 24Na+ influx. Long-term (7 hr) treatment with PMA, which suppresses protein kinase C activity, restored ANG's ability to stimulate 45Ca++ influx. The stimulating effects of ACh on 24Na+ and 45Ca++ influxes were not affected by short- or long-term treatment of the cells with PMA. Our results suggest that ANG desensitization involves protein kinase C inhibition of a step in the stimulus-response chain that is subsequent to phospholipase C-activation.

The contractile responses of the rabbit isolated coeliac and mesenteric arteries to five agonists (angiotensin, adrenaline, histamine, acetylcholine and 5-hydroxytryptamine), but not to K+, were potentiated by indomethacin (8.4 microM) The potentiation was similar whether indomethacin was added 1 h before or during the response to the agonist. The agonists that were more potentiated by indomethacin were also more dependent on the Ca2+ concentration in the medium, for their contractile action. Prostaglandin E2 in low concentrations (micromolar) did not affect the resting tone but relaxed the agonist-contracted arteries both in normal and in Ca2+-free medium. No prostaglandin E (PGE)-like substances were detected in the perfusate of arteries contracted by angiotensin. Reduction of the external Na+ concentration to 80 mM resulted in potentiation of the responses to agonists (angiotensin and adrenaline), but not to K+, and in this Na+-deficient medium potentiation by indomethacin was greatly reduced. These results suggest that potentiation by indomethacin of the arteries' responses to vasoactive substances may result from that drug's inhibitory action on sodium influx and consequent increase in calcium entry through receptor-operated channels.

The effect of modifications of the N-terminal end of the angiotensin II (AII) molecule on its ability to induce tachyphylaxis in the isolated rabbit aorta was studied by analyzing the effects of several analogs of AII. No tachyphylaxis to AII was observed, but (1-sarcosine)-AII, (1-guanido-acetic)-AII and (1-glycine)-AII induced marked tachyphylaxis. (1-Betaine)-AII, (2-lysine)-AII, (2-ornithine)-AII and (1-sarcosine,2-lysine)-AII were unable to induce tachyphylaxis in the rabbit aorta. A positively charged N terminus and the guanidino group of the Arg2 side chain appear to be needed for the manifestation of the tachyphylactic property, while the Asp1 side chain is responsible for the absence of tachyphylaxis to AII. It is proposed that the analogs that are able to induce tachyphylaxis, after binding to the receptor to produce the agonistic response, induce a slow conversion of the hormone-receptor complex into a tachyphylactic state, and that this conversion is promoted by the interaction of the N terminus and the guanidino group of the analog with their complementary sites.

The responses of rabbit mesenteric or coeliac artery rings to angiotensin II or adrenaline (but not to K+) were enhanced by endothelium destruction (by rubbing). Potentiation by indomethacin of the response to the agonists was observed in rubbed rings but not in intact ones. Both angiotensin II and adrenaline (in the presence of propranolol and prazosin) induced endothelium-dependent relaxation of the arteries. Rubbed rings, but not intact ones, contracted when Ca2+ was added to a previously Ca2+-free medium containing angiotensin II or adrenaline. The vasoconstrictor response appears to be modulated by the regulation of receptor-operated Ca2+ channels through EDRF released by the endothelium and by some cyclo-oxygenase product at the level of the smooth muscle cell membrane.

The homologous desensitization induced by angiotensin II analogues in the guinea-pig isolated ileum was studied. Desensitization assessed by the loss of response on repeated treatment showed [Sar1]angiotensin II to be a strong desensitizer whereas no desensitization to [Lys2]angiotensin II was detected. However, prolonged treatment with either analogue desensitized the tissue, indicating that [Lys2]angiotensin II-induced desensitization was reversed faster. A correlation was found between the degree of desensitization caused by repeated treatment and the time for half-relaxation after washout of the first treatment, but the relaxation after washout became faster in the desensitized state. In experiments designed to study competition between the agonistic and desensitizing properties of angiotensin II analogues, high concentrations of [Lys2]angiotensin II blocked the agonistic but not the desensitizing effect of lower concentrations of [Sar1]angiotensin II. It is concluded that desensitization is due to the interaction of angiotensin II with a regulatory site on the receptor.

The structural requirements for the production of angiotensin tachyphylaxis in the guinea-pig ileum were studied by analyzing the tachyphylactic properties of the following synthetic analogues of angiotensin II (AII): [1-sarcosine]AII, [1-betaine]AII; [1-guanidinoacetic]AII; betainyl-AII; [2-lysine]AII; [2-ornithine]AII. In the non-atropinized ileum, no tachyphylaxis was observed with any of the following analogues: [2-lysine]AII, [2-ornithine]AII, [2-ornithine]AII, [1-betaine]AII and betainyl-AII. [1-Guanidinoacetic]AII induced tachyphylaxis, but to a smaller degree than AII, while [1-sarcosine]AII was significantly more tachyphylactic than AII. Similar results were obtained in the atropinized ileum, except that moderate tachyphylaxis was also observed with betainyl-AII and [1-betaine]AII. The analogues with lysine or ornithine residues in position 2 did not induce tachyphylaxis under any of the conditions studied. It is concluded that, besides the protonated N-terminal amino group, the guanidino group of the Arg2 side-chain is essential for the manifestation of angiotensin tachyphylaxis in the guinea-pig ileum.

The role of the tyrosine side-chain in the smooth muscle contracting activity of angiotensin III was investigated by determining intrinsic activities and ED50 values of [4-(3-chlorotyrosine)]angiotensin II and [4-(3-benzyltyrosine)]angiotensin II in the isolated guinea-pig ileum and rat uterus. [4-(3-chlorotyrosine)]angiotensin II activity was compared with that of angiotensin II at different pH values, in which the ratio of their degrees of phenolic ionization varied. The results indicated that deprotonation of the phenolic group hinders binding to smooth muscle cell receptors, but not triggering of the response by the hormone-receptor complex. Steric hindrance by the benzyl substituent in [4-(3-benzyltyrosine)]angiotensin II reduced both receptor-binding and triggering of the response.

The role of ion fluxes in angiotensin II (AII) desensitization (tachyphylaxis) was investigated by studying Na+ and Ca2+ translocation in cultured vascular smooth muscle cells from the rat aorta. The effects of AII were compared to those of [1-sarcosine]-AII (Sar1-AII), an analogue which also induces tachyphylaxis, and [2-lysine]-AII (Lys2-AII), an analogue that does not show this property. Maximally effective concentrations of the three peptides induced a rapid and transient increase in 45Ca2+ efflux, a rapid and sustained decrease in total cell Ca2+ and an increased Na+ permeability. Repeated treatments, at short intervals, with either of the three peptides abolished the effect on Ca2+ efflux, and this desensitization was slowly reversible. A 30-min rest period was sufficient for full recovery of the response of cells that were desensitized by Lys2-AII, whereas the recovery from AII or Sar1-AII-desensitization was still not complete after 60 min. Our results suggest that the difference in the behaviour of the "tachyphylactic" AII and Sar1-AII and the "non-tachyphylactic" Lys2-AII lays not in the production of different signals upon binding to the receptor, but in a difference in the hormone-receptor interaction itself.