3-d image analysis of fluorescent drug binding.

Fluorescent ligands provide the means of studying receptors in whole tissues using confocal laser scanning microscopy and have advantages over antibody- or non-fluorescence-based method. Confocal microscopy provides large volumes of images to be measured. Histogram analysis of 3-D image volumes is proposed as a method of graphically displaying large amounts of volumetric image data to be quickly analyzed and compared. The fluorescent ligand BODIFY FL-prazosin (QAPB) was used in mouse aorta. Histogram analysis reports the amount of ligand-receptor binding under different conditions and the technique is sensitive enough to detect changes in receptor availability after antagonist incubation or generic manipulations. QAPB binding was concentration dependent, causing concentration-related rightward shifts in histogram. In the presence of 10 microM phenoxybenzamine (blocking agent), the QAPB (50 nM) histogram overlaps the autofluorescence curve. The histogram obtained for the 1D knockout aorta lay to the left of that control and 1B knockout aorta, indicating a reduction in 1D receptors. We have shown, for the first time, that it is possible to graphically display binding of a fluorescent drug to a biological tissue. Although our application is specific to adrenergic receptors, the general method could be applied to any volumetric, fluorescence-image-based assay.

Acute and chronic captopril, but not prazosin or nifedipine, normalize alterations in adrenergic intracellular Ca2+ handling observed in the mesenteric arterial tree of spontaneously hypertensive rats.

The effect of hypertension and acute (36-h) or chronic (from age 6 to 16 weeks) antihypertensive treatment with prazosin (2 mg kg(-1) per day), nifedipine (50 mg kg(-1) per day), or captopril (50 mg kg(-1) per day) on Ca2+ mobilization due to alpha1-adrenoceptor activation was analyzed in functional studies using arterial rings [four conductance/distributing vessels: aorta, main mesenteric, iliac, and tail arteries and two resistance vessels; first and second small mesenteric artery branches obtained from spontaneously hypertensive rats (SHR, 6 and 16 weeks old) and age-matched Wistar Kyoto rats (WKY)]. Maximal response to noradrenaline in the presence of extracellular Ca2+ is not affected by hypertension or by the antihypertensive treatment. The extracellular Ca2+-independent contractile responses increased with age in iliac, tail, and small mesenteric arteries (SMA) and were further increased in SHR in SMA from both young and adult animals and in the main mesenteric artery of adult SHR. In main mesenteric artery, this increased contraction in SHR was associated with a higher increase in cytosolic [Ca2+] mobilized by noradrenaline without changes in the total stored Ca2+. Acute or chronic treatment with captopril abolished the differences observed between WKY and SHR in the noradrenaline-induced contraction in mesenteric arteries loaded in Ca2+-free medium. In contrast, animals acutely treated with prazosin or chronically treated with either prazosin or nifedipine exhibit the same differences in Ca2+ handling than untreated rats. In conclusion, these differences are not a consequence of increased blood pressure but precede it and can only be normalized by inhibition of the rennin-angiotensin system.

Effect of divalent cations on KCl- and noradrenaline-induced contractile responses in rat aorta after nifedipine treatment.

Nifedipine (1 microM) relaxed the sustained contractile responses induced by 1 microM noradrenaline or 60 mM KCl in rat aortic strips. After washing, a second addition of the spasmogens gave smaller tonic contractions than the first one. Even more, a third addition of KCl also gave a smaller contraction than the first one, but a complete recovery of the contractile response to noradrenaline was obtained by a third addition of this agonist. Application of cumulative amounts of Ca2+ or Ba2+ (2.4-24 mM) on the residual contraction in response to these agents after nifedipine treatment, but in the absence of the blocker, restored the magnitude of the contractile responses. Addition of cumulative amounts of Mg2+ (2.4-24 mM) did not modify or even relax the contractile responses to KCl and noradrenaline, respectively.

The 5-HT and alpha-adrenoceptor antagonist effect of four benzylisoquinoline alkaloids on rat aorta.

The action of four benzylisoquinoline alkaloids (two aporphines-glaucine and apomorphine, a benzylisoquinoline-papaverine and a bisbenzyltetrahydroisoquinoline-antioquine) on 5-HT-induced contraction in rat thoracic aorta has been examined and compared with that of the control drugs: ketanserin, nifedipine, prazosin and phentolamine. The relaxant action on 5-HT-induced contraction was contrasted with that on the contraction induced by noradrenaline and KCl. The results obtained with control drugs show that ketanserin has clear selectivity for 5-HT receptors, whereas prazosin and phentolamine have high selectivity for the alpha1-adrenoceptor and nifedipine seems to have a more potent effect on KCl-induced contraction than on that induced by 5-HT or noradrenaline. The contraction evoked by 5-HT (10 microM) was inhibited in a concentration-dependent manner by all the alkaloids. The order of potency was: papaverine = glaucine > apomorphine > antioquine. Papaverine had a non-specific relaxant action on 5-HT-, noradrenaline- and KCl-induced contraction, antioquine had a weak relaxant action on the agonist assays, and glaucine and apomorphine inhibited noradrenaline- and 5-HT-induced contraction more potently than they inhibited the K+-depolarized response. These results indicate that the aporphines assayed, S-glaucine and R-aporphine, had selective action against agonist (noradrenaline or 5-HT)-induced contraction rather than against KCl-depolarization of rat aorta. In contrast papaverine, a benzylisoquinoline alkaloid, relaxes all agents used non-selectively as could be expected from the lack of specificity that characterizes this alkaloid.

Alpha-adrenoceptor interaction of tetrandrine and isotetrandrine in the rat: functional and binding assays.

The action of 1S,1'S-tetrandrine, a bisbenzyltetrahydroisoquinoline alkaloid, on alpha1-adrenoceptors has been compared with that of its isomer 1R,1'S-isotetrandrine. The work includes binding assays to analyse the affinity of these products for the [3H]prazosin binding site of rat cerebral cortical membranes and functional studies on rat isolated aorta to examine the effects of both alkaloids on intracellular calcium processes related or not to alpha-adrenoceptor activation. A radioligand receptor-binding study showed that both compounds interacted with the alpha1-adrenoceptors displacing [3H]prazosin from the specific binding site. The Ki values (inhibition constants) were 0.69+/-0.12 and 1.6+/-0.4 microM for tetrandrine and isotetrandrine, respectively. The functional studies showed that both alkaloids concentration-dependently inhibited noradrenaline-induced contraction in Ca2+-free solution (IC50 values, i.e. the concentrations needed to induce 50% inhibition, were 252.8 and 174.9 microM for tetrandrine and isotetrandrine, respectively), the spontaneous contractile response elicited by extracellular calcium after depletion of noradrenaline-sensitive intracellular stores (increase in resting tone; IC50 values 11.6 and 19.6 microM for tetrandrine and isotetrandrine, respectively) and the refilling of intracellular Ca2+ stores sensitive to noradrenaline (IC50 values 7.4 and 14.9 microM for tetrandrine and isotetrandrine, respectively). The results show that tetrandrine and isotetrandrine interact with alpha1-adrenoceptors by displacing the [3H]prazosin binding site and that both compounds inhibit mainly the Ca2+-dependent process and have less action on alpha1-adrenoceptors. Tetrandrine is more potent than isotetrandrine.

The effect of S-(+)-boldine on the alpha 1-adrenoceptor of the guinea-pig aorta.

1. The cardiovascular activity of S-(+)-boldine, an aporphine alkaloid structurally related to papaverine, was determined. The work includes functional studies on guinea-pig isolated aorta contracted with noradrenaline, caffeine, KCl or Ca2+, and on guinea-pig trachea contracted with acetylcholine or histamine. 2. S-(+)-boldine inhibited in a concentration-dependent manner the contractile response evoked by noradrenaline (10 microM) in guinea-pig aorta (IC50 = 1.4 +/- 0.2 microM) while the KCl depolarizing solution (60 mM)- or the Ca2+ (1 mM)-induced contractions were only partially affected by boldine up to 300 microM. In contrast, papaverine relaxed noradrenaline (NA), KCl or Ca2+ induced contractions showing similar IC50 values in all cases. S-(+)-boldine had a greater potency on the contraction elicited by NA whereas papaverine acted in a non-selective manner. 3. S-(+)-boldine was found to be an alpha 1-adrenoceptor blocking agent in guinea-pig aorta as revealed by its competitive antagonism of noradrenaline-induced vasoconstriction (pA2 = 5.64 +/- 0.08), and its potency was compared with that of prazosin (pA2 = 8.56 +/- 0.24), a known potent alpha 1-adrenoceptor antagonist. In contrast, papaverine caused rightward shifts of the NA concentration-response curves with depression of maximal response indicating that it acts as a non-competitive antagonist. 4. Contraction of guinea-pig aorta induced by caffeine (60 mM) in a Ca(2+)-containing Krebs solution was not affected by a 60 min incubation period with different doses of S-(+)-boldine (1-300 microM). Papaverine inhibited partially this caffeine-induced contraction at the maximal dose used (100 microM). 5. Inositol phosphates formation induced by noradrenaline (10 microM) in guinea-pig thoracic aorta was inhibited by S-(+)-boldine (30 microM) but not by papaverine (10 microM). 6. Contractions of guinea-pig trachea caused by acetylcholine (100 microM) or histamine (10 microM) were not modified by S-(+)-boldine (0.1-100 microM). 7. These results provide evidence that S-(+)-boldine, an aporphine alkaloid, has interesting properties as an alpha 1-adrenoceptor blocker in vascular smooth muscle, and acts as a competitive antagonist of the alpha 1-adrenoceptor present in the guinea pig aorta.

Effect of inhibition of the electrogenic Na+/K+ pump on the mechanical activity in the rat uterus.

The effects of ouabain and K(+)-free solution were studied in estrogen-primed rat uterine strips under resting tone or repeatedly stimulated with KCl, acetylcholine or oxytocin applied for 20 minutes at 60 minute intervals. These effects were compared with those of the K+ channel opener cromakalim. In preparations under resting tone, ouabain (0.1 mM and 0.3 mM) induced rhythmic contractions which disappeared after 20-30 minutes whereas at a higher concentration (1 mM) it evoked a rapid, phasic response followed by a small tonic contraction. Exposure of the strip to a K(+)-free solution induced either rhythmic waves, which ceased after 8-10 minutes, or a single phasic contraction which was followed by a small and slow increase in the resting tone (54 +/- 10 mg after 180 min exposure). Nifedipine (0.3 microM) abolished the rhythmic or phasic component of these responses but failed to modify the late small tonic contraction induced by ouabain 1 mM or by K(+)-free solution. Ouabain (0.1-1 mM) or K(+)-free-evoked responses disappeared after short (4 min) or prolonged (60 min) exposure to a Ca(2+)-free, 3 mM EGTA-containing solution. Cromakalim (10 nM-0.1 mM) did not induce any variation in the resting tone either in the presence or in the absence of Ca2+ in the medium. In strips repeatedly stimulated with acetylcholine (0.1 mM) or oxytocin (1 microM), ouabain (0.3 mM), K(+)-free-solution and cromakalim (10 microM) reduced the amplitude of the initial, phasic response and progressively decreased the oscillatory component of the response to these agonists. Conversely, the successive responses evoked by KCl 60 mM in similar experimental conditions were not affected by ouabain or cromakalim. Ouabain (0.3 mM), K(+)-free solution and cromakalim (10 microM) decreased the Ca(2+)-independent, maintained contractions induced by acetylcholine or oxytocin after prolonged exposure to a Ca(2+)-free, EGTA-containing medium. These inhibitory effects were partially or completely reversed in the presence of the non-selective potassium channel blocker tetraethylammonium (10 mM) or in a Ca(2+)-free solution containing 60 mM K+. In conclusion, these results suggest that the response induced by ouabain or K(+)-free solution in estrogen-primed rat myometrium involves Ca2+ influx through potential-operated calcium channels but not Ca2+ release from intracellular stores. In addition, our results show that prolonged exposure to ouabain or K(+)-free medium decreases membrane receptor-mediated responses in rat uterus. This inhibitory effect seems to be the result, at least in part, of a decrease in the cytosolic level of K+, due to the inhibition of the electrogenic Na+ pump.

Relaxant activity of three aporphine alkaloids from Annona cherimolia on isolated aorta of rat.

In the present study we tested the relaxant effect of three aporphine alkaloids--roemerine, anonaine and dehydroroemerine--isolated from the roots of Annona cherimolia, on isolated strips of rat thoracic aorta. All compounds completely relaxed KCl- and noradrenaline-induced contractions with different potencies depending on their structural characteristics. The experiments, carried out in Ca(2+)-free medium using two different agonists (noradrenaline and caffeine) which mobilize calcium intracellularly by different mechanisms of action, showed that the alkaloids made no contribution to intracellular calcium processes. The present study provides evidence that the relaxant effects produced by aporphine alkaloids may be due to the blockade of calcium movements across the cell membrane, mainly through voltage-operated channels, and to the disruption of alpha 1-adrenoceptors connected to receptor-operated channels.

Vasodilator effects of liriodenine and norushinsunine, two aporphine alkaloids isolated from Annona cherimolia, in rat aorta.

The effect of two aporphines, liriodenine and norushinsunine, isolated from Annona cherimolia, were studied in the rat aorta in order to examine their mechanism of action. Both alkaloids (10(-7) - 10(-4) mol/l) showed relaxant effects on the contractions elicited by 10(-6) mol/l noradrenaline (NA) or 80 mmol/1 KCl, but, while liriodenine showed a nonspecific relaxant action on both spasmogens, norushinsunine was more potent on KCl-induced contraction. In Ca2+ free medium, both alkaloids (0.1 mmol/l) inhibited the responses elicited by NA, but not those elicited by caffeine. This inhibitory action occurred when the alkaloids were present during the release of the Ca2+ internal stores or during the refilling process. These results suggest that the two aporphines show a relaxant action in rat aorta which is mediated by an interaction with alpha1-adrenoceptors and an alteration of the Ca2+ entry via voltage-operated channels. Norushinsunine exhibits a certain degree of selectivity as an L-type Ca2+ channel blocker.

Effect of different treatments in calcium-free medium on basal tone and contractile responses of guinea pig tracheae.

Acetylcholine (ACh; 0.1 mmol/l) and KCl (80 mmol/l) induce a biphasic contractile response in isolated guinea pig tracheae maintained at 37 degrees C either in the presence or absence of extracellular Ca2+. Exposure of the tissue to Ca(2+)-free solution evokes a significant decrease in basal tone and the sources of Ca2+ appear to be decreased by prolonged agonist stimulation, and even more by successive agonist stimulation. After an incubation period of 20 min in Ca(2+)-containing solution, the response is restored. Mg(2+)-depletion in Ca(2+)-free medium increased the contractile response to ACh, but not to KCl, and delayed the tonic component of the next contraction elicited in Ca(2+)-containing solution.

Different actions of some relaxant agents acting via the cAMP system in rat uterus.

The influence of propranolol, isoprenaline, papaverine and caffeine on basal tone and contractile responses to spasmogens (oxytocin, KCl) was investigated in the presence and the absence of external calcium in estrogen-treated rat uterus. Isoprenaline, papaverine and caffeine relaxed precontracted uterus and caffeine also decreased the basal tone of uterine muscle in calcium-containing or calcium-free solution. Propranolol had a dual activity in calcium-free medium: lower concentrations contracted the sustained contraction elicited by oxytocin, whereas the highest concentration partially relaxed it. In calcium-containing solution the highest dose of propranolol partially inhibited KCl-induced contractions.

Intervention of two voltage-dependent calcium-entry pathways in the contractile response to acetylcholine and KCl in rat uterus.

The contractile response of rat uterine smooth muscle was investigated. Verapamil and diltiazem concentration-dependently relax the sustained contractions induced by KCl (56 mmol/l) or acetylcholine (10(-4) mol/l). This inhibitory effect was not not freely reversed by washing the tissue and subsequently no contractile response was obtained in depolarized tissue, but a lower biphasic response (phasic and tonic) to acetylcholine was observed. Addition of cumulative concentrations of CaCl2 (1.2-19.2 mmol/l) induced a partial recovery of the contractile response to acetylcholine or KCl, but addition of MgCl2 (1.2-19.2 mmol/l did not. When the channel was reactivated by a third addition of KCl or acetylcholine after treatment with Ca2+, both spasmogens-induced phasic contractions recovered towards the initial configuration but the tonic component was not restored under any conditions. No recovery of mechanical response could be observed after Mg2+ treatment.

Influence of the absolute configuration on the vascular effects of tetrandrine and isotetrandrine in rat aorta.

The relaxant action of (1S, 1'S) tetrandrine and its isomer (1R, 1'S) isotetrandrine were examined in rat aortic strips, in presence or absence of extracellular calcium. Both alkaloids relax, concentration dependently, the contractile response elicited by depolarizing solution (KCl 80 mM) or noradrenaline (1 microM). Tetrandrine, however, showed a selectivity of action towards the KCl-induced contraction while isotetrandrine did not. In Ca(2+)-free solution, both alkaloids inhibited the contraction induced by noradrenaline, but they did not affect the transient contraction due to caffeine then this effect is not attributable to direct inhibition of the smooth muscle contractile elements. The refilling of intracellular calcium stores sensitive to noradrenaline or caffeine was significantly inhibited by both alkaloids.

The effect of EDTA on contractile responses of guinea-pig trachea in calcium-free medium and on the recovery of the contractile responses in calcium-containing solution.

1. Acetylcholine (0.1 mmol/l) and KCl (80 mmol/l) induce contractile responses in guinea-pig trachea. 2. In the absence of extracellular Ca, contractile responses to ACh or KCl decrease as the preincubation time or the EDTA concentration increases. 3. Exposure of the tissue to Ca-free medium decreases the basal tone. 4. Prolonged incubation in the presence of EDTA (1 mmol/l) promotes a delay in the recovery of the tonic component of the contraction elicited subsequently by acetylcholine and KCl in physiological salt solution, and abolishes the phasic one.

Actions of alpha-adrenoceptor blocking agents on two types of intracellular calcium stores mobilized by noradrenaline in rat aorta.

In isolated rat aortic strips noradrenaline induces a biphasic contractile response in Ca-free medium, associated with two different intracellular calcium pools, one of which is common to caffeine. We analyzed the mechanisms involved in the depletion and repletion of both intracellular Ca pools sensitive to noradrenaline in different experimental procedures in presence of prazosin, phentolamine and yohimbine. At 37 degrees C the alpha-adrenergic blocking agents inhibited contractile responses to noradrenaline in Ca-free medium, with prazosin being highly selective. alpha 2-adrenoceptors probably do not participate in the release of Ca from internal stores, as no contractile response was observed after addition of clonidine in Ca-free medium. This indicates that noradrenaline-induced Ca-release from internal stores is mainly due to activation of alpha 1-adrenoceptors. At 25 degrees C, these compounds failed to inhibit caffeine-induced contraction in Ca-free medium, but abolished the release of Ca from an intracellular store only sensitive to noradrenaline. This effect is attributable to a blockade of alpha 1-adrenoceptors and/or inhibition of receptor-mediated signal transduction.

Evidence that depletion of internal calcium stores sensitive to noradrenaline elicits a contractile response dependent on extracellular calcium in rat aorta.

1. Noradrenaline 1 microM induced a contractile response in rat isolated aorta in the presence or in the absence of extracellular Ca2+ with depletion of intracellular Ca2+ stores. Thereafter, during incubation in the presence of Ca2+, an increase in the resting tone was observed. Such a contractile response did not occur after exposure to caffeine or 5-hydroxytryptamine. 2. This increase in tension was inhibited in a concentration-dependent manner by alpha-adrenoceptor antagonists (prazosin, phentolamine and yohimbine), the non-specific relaxing compound, papaverine and by the Ca(2+)-entry blocker, nifedipine. Therefore, this contractile process is related to depletion of Ca2+ stores sensitive to noradrenaline and is linked to Ca2+ entry through voltage-operated Ca2+ channels and alpha-adrenoceptors. 3. Phentolamine and yohimbine did not block the Ca2+ refill pathway; prazosin and nifedipine inhibited the reuptake of Ca2+ by an internal store sensitive only to noradrenaline; papaverine inhibited the refilling of caffeine- and noradrenaline-sensitive Ca(2+)-stores.

Participation of intracellular calcium stores in serotonin-induced contractions in rat aorta.

Serotonin 1 mumol/l induces a contractile response in the isolated rat aorta in both the presence or absence of extracellular Ca. The present study analyzes the influence of temperature and caffeine on subsequent serotonin-induced contractions. In Ca-free medium, the contraction elicited by serotonin was higher at 25 degrees C than at 37 degrees C. In addition, the existence of two independent intracellular Ca pools releasable by serotonin, one of them also sensitive to caffeine, is postulated. The results also showed that addition of serotonin decreases the contractile response to this agonist in Ca-free medium.

Modulatory role of magnesium on the contractile response of rat aorta to several agonists in normal and calcium-free medium.

Acute withdrawal of external Mg2+ increased basal tone of rat isolated aorta incubated in the presence of Ca2+. Above normal levels of Mg2+ (1-4 mM) inhibited basal tone while much higher levels of the divalent cation (64-256 nM) evoked contractile responses regardless of the presence of Ca2+. Contractile responses to noradrenaline (1 microM) and KCl (80 mM) were inhibited by addition of cumulative concentrations of Mg2+. Acetylcholine-induced contractions in the presence of physiological concentrations of Mg2+ (1 mM) decreased gradually to the basal tone, but a sustained contraction was observed in the absence of this ion. In Ca(2+)-free medium, acetylcholine-induced phasic responses indicate the existence of an acetylcholine-sensitive Ca2+ store. KCl induced contraction only in Krebs solution, although a small residual contraction could be observed in Ca(2+)-free medium in some experiments. Mg(2+)-depletion in the extracellular medium increased contractile responses induced by acetylcholine and KCl in Ca(2+)-free medium. These results suggest that extracellular Mg2+ modulates basal tone, Ca2+ channels and responsiveness to various agents in the absence of Ca2+.

Effects of different agents on the contractile response elicited by extracellular calcium after depletion of internal calcium stores in rat isolated aorta.

Noradrenaline, 1 microM, induced a sustained contractile response in rat isolated aorta in the presence and in the absence of extracellular Ca2+. After depleting the noradrenaline-sensitive intracellular Ca2+ stores, an increase in the basal tone of the aorta was observed during the incubation period in the presence of Ca2+ and in the absence of the agonist. We have tested the possible pathways through which Ca2+ enters the cell to refill the previously depleted Ca2+ pools, a process that is accompanied by an increase in tension. The magnitude of this increase does not depend on the presence of Mg2+ in the extracellular medium nor on the temperature, suggesting that it is mediated by an event that does not depend on intracellular energy or Ca2+, Mg(2+)-ATPase. It is inhibited in a concentration-dependent manner by an unspecific relaxing compound, caffeine, and an organic Ca2+ entry blocker, verapamil, but not by an inorganic Ca2+ entry blocker, lanthanum. Caffeine (10 mM) and verapamil (10(-5) M) completely inhibited the increase in the resting tone, but only verapamil abolished the refilling of the noradrenaline-sensitive Ca2+ pools, indicating that the extracellular Ca2+ enters the cell through voltage-operated Ca2+ channels. Caffeine inhibited the increase in the resting tone without blocking the refilling process of the stores at 37 degrees C, but at 25 degrees C a partial inhibition of the repletion of internal Ca2+ pools was observed. These results confirm previous work that showed a temperature-dependent activity of caffeine.

Amplifying effect of serotonin on contractile responses in rat aorta and depletion of intracellular Ca-stores.

1. Serotonin, 1 microM, induces a contractile response in isolated rat aorta in the presence or absence of extracellular Ca. 2. In Ca-free media, the fast phasic contraction is lower in magnitude and further addition of serotonin evokes no response. 3. Recovery of the contractile response in Ca-free medium is obtained by a 40 min incubation in Ca-containing solution. 4. In Ca, Mg-free medium, the response to serotonin is significantly higher than that obtained in the presence of Mg. 5. An amplifying effect of serotonin on the contractile responses induced by serotonin itself or by noradrenaline was observed in Ca-containing but not in Ca-free solution.