Foscarnet inhibits vascular smooth muscle contraction

Foscarnet inhibits vascular smooth muscle contraction

Life Sciences, Vol. 53, pp. 1227-1234 Printed in the USA Pergamon Press FOSCARNET INHIBITS VASCULAR SMOOTH MUSCLE CONTRACTION Vasilis Paspaliaris, X...

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Life Sciences, Vol. 53, pp. 1227-1234 Printed in the USA

Pergamon Press

FOSCARNET INHIBITS VASCULAR SMOOTH MUSCLE CONTRACTION Vasilis Paspaliaris, Xianghua Mai and David D. Leaver Department of Pharmacology, The University of Melbourne, Parkville, Victoria 3052, Australia. (Received in final form July 30, 1993) Summary Foscarnet inhibited noradrenaline and calcium-mediated contractions of the isolated perfused tail artery of the rat. When the noradrenaline contractile response was split into two components, where the first was due to the release of calcium from intracellular stores and the second to the influx of calcium from the extracellular fluid, foscarnet (30 #M) inhibited only the first component of the response. Foscarnet did not inhibit the calcium influx component of the noradrenaline contraction, nor did it affect the inhibition of this component by the L-type calcium channel antagonists verapamil and nicardipine. These results indicate that foscarnet inhibits vascular smooth muscle contraction by inhibiting calcium release from intracellular stores.

Monophosphonates, unlike bisphosphonates, are compounds which have one P-C bond and occur naturally throughout the animal world. Their biological role is still unknown but one possibility is that they increase resistance, of the molecule to which they bind, to enzymatic degradation (1). They are known antimetabolites of pyrophosphatases, the glycolytic pathway, lipid processes, and of nucleotide related processes (such as protein synthesis, nucleotidases, polymerases and phosphorylases, kinases and related systems) (2). Recent reports suggest that bisphosphonates act as calcium (Ca) channel antagonists (3-4) and that substitution of a monophosphonate group in, for example, a dihydropyridine compound or a non-Ca channel antagonist compound potentiates or induces the Ca channel antagonism of such compounds, respectively (5-11). We recently reported that a bisphosphonate, clodronate (dichloromethylenebisphosphonate), can inhibit vascular smooth muscle (VSM) contraction by inhibiting the intracellular Ca mobilization and extracellular Ca influx that occurs during agonist-induced contraction (4). Moreover, clodronate was also shown to inhibit the action of L-type Ca channel antagonists in VSM (4). In this study, we examined whether the monophosphonate, foscarnet, (i) inhibited noradrenaline (NA) and high potassium(K)-mediated (2.5 mM Ca) contractions in the VSM of the isolated perfused rat tail artery. Foscarnet (phosphonoformic acid), is an inhibitor of herpes virus-induced DNA polymerases 0024-3205/93 $6.00 + .00 Copyright © 1993 Pergamon Press Ltd All rights reserved.

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and human immunodeficiency virus reverse transcriptase (12). It is used therapeutically in acquired immunodeficiency syndrome (AIDS) patients with herpes simplex virus and cytomegalovirus infections (13). Foscarnet has also been found to act as a selective inhibitor of the Na + gradient-dependent uptake of Pi by rat intestinal brush border membrane (14) and renal brush border membrane probably by directly inhibiting the Na+/Pi co-transporter (15). In this study, foscarnet was shown to inhibit NA- and K-mediated contraction in VSM and shown to inhibit intracellular Ca release. However, in contrast to clodronate, foscarnet was not detected to have any inhibitory effect on either Na-mediated extracellular Ca influx or the action of Ltype Ca channel antagonists. Malerials and Melhods

Tissue preparation Male Sprague-Dawley rats (250-300 g) were killed by decapitation, and a 1.5-2 cm length segment of the proximal end of the rat tail artery was removed, cleaned of connective tissue, cannulated, and mounted vertically for perfusion and superfusion with physiological salt solution (PSS) (16). The PSS was of the following composition (mM): NaCI 125, KC1 5, CaC% 2.5, MgC% 1, NaH2PO4 1.2, glucose 11, and EDTA 1, pH 7.2, bubbled with 95%/5% O J C O 2 at 37°C. Perfusion pressure was measured with a pressure transducer connected to the perfusion pathway. Vasodilatation and vasoconstriction were measured as decreases and increases in perfusion pressure, respectively. All arteries were equilibrated by perfusion with PSS for 30 min before drug addition. The resting perfusion pressure after equilibration was 33 + 7 mmHg. Arteries with a perfusion pressure below 20 mmHg and above 40 mmHg after equilibration were discarded.

NA- and K-mediated contractions The effect of foscarnet on the NA concentration-response relationship was examined initially by perfusing the artery with PSS containing 1-300 nM NA and then by repeating the procedure with PSS containing foscarnet (1, 3, 10, 30 or 100/~M). The effect of foscarnet on K-mediated contractions was examined in arteries perfused with a Ca-free, K (60 mM)-depolarizing PSS. K-mediated contractions were induced by adding various concentrations of Ca (0.1-7 mM) to the perfusate first without and then with foscarnet present in the perfusate (1-100 /~M). All concentration-response curves were produced by non-cumulative additions of NA, Ca or foscarnet.

lntracellular and extracellular Ca mobilization technique The effect of foscarnet on NA-mediated intracellular and extracellular Ca mobilization was examined using the technique established by Manzini et al (17). Briefly, this involves perfusing the artery with a Ca-free, K-depolarizing PSS until a steady state level of contractility is attained. Noradrenaline (50 nM) is then added to the perfusate, where a transient contractile response as a result of the release of intracellular Ca is initiated. Immediately after this contractile response has dissipated, the perfusing solution is changed to normal PSS and this change initiates another transient contractile response due to the influx of the now available extracellular Ca through voltage- and receptor- operated Ca channels. Thus, this technique enables differentiation between the component of the NA-mediated contraction due to the

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mobilization of intracellular Ca and the component due to the influx of extracellular Ca. The ability of foscamet (30 t~M) to affect Ca channel blockade by nicardipine (1, 3, 10 and 30 riM) and verapamil (300 nM, 1 and 3 #M) on the NA response was also examined with the Manzini technique. In the absence and presence of foscarnet, the Ca channel antagonists were added to the perfusate for 3 min before NA challenge. When the effect of foscarnet was examined it was added to the perfusate 10 min prior NA challenge.

Drugs Compounds investigated were trisodium phosphonoformate hexahydrate (Astra); (-)norepinepherine acid tartrate (Winthrop Laboratories); Verapamil hydrochloride and nicardipine hydrochloride (Sigma). Nicardipine was dissolved in a mixture of one part propylene glycol in four parts distilled water.

Statistical analysis Results are expressed as mean + SE. Statistical analysis of means and samples was performed using Student's t-test (two-tailed), where P < 0 . 0 5 was taken as an index of statistical significance. Concentration-response curves were analyzed by linear regression analysis in which parallelism, linearity, and coincidence were calculated between control and drug curves by analysis of variance. The ECs0 values (molar concentration producing 50% of the maximal response) together with standard errors were calculated by linear regression analysis. Results

Effect of foscarnet on NA-mediated contraction Foscarnet inhibited NA-mediated contraction (FIG. 1), in which the concentration-response curve for NA was shifted to the right and the response depressed. The concentration-response curves developed to NA in the presense of 30 and 100 tzM foscarnet were significantly different from control, whereas lower concentrations (1, 3, and 10 #M) of foscamet showed no significant inhibition.

Effect of foscarnet on K-mediated contraction As with the NA concentration-response curve, the Ca concentration-response curve, developed in Ca-free, K-depolarizing PSS, was shifted to the right and the maximal response was depressed in the presense of foscamet (FIG. 2). Significant inhibition, though, of the Ca concentration-response curve was seen at 10, 30, and 100 /xM of foscarnet. Foscarnet concentrations of 1 and 3 #M did not significantly attenuate the Ca concentration-response curves.

Effect of foscarnet on NA-mediated intracellular and extracellular Ca mobilization Foscarnet (30 ~M) reduced NA-mediated (50 and 100 nM) contractions in Ca-free, Kdepolarizing PSS, whereas the response following re-perfusion with normal PSS was the same as control (FIG 3). This suggests that foscamet inhibited the component of the NA contraction that was attributable to the initial intracellular Ca mobilization and not the component of the

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response due to extracellular Ca influx. Both nicardipine (1, 3, 10 and 30 nM) and verapamil (300 nM, 1 and 3/xM) had no effect on the NA-induced (50 nM) contractions in Ca-free, Kdepolarizing PSS, but both markedly reduced the contraction following reperfusion with PSS (FIG 3). The response to NA (50 nM) in the presence of the Ca channel antagonists and foscamet (30/~M) was reduced in the Ca-free, K-depolarized PSS phase, similar to the effect seen with foscarnet alone, and following reperfusion with normal PSS the same inhibition was seen as that with the Ca channel antagonists alone (FIG 3). Hence, the inhibitory action of verapamil and nicardipine on the second component of the NA contraction, which is attributable to a block in Ca influx of extracellular Ca, and which was prevented with the bisphosphonate clodronate (4), was not prevented by the monophosphonate foscarnet.

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Effect of foscarnet on the noradrenaline (NA) concentration-response curve. The ECso of NA (O) increased from 12.1 + 1.7 nM (n=12) to 26.7 + 5.7 nM (n=5), and 84.8 +__ 5.0 nM (n=5) in the presence of 30 ([]) and 100 ~M (1) foscarnet, respectively. The curves for 30 tzM and 100 ttM foscarnet depart from coincidence and are parallel to control (p < 0.05). The average of the magnitude of contraction due to 300 nM NA (405 __+45 mmHg) was taken as the 100% response. Discussion Stimulation of postjunctional al-adrenoceptors in VSM by NA activates phospholipase C, which generates inositol-l,4,5-triphosphate (IP+), which in turn causes an increase in cytosolic Ca by releasing Ca from the endoplasmic reticulum store (18-21). This increase in cytosolic Ca is accompanied by a transient contraction (22) followed by the opening of receptor- and voltageoperated channels which leads to an influx of extracellular Ca causing a more sustained contraction of the VSM (23-26). When the rat tail artery is perfused with a Ca-free, Kdepolarizing solution, the introduction of Ca produces influx of extracellular Ca through

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voltage-operated channels, opened by the depolarizing action of the high K solution, which mediates the intracellular enzymatic processes involved in contraction (27). It was of interest therefore to discover whether foscarnet inhibited both the NA- and K-mediated contractions by inhibiting the propagation of Ca through Ca channels and/or by inhibiting the NA-mediated intracellular Ca release. To investigate these possibilities, we used the Manzini technique (17), which splits the NA-mediated contraction in two components, the first contraction being due to the second messenger cascade that releases Ca from intracellular stores, and the second contraction being due to the entry of extracellular Ca through Ca channels. Surprisingly, foscarnet did not affect the component of the NA response attributable to extracellular Ca influx, but significantly affected the response attributable to intracellular Ca mobilization. Foscarnet has been shown to enter cells and to have inhibitory effects on intracellular enzymatic processess (2,12,28,29), and this intracellular action on VSM contraction on the release of Ca, suggests that foscarnet may inhibit one or more of the processes in the second messenger cascade. To date, we have shown that foscarnet does not inhibit the activity of protein kinase A or protein kinase C isolated from rat cardiac and vascular smooth muscle (unpublished data).

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Effect of foscarnet on the Ca concentration-response curve in Ca-free, Kdepolarizing PSS. The ECs0 of Ca (O) increased from 0.93 + 0.15 mM (n = 15) to 1.33 __+0.60 mM (n=8), 1.61 + 0.53 mM (n=7), 2.54 __+ 0.67 mM (n=7) in the presence of 10 (A), 30 (IS]) and 100 #M ( I ) , respectively. All three curves depart from coincidence and are parallel to control (p < 0.05). The average of the magnitude of contraction due to 7 mM Ca (325 __+ 25 mmHg) was taken as the 100% response.

We have reported that the bisphosphonate clodronate has a unique effect on VSM, in that, it inhibits not only the propagation of Ca through Ca channels but also the action of L-type Ca channel blockers (4). Similarly, it has been reported that the compound SR-7037 (tetrabutyl2(2-phenoxyethyl)-l,3-propylidene diphosphonate) completely displaced dihydropyridine

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([3H]PN200-110), phenylalkylamine ([3H]D888), and benzothiazepine ([3H]diltiazem) ligands from brain L-type Ca channels (3). Moreover, it has been also shown that the phosphonate KB-944 (diethyl 4-(benzothiazol-2yl) benzylphosphonate) has Ca channel antagonist properties and, it was claimed, that the diethylphosphonate group plays an important role in affording vasodilator activity (8-11). Also, the introduction of a cyclic phosphonate group at position 5 of 1,4-dihydropyridine moiety, increased the potency and duration of action of the 1,4dihydropyridine in vivo and in vitro (5-7).

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The effect of various treatments of the two components of the noradrenaline (NA) response. The first component (F1) represents the mobilization of intracellular Ca, and the second component (B) represents Ca influx from the extracellular pool. NA (50 nM) was used to initiate contraction. Foscarnet (Fosc; 30 ~M) significantly inhibited the first component ( p < 0.05; n =9), whereas nicardipine (Nic; 10 nM) and verapamil (Ver; 1 /zM) significantly inhibited the second component of the ( P < 0.05; n=6). Foscarnet together with either nicardipine (Fosc + Nic) or verapamil (Fosc + Ver) significantly inhibited both the first component ( P < 0.05; n=6), as seen with foscarnet alone, and the second component of the response ( p < 0.05; n=6), as seen with the two Ca channel antagonists alone. There was no significant effect of foscarnet on the other concentrations of the Ca channel anatagonists (data not shown). 0% inhibition for the first component of the response was taken as that seen with 50 nM NA challenge (63 __+ 12 and 101 ± 25 mmHg, respectively; n = 17). This evidence suggests that the phosphonate group(s) attached to a larger compound may be responsible for the effect on L-type Ca channels. Foscarnet had no inhibitory effect on NAmediated Ca influx, nor did it inhibit the action of L-type Ca channel antagonists in VSM.

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Thus, it seems that the compounds the phosphonate group(s) are attached to are necessary for the binding to L-type Ca channel, and that this binding is essential for the localization and interaction of the phosphonate group with the channel. In the case of the bisphosphonates, presumably the physico-chemical nature of the molecule and its resistance to enzymatic degradation is enough to assist the interaction of the bisphosphonate group with the channel and/or its associated events.

Acknowledgements The authors would like to thank Mr. T. Tiganis, Dr. J.M. Moseley and Dr. P.J. Robinson for their helpful assisstance and comments. The study was supported by a Postgraduate Biomedical Research award from the NHMRC of Australia.

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