Modification of adrenergic reactivity in rat tail artery by dietary lipids and calcium channel antagonists

Gen. Pharmac. Vol. 27, No. 5, pp. 895-900, 1996 Copyright © 1996 Elsevier Science Inc. Printed in the USA.

ISSN 0306-3623/96 $15.00 + .00 SSDI 0306-3623(95)02096-9 All rights reserved ELSEVIER

Modification of Adrenergic Reactivity in Rat Tail Artery by Dietary Lipids and Calcium Channel Antagonists Bogustaw Okopidn* and Henryk I. Trzeciak DEPARTMENT OF PHARMACOLOGY,SILESIAN ACADEMY OF MEDICINE, UL. MEDVK6W18, 40-752 KATOW]CE,POLAND

ABSTRACT. 1. The antiatherosclerotic activity of dihydropyridines (DHP), potent calcium antagonists, was studied with respect to prevention of hypercontractility of perfused rat tail arteries. 2. Used for 1 month, atherogenic diet increased pressor responses to norepinephrine (NE) in Ca 2+free physiological salt solution (PSS), and PSS containing Ca g+. 3. When nifedipine (NIF) or nitrendipine (NIT) was administered simultaneously with an atherogenic diet, the contractile activity of NE in CaZ+-free PSS was attenuated. Moreover, vasoconstrictor responses to NE in PSS containing Ca 2+ were inhibited after 1-month treatment with N I T and nimodipine (NIM). 4. NIF, N I T and NIM prevented atherosclerosis-induced vascular hyperreactivity to (x-adrenoceptor agonists in rat tail artery, tEN PHARMAC27;5:895--900, 1996. KEY WORDS. Nifedipine, nitrendipine, nimodipine, atherogenic diet, vascular smooth muscle contractility, rat tail artery

INTRODUCTION Hyperlipidemic diets are often used to induce experimental atherosclerosis in animals. When compared to the control, atherosclerotic arteries show more pronounced contractile reactivity to catecholamines and other vasoconstrictors (Henry and Yokoyama, 1980; Rosendorf et al., 1981; Heistad etal., 1984; Hof and Hof, 1988; Broderick et al., 1989; Lopez et al., 1990). A correlation was recently demonstrated between the intracellular free Ca 2+ concentration (Cain) and the strength of vascular smooth muscle (VSM) constriction both for perfused vessel and arterial helical strip contraction (Abe et al., 1990; Thorin-Trescases et al., 1990). On the other hand, Cam also stimulates proliferation, migration and phenotypic transformation of VSM cells during atherogenesis (Schmitz et al., 1991). This is why the idea emerged of using vasorelaxant calcium antagonists for atherogenesis inhibition. Henry and Bentley (1981) were the first to find evidence for diminished lipid and calcium accumulation in arteries of rabbits fed an atherogenic diet and simultaneously treated with NIF. Further studies were aimed at confirmation of antiatherosclerotic potency of calcium channel blockers (Parmley et al., 1985; Weinstein and Heider, 1989; Fleckenstein-Ortin et al., 1994). However, the results did not allow explicit evaluation of atherogenesis inhibition after treatment with calcium antagonists. Therefore, the purpose of the present study was to assess the effect of atherogenic diet and long-term treatment with NIF, NIT and NIM on (x-adrenergic contractility in isolated rat tail artery. The contractile response to NE in perfused tail artery was evaluated in PSS or Ca2+-free PSS. The use of Ca2+-free I~SS made it possible to utilize the intracellular Ca 2+ store that had been gained during prolonged atherogenic diet administration. *To whom correspondenceshould be addressed. Received 19 April 1995.

MATERIALS AND METHODS

S t u d y design Experiments were performed on male Wistar rats bred in the Central Experimental Animal Farm of the Silesian Academy of Medicine. The initial body weight was 170-183 g. The rats were fed the atherogenic diet previously shown to elevate serum cholesterol and containing (in g/kg): butter 400, casein 200, sucrose 169, cholesterol 5, cholic acid 2, choline chloride 1 and nutrients (Gresham and Howard, 1960). The diet (5 g per rat) was administered daily in the morning for 1 month. For the remainder of the day, the rats were maintained on a standard diet. The rats were housed in an animal room at a constant temperature (21-24°C), humidity (50-55%), and alternative 12/12-hr light-dark cycle, and had access to tap water throughout the experiment. NIF, NIT and NIM were administered twice daily as suspensions of 3 % aqueous solution of gum tragacanth through a gastric tube. The suspensions were prepared fresh twice daily in a darkroom to avoid photodegradation. Both in rats fed an atherogenic and a standard diet, each drug was used in two doses: NIF 3 and 10 mg/kg (NIF3; NIF10), NIT 10 and 30 mg/kg (NIT10; NIT30) and NIM 15 and 50 mg/kg (NIM15; NIM50). Control animals fed the same type diet received only 3% aqueous solution of gum tragacanth in a dose of 5 ml/kg twice daily through a gastric tube.

P e r f u s e d artery e x p e r i m e n t Rats weighing 220-270 g were killed after 1 month by direct heart puncture under ether anesthesia. Blood samples were taken for serum cholesterol assessment. Rat tail artery was excised and gently cleaned of blood and adherent tissue. The endothelium was removed mechanically according to the commonly applied and verified procedure (Abe et al., 1990). The proximal segment (2-3 cm) of the tail artery was cannulated and mounted vertically under 0.5 g tension in an organ bath (10 ml; Nicholas, 1969; Medgett and Ra-

896 janayagam, 1984). During the first stage of in vitro experiments, all arteries were stabilized in Ca2+-free PSS (37°C, 5% CO2 in O2) by gradually increasing perfusion from 0.5 to 4 ml/min until perfusion pressure of 3-6 kPa was reached (~"40-60 min), Further experiments were performed exclusively on the arteries that fulfilled the above-mentioned criterium. After the contractility of the tail artery had been stabilized, Ca>-free PSS was quickly changed to Ca>-free PSS containing NE (10 IxM) and ascorbic acid (240 I-M) as the antioxidant. The constriction of the tail artery in response to NE was measured as an increase in perfusion pressure (Statham P23 ID transducer) at a constant flow of perfusion fluid (4 ml/min). In Ca 2+free PSS, intracellular organelles were the only source of Ca 2+ for the contractile proteins of myocytes (Karaki and Weiss, 1988). After ~ 2 0 min, arterial relaxation was evidenced by a decrease in perfusion pressure. This may have been caused by Cain depletion after NE-induced cq-adrenergic stimulation. Activation of eq-adrenoceptors not only makes the Cain available, but also facilitates the influx of extracellular calcium ( C a J into myocytes (Abe et al., 1990). Therefore, CaC12 (2.5 raM) was added to the organ bath to reestablish contractile responses to NE, which was always present in Ca2+-free PSS (Sk/irby et al., 1984). Thus, in the artery with previously depleted Cam pool, ~x-adrenergic contractile response caused by CaCl2 depended only on Ca~, influx. After experiments in Ca2+-free PSS had been completed, the tail artery was washed and allowed a further 60-min period of equilibration in PSS to refill Ca,n store (Karaki and Weiss, 1988). Finally, the cumulative concentration-response curves to NE (10 nM-10 IzM) were obtained by increasing the organ bath concentration of the agonist in approximately three-fold steps, allowing equilibration to be attained at each concentration.

Drugs and solutions Nifedipine, nitrendipine, nimodipine and ascorbic acid were obtained from "Polls (Poland). All drugs used in the perfused artery experiment were purchased from Sigma (St. Louis, MO). PSS was composed of (mM) NaC1 118, KC1 4.7, NaHCO3 25, KH2PO4 1.2, MgSO4 1.2, CaC12 2.5 and glucose 11 at pH 7.4. Cocaine (1 I~M), propranolol (1 I~M) and yohimbine (50 nM) were added to the PSS to block neuronal uptake of NE, 15- and postjunctional cq-adrenoceptors, respectively. Moreover, indomethacin (30 IxM) was used to inhibit prostaglandin biosynthesis. Ca2+-free PSS was prepared by omitting CaCl2 from PSS and adding EGTA (3 raM).

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FIGURE 1. Effect of 1-month treatment with nifedipine (NIF), nitrendipine (NIT) and nimodipine (NIM) on body weight of rats fed a standard or an atherogenic diet. Doses of drugs (mg/kg) administered orally twice a day are given in the legend. Control animals received only 3% aqueous solution of gum tragacanth (5 ml/kg). Results are mean values (n= 12 in each group); vertical error bars are omitted for clarity. *Indicates significantly smaller weight compared with weight of rats fed an atherogenic diet from control, (O) NIF10 (&) and NIT10 (D) groups (P<0.05). O, control (standard); Q, control (atherogenic); A, NIF3; m, NIT3 0; V, NIM15; T, NIM50.

Data analysis The results are means_+SE. Statistical analysis was performed using the Newman-Keuls test for multiple comparison of means; P<0.05 was considered statistically significant. The value of NE concentration necessary for half-maximal contraction (ECs0) was calculated by linear regression analysis of the 2 0 4 0 % region of each concentration-response curve. Values represent geometric mean (with 95% confidence limit) of EC50 in each group. RESULTS

Body weight and serum cholesterol level Initial body weight was approximately the same for all fourteen groups of rats (170-183 g). The weight of control rats fed a standard diet decreased significantly on days 24 and 30 compared to the corresponding weight of animals fed an atherogenic diet from the control and NIF10 as well as N1T10 groups (Fig. 1). Although the absolute weights of the animals fed an atherogenic diet did not differ significantly at the end of 1-month DHP treatment, weight gain

was different. We observed that l-month administration of NIT (30 mg/kg) and NIM (50 mg/kg) significantly attenuated the increase of weight (69.9-+2.5 and 64.5-+3.3 g, respectively) when compared to atherogenic control (81.6-+7.8 g, P<0.05, n= 12 in each group). Total serum cholesterol concentration in control rats fed an atherogenic diet was significantly increased (422_+52 mg/dl) when compared to control levels in rats fed a standard diet (70_+5 mg/dl, P<0.01, n= 12). Moreover, total serum cholesterol concentrations in groups fed the same type diet were not significantly affected by DHP treatment.

NE-induced maximum perfusion pressure in Ca2+-free PSS The maximum increase of perfusion pressure (APma×) in arteries of control rats fed a standard diet stimulated by NE (10 IxM) was 12.9_+2.1 kPa (n=12). The value of APmax in arteries of control

Vasoprotective Action of Dihydropyridines

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FIGURE 2. Effect of 1-month treatment with nifedipine (NIF), nitrendipine (NIT) and nimodipine (NIM) on maximal increase of perfusion pressure (APm~) in rat tail arteries perfused with Ca2+-free physiological salt solution (PSS) contained norepinephrine (10 IxM). Doses of drugs (mg/kg) administered orally twice a day are given under abscissas of graphs. Control animals received only 3% aqueous solution of gum tragacanth (5 ml/kg). Results are mean values (n = 12 in each group); vertical error bars show SE. *P<0.05 and **P<0.01 indicate significantly smaller APr~ than in arteries from control rats fed a standard (top) or an atherogenic diet (bottom).

FIGURE 3. Effect of 1-month treatment with nifedipine (NIF), nitrendipine (NIT) and nimodipine (NIM) on maximal increase of perfusion pressure (APmJ in rat tail arteries previously depleted of intracellular free Ca z+ concentration and perfused with Ca z+free physiological salt solution (PSS) containing norepinephrine (NE, 10 p~M). CaCI2 (2.5 mM) was added to the organ bath to reestablish contractile response in arteries to NE in Ca2+-free PSS. Doses of drugs (mg/kg) administered orally twice a day are given under abscissas of graphs. Control animals received only 3% aqueous solution of gum tragacanth (5 ml/kg). Results are mean values (n= 12 in each group); vertical error bars show SE.

rats fed an atherogenic diet was significantly increased (19.7-+4.2, P<0.01, n=12) compared to the values of APm~x in groups fed a standard diet. Administration of NIF (3 and 10 mg/kg) and NIT (10 and 30 mg/kg) significantly diminished maximum constriction in tail arteries of rats fed an atherogenic diet in comparison with the control (Fig. 2 - down graph). A decrease in APma~value was also observed in arteries of rats from groups NIF10, NIT10 and NIT30 that had been kept on a standard diet (Fig. 2 - upper graph). Long-term treatment with NIM (15 and 50 mg/kg) produced no changes in arterial vasoconstriction of animals fed a standard or an atherogenic diet (Fig. 2). During the next stage of the experiment, CaCI2 (2.5 mM) was added to the bath solution to reestablish the contractile response to NE in CaZ+-free PSS. In tail arteries of control rats fed a standard diet, CaCI2 (2.5 mM) stimulated an increase in APm~xof 12.0-+1.9

kPa (n = 12). In arteries of atherogenic control rats, the constriction to CaC12 (2.5 mM) was significantly increased (16.1_+3.8 kPa, P<0.05, n = 12). The CaCl2-trigerred vasoconstriction in tail arteries of rats fed a standard or an atherogenic diet was not significantly affected by DHP treatment (Fig. 3).

NE.incluced contractile responses in PSS containing Ca 2+ In tail arteries of control rats fed a standard diet, NE (10 IxM) stimulated the maximal constriction (Emax)of 18.2_+ 1.2 kPa (n = 12). The value of Em~xwas significantly higher for the tail arteries of control rats fed an atherogenic diet ( 2 4 . 0+- 1.7 kPa, n= 12) compared to the values of Ema. in groups fed a standard diet. Contractile response to NE (10 I~M) in groups fed an atherogenic diet (i.e.

898

B. Okopiefi and H. I. Trzeciak

TABLE 1. Effect of 1-month treatment with nifedipine (NIF), nitrendipine (NIT) and nimodipine (NIM) on contractile response to norepinephrine (NE, 10 nM-10 ~M) in tail arteries of rats fed a standard or an atherogenic diet Diet

Treatment

EC50 (nM)

Em~ (kPa)

Standard

Control NIF 3 NIF 10 N I T 10 N I T 30 NIM 15 NIM 50

610 720 510 520 710 570 570

(490-770) (590-960) (390~80) (440-630) (540-990) (470-740) (440-760)

18.2 18.1 17.9 18.0 18.1 18.3 18.8

- 1.2 + 0.9 _+ 1.5 -+ 0.7 + 1.8 _+ 0.9 + 1.2

Atherogenic

Control NIF 3 NIF 10 NIT 10 NIT 30 NIM 15 NIM 50

300 260 280 270 340 280 390

(220400) (210-350) (220-390) (220410) (2504-80) (1904-00) (260-560)

24.0 24.3 23.9 22.0 20.3 23.6 19.8

+ + + + _+ _+ -+

1.7 1.2 1.0 1.5 1.4" 1.2 1.5"*

Doses of dmgs (mg/kg) administered orally twice a day are given in the 2nd column. Control animals received only 3% aqueous solution of gum tragacanth (5 ml/kg). The value of NE concentration necessary for halfmaximal constriction (ECs0) was calculated following curve-fitting for each experiment. Values represent geometric mean (with 95% confidence limit) of ECs0 (n = 12 in each group). The result of maximal constriction to NE (10 ~tM, E~) was taken from each concentration-response curve. Values represent mean + SE of Emax (n = 12 in each group). *P < 0.05 and **P < 0.01 indicate significantly smaller Em~ than in other groups fed an atherogenic diet, except NIT10.

NIT30 and NIM50) was significantly decreased compared to the other groups fed an atherogenic diet, except NIT10 (Table 1). No significant differences were found in Emaxto NE (10 I~M) between any of the standard diet-fed groups (Table 1). The value of NE concentration necessary for half-maximal contraction (ECs0) in arteries of rats fed an atherogenic diet was 300 ( 2 2 0 ~ 0 0 ) nM (n=12), and it was markedly lower than the ECs0 value for arterial vasoconstriction of control rats fed a standard diet [610 (490-770) nM, n = 12, statistical analysis was not performed because of various E~ax values in these groups]. Similar discrepancies in the ECs0 values were observed between the other atherogenic diet-fed groups and rats kept on a standard diet (Table 1). However, no differences were noted in the EC50 values between any of the DHP-treated groups fed the same type diet (Table 1). DISCUSSION We have observed that several weeks of dietary lipid treatment were sufficient both to induce hypercholesterolemia and enhance vascular reactivity to pressor agents (Galle et al., 1990; Merkel et al., 1990). Excessively long administration of atherogenic diet results in the formation of atherogenic plaques that mechanically impair the contractility of the vascular wall and make arteries useless for functional studies (Fleckenstein-GrOn et al., 1991 ). Some investigators reported arterial contractility changes that preceded the formation of typical atherosclerotic plaques (Wines et al., 1989; Merkel and Bilder, 1992). Our previous and present studies showed a higher increase of perfusion pressure to NE in tail arteries of rats fed a high-fat diet than in those of rats fed a standard diet (Fig. 2; Trzeciak et al., 1993 ). The atherogenic diet was biochemically confirmed to induce hypercholesterolemia. We suspect that, owing to dietary-induced hypercholesterolemia, arterial myocytes may have stored a larger

amount of Ca 2+ than did standard control VSM cells. Therefore, we investigated the contractility of the tail artery of rats fed an atherogenic diet and treated with calcium antagonists such as DHP in two doses. Both smaller and larger doses of DHP were equipotent in reducing blood pressure in rats (Ohtsuka et al., 1989). Long-term administration of NIF and N I T attenuated the contractile response of VSM to NE and, as expected, this attenuation was more pronounced in arteries of animals fed an atherogenic diet (Fig. 2). The results are in accordance with the basic mechanism of DHP activity, which consists in the blockage of Ca > influx to myocytes. The less pronounced vasodilatory effect of NIM, when compared to NIF and NIT, might be accounted for by its lesser affinity to peripheral vessels (Fig. 2; Ohtsuka et al., 1989). Enhanced Ca > permeability through myocyte membranes was observed in aortas of rabbits fed a high-cholesterol diet (Strickberger et al., 1988). Therefore, we performed an in vitro experiment with a Caex pool as the source of Ca > for contractile proteins of smooth muscle cells. The ~-adrenergic contractility of arteries previously devoid of Ca,n was investigated with the use of CaC12 added to the organ bath. The method allowed observation of hypersensitivity to NE in tail arteries of rats fed an atherogenic diet (Fig. 3). This effect was not influenced by DHP treatment (Fig. 3). To determine whether or not dietary lipid treatment could also induce postsynaptic modifications in tail artery c~-adrenoceptors, we examined the vasoconstrictor action of N E (10 nM-10 p.M). Arteries from rats receiving an atherogenic diet showed greater constriction to maximal NE concentration (Table 1). Similar results concerning pressor effects of exogenous NE in tail arteries of rats fed a diet rich in saturated fatty acids were obtained by Panek et al., (1985). The conditions in which our in vitro experiment was performed excluded any immediate effect of cholesterol excess on vascular calcium storage. However, damage to cell membrane structure due to long-term hypercholesterolemia may have enhanced Ca > influx to myocytes during the period of stabilization in PSS and may have brought about arterial hypercontractility. Cholesterolmediated injury of VSM may have been caused by increased membrane expression or more pronounced activity of calcium channels. The altered action of calcium channels resulting from excessive cholesterol was confirmed experimentally (Bialecki and Tulenko, 1989). In the arteries of rats from groups NIT30 and NIM50 receiving an atherogenic diet, we found diminished E ..... values (Table 1). The decreased contractility of the VSM in rats treated with DHP could not have resulted from calcium channel blockage by the drugs during the prolonged stage of the experiment. The observed changes in vascular reactivity seem to have been caused by intracellular effects of DHP, which were found in vitro at concentrations several hundred times higher than therapeutic doses (Walsh et al., 1988). O n the other hand, considering their lipophilic nature, NIT and NIM, when administered for a 1-month period, could accumulate in rat myocytes. In fact, NIM and NIT, as opposed to NIF, had the ability to penetrate into muscle cells (Pang and Sperelakis, 1984). The lipophilicity of particular DHP compounds does not correlate with their affinity to determined segments of the vascular bed (Borchard, 1994). Hence, the vasorelaxant action of N IM, obtained during Cain removal from the arteries in Ca2+-free PSS, was less pronounced when compared to those of NIF and NIT (Fig. 2). Some authors also found a vasodilatory effect of DHP without simultaneous calcium channel blockage by the compounds (Rinaldi et al., 1987). Moreover, the ECs0 value for vasoconstriction was markedly reduced in atherogenic diet groups. These results are consistent with previous observations that a decrease in ECs0 for contraction of

Vasoprotective Action of Dihydropyridines atherosclerotic arteries was obtained for ergonovine (Henry and Yokoyama, 1980). Because differences were evident in ECs0 values between rats fed an atherogenic or a standard diet, a specific alteration in a-adrenoceptor number or function may have occurred. A n increased number of cx-adrenoceptors in aortas of rabbits fed a cholesterol-rich diet had been detected earlier (Nanda and Henry, 1982). Another possible explanation for the diminished ECs0 value in the studies on blood vessels of animals fed atherogenic diets might be an increase in the number of smooth muscle myocytes. Numerous reports described mitogenic effects of LDL, endothelins, and other local growth factors observed in early atherosclerotic lesions (Ross, 1993). O n the other hand, the decreased membrane fluidity resulting from cholesterol accumulation in myocytes may alter the lipid microenvironment of cx-adrenoceptor molecules (Loh and Law, 1980). Incorporation of cholesterol into the myocyte membrane and sensitization of vasculature in atherosclerotic arteries by accumulated oxidized lipoproteins may render VSM more sensitive to c~-adrenergic stimuli (Galle et al., 1990). However, inhibited generation or facilitation of peroxide radicals removal due to DHP administration might have delayed the process of lipid peroxydation within the cell membrane housing calcium channels and c¢-adrenoceptors (Janero and Burghardt, 1989). The immediate DHP antagonism towards cq-adrenoceptor was shown in the brain and tail artery of rats in the presence of very high concentrations of NIF (Thayer et al., 1985). SUMMARY Dihydropyridines showed vasoprotective effects in atherogenesis induced by dietary lipid treatment for 1 month. As a result, contractile responses to NE in perfused rat tail artery were attenuated without any changes in serum cholesterol. Mechanisms responsible for the antivasoconstrictor action of DHP in atherosclerotic arteries are not fully understood and need further evaluation. The most important mechanism seems to be calcium channel blockage that leads to inhibition of overloading VSM cells with Ca 2+. Moreover, the intracellular effects ofdihydropyridine treatment, unrelated to their channel-gating properties, cannot be ruled out. NIF and NIT, which had high affinity to VSM, diminished the contractile activity of NE in Ca2+-free PSS. Moreover, vasoconstrictor responses to NE in PSS containing Ca 2+ were inhibited after I-month treatment with NIM and NIT, which easily penetrated into myocytes. It can be concluded that nifedipine, nitrendipine and nimodipine prevented atherosclerosis-induced vascular hyperreactivity to o~-adrenoceptor agonists in rat tail artery.

The study was supported by research grant no. 4 0290 91 O1 from the Committee of Scientific Research. We wish to express our gratitude to Prof. Dr. Leszek Szadujkis-Szadurski for the development and implemenzation of the perfused artery experiment. The skillful and expert animal care of Iwona Lukaszek is gratefully acknowledged.

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