vasoactive properties of phenylpropanolamine (d, i-norephedrine) and its enantiomers in isolated rat...

Drug Development Research 23: 159-169 (1991)

Vasoactive Properties of Phenylpropanolamine (d,l-Norephedrine) and Its Enantiomers in Isolated Rat Caudal Artery David A. Johnson and Timothy J. Maher

Department of Pharmacology, Massachusetts College of Pharmacy, Boston, Massachusetts

ABSTRACT

Johnson, D.A. and T. J. Maher: Vasoactive properties of phenylpropanolamine (d,l-norephedrine) and its enantiomers in isolated rat caudal artery. Drug Dev. Res. 23: 159-169, 1991.

The sympathomimetic properties of PPA and its individual component enantiomers d- and I-norephedrine (NOR) were examined in isolated perfused/superfused caudal arteries from Sprague-Dawley (SD) rats. Segments of caudal artery were cannulated, mounted in a tissue bath, and perfused with a modified Kreb's solution. PPA, d- and I-NOR were alternately injected into the perfusion stream, and vasoconstriction was measured as the rise in perfusion pressure. Various drugs were added to the perfusion buffer to determine the activity of PPA, d-, and I-NOR at specific adrenoceptor subtypes and to determine whether the actions of these vasoconstrictors were influenced by calcium channel antagonists, cocaine, or reserpine. The results demonstrated that the norephedrines acted as partial agonists primarily via direct stimulation of postjunctional alpha,-adrenoceptors with relatively low potency; however, I-NOR did have significant activity at beta-adrenoceptors as well. When the enantiomers of PPA were compared, d-NOR had only minor vasoconstrictor activity, relative to I-NOR, which was well over 100 times less potent than the endogenous

Received final version February 14, 1991; accepted February 19, 1991.

Address reprint requests to Timothy J. Maher, Dept. of Pharmacology, Massachusetts College of Phar- macy, 179 Longwood Avenue, Boston, MA 02115.

David A. Johnson is now at Interneuron Pharmaceuticals Inc., One Ledgemont Center, 99 Hayden Avenue, Suite 340, Lexington, MA 02173.

0 1991 Wiley-Liss, Inc.

160 Johnson and Maher

Key words: syrnpathornirnetics, adrenoceptors, partial agonists

INTRODUCTION

Phenylpropanolamine (PPA), the racemic mixture of the enantiomers d- and 1-norephedrine (NOR), is a chemical analogue of the endogenous sympathomimetic amines. In the United States PPA, utilized for nearly 75 years, is currently found in over 150 medications with approximately 6 billion doses consumed annually [Lasagna, 19881. Although occasion- ally prescribed as a mydriatic agent, and to treat urinary incontinence and retrograde ejacu- lation, the major application of PPA is as a constituent ingredient of non-prescription nasal decongestants and appetite suppressants.

The ability of PPA to act as a nasal decongestant has been traditionally attributed to its ability to constrict small blood vessels of the nasal mucosa via stimulation of postjunctional alpha,-adrenoceptors [Weiner, 19801. However, many of the preliminary pharmacological studies of the actions of PPA were carried out prior to a substantial understanding of adrenoceptor function. Furthermore, most studies of the pharmacologic activities of PPA were either performed in whole animal preparations or nonvascular smooth muscle tissues other than isolated blood vessels. These studies demonstrated that the potency and activity of PPA were dependent upon the particular tissue under investigation [Trendelenburg et al., 1962a,b]. Moreover, the activity of the individual enantiomers of PPA have not been thoroughly studied in isolated tissues.

Periodically reports have appeared in the literature which have described toxicity associated with ingestion of PPA [Larson and Rogers, 19861. As a consequence of these reports, PPA has become unavailable in an over-the-counter form in several European countries, and several states of Australia. In many of these cases, however, the actual ingredients of the medications in question most likely were not PPA at all, but rather, structurally related compounds with significantly different pharmacological properties [Horowitz et al., 1979; Moya-Huff et al., 19871.

There were several main goals in this study: first to determine the relative potency and activity of PPA and its enantiomers in the isolated rat caudal artery relative to that of the endogenous neurotransmitter, 1-norepinephrine (NE); second, since it has been reported that the activity of PPA may be mediated either through direct stimulation of postjunctional adrenoceptors and/or indirectly by the release of NE from sympathetic nerve terminals [Trendelen- burg et al., 1962a,b; Liebman, 19611, experiments were designed utilizing noradrenergic uptake blockers, and pretreatment with the monoamine-depleting agent reserpine, to determine whether the vasoactive properties of PPA were related to direct or indirect activity at the neuroeffector junction; and third, the activities of PPA at both alpha,- and alpha,-adrenoceptors were determined by using the selective alpha-adrenoceptor antagonists corynanthine and yohimbine.

The beta-adrenergic activity of PPA has not been well established. Beta activity has been demonstrated previously in this laboratory utilizing whole animal preparations [Moya-Huff and Maher, 19871. Moreover, Wellman [I9841 demonstrated PPA has an effect on rat brown adipose tissue thermogenesis which is mediated by beta-adrenoceptors. However, experiments in isolated vascular tissue have not been performed. Thus, experiments were designed in which the activity of PPA and its constituent enantiomers were determined in the presence and absence of selective and non-selective beta-adrenoceptor antagonists. To investigate whether PPA utilizes intracellular or extracellular calcium to produce vasoconstriction, additional experiments were included to determine the effect of calcium channel blockade on vasoconstriction induced by PPA.

Vasoactive Properties of PPA 161

MATERIALS AND METHODS

The isolated artery techniques used in this study were a modification of the procedures described by Medgett and Langer [1983]. Male SD rats (Charles River, Willmington, MA) were housed in steel mesh cages, in a temperature-controlled room, on a 12/12 hour light/dark cycle and were fed standard laboratory chow ad libitum. On the day of an experiment animals weighing between 250 and 350 g were anesthetized with chloralose (50 mg/kg) and urethane (500 mg/kg) via intraperitoneal injection. An incision was made along the ventral aspect of the tail for two-thirds of its length beginning at the base. The caudal artery was exposed and carefully dissected away from the surrounding connective tissues, and a proximal segment, approximately 5 cm in length, was sutured, removed, and placed in a petri dish containing a modified Kreb's buffer. The buffer had the following composition (mM): NaCI, 118; KC1, 4.7; NaHCO,, 25; NaH,PO,, 1 .O; MgCl,, 1.2; CaCl,, 1.3; glucose, 11.1; ascorbic acid, 0.1 and was gassed with 5% CO, and 95% 0,. The rat was then euthanized by cervical dislocation. The proximal end of the artery was cannulated with polyethylene 100 tubing, tapered at the tip. Following cannulation, the artery was sutured 2 cm from the tip of the cannula, and an incision was made just proximal to the end of the suture, thus allowing the exit of buffer from the vessel, as well as superfusion of the extraluminal surface. The cannula was attached, and the distal suture of the artery was tied to an L-shaped glass rod which was mounted within a 10 ml tissue bath (Metro Scientific, Farmingdale, NY).

The arterial segment was allowed to equilibrate in the bath for at least 15 min prior to perfusion (Buchler peristaltic pump) with warmed (37°C) buffer at an initial flow rate of 1 ml/min, which 30 min later was increased to 2 ml/min. Following equilibration, tissue viability was tested by injection of 50 pl of 1 X lop2 M PPA into the perfusion stream via a side-port in the tubing 15 cm proximal to the artery. If the tissue response was found to be satisfactory, non-cumulative dose response curves for I-, dJ-, and d-NOR were performed by alternately injecting 500 pl of each drug, in concentrations ranging from 1 X to 3.3 X M for I- and d,l-NOR and 3.3 X lop3 to 1 X lo-' M for d-NOR. Following injection, tissues were allowed to return to baseline tension before the next higher dose. Vasoconstriction was measured as the increase in perfusion pressure at constant flow, using a Statham pressure trans- ducer (Hato Rey, PR), coupled to a Grass model 7 polygraph (Quincy, MA). To prevent damage to tissues, perfusion pressure was not permitted to exceed 200 mm Hg.

Relative Potency and Intrinsic Activity of the Norephedrines

The relative potency and intrinsic activity of I , d,L, and d-norephedrine were determined by performing a dose-response curve with the endogenous neurotransmitter NE. Cocaine (1 X

M), was added to the buffer to eliminate possible indirect activity of the norephedrines. The results were expressed as the increase in pressure relative to that produced by 50 pl of methoxamine (1 x lo-, M), a well-accepted nonmetabolizable alpha,-adrenoceptor agonist. The potency and activity of a diastereomer of PPA, d-norp-seudoephedrine (d-NORP), was also investigated.

Activity at Adrenoceptor Subtypes

The activities of the norephedrines at adrenoceptor subtypes were determined by con- structing dose-response curves with the inclusion of specific adrenoceptor antagonists in the perfusion buffer, thus responses from activation of adrenoceptor subtypes not under exami- nation could be eliminated. Cocaine (1 X lop5 M) was added to block possible indirect activity. The following antagonists were added to the buffer to study the activity of the norephedrines (receptor, antagonist, concentration [-log M): alpha,, propranolol, 6; alpha,, yohimbine, 7; alpha,, propranolol, 6; alpha,, corynanthine, 5; beta,, metoprolol, 6; beta, propranolol, 6; gamma, propranolol, 6; gamma, yohimbine, 7; gamma, corynanthine, 5 . The response to adrenoceptor blockade was measured as the shift of the dose-response curve to the


right, in the case of alpha-adrenoceptor blockade, or to the left for beta-adrenoceptor blockade. Possible gamma receptor activity was measured as the tissue response to the agonists when both alpha- and beta-adrenoceptors were blocked.

Direct vs. Indirect Activity

In order to evaluate whether PPA has indirect activity, vesicular stores of NE were depleted by pretreating the rats 18 hr before the removal of tail arteries with an IP injection of reserpine ( 5 mgikg). The effectiveness of the treatment was confirmed following the dose- response curve for PPA by an injection of the indirect adrenoceptor agonist tyramine (50 ~ 1 , 1 X M). If there were no responses to tyramine, the reserpine treatment was deemed effective. Control responses for tyramine were carried out in tissues which had not been pretreated with reserpine.

Calcium Channel Antagonists

Dose-response curves for PPA were constructed with the calcium channel antagonists amlodipine and verapamil added to the buffer (which included ethanol 0.1 % to aid in disso- lution) in concentrations of 1 X lo-' and 1 X M, respectively. A separate series of control tissues were performed in buffer to which ethanol 0.1% had been added.

Statistics

The statistical evaluation of results utilized the Pharmacologic Calculation System statistical package (Microcomputer Specialists, Philadelphia, PA). Results are expressed as the mean % S .E.M., and shifts in dose-response curves were considered significant if the relative potency 95% confidence limit did not encompass unity. Tests for parallelism were performed from the regressions of dose-response curves. Differences in maximal responses between groups were evaluated by unpaired Student t-test for two groups or analysis of variance (ANOVA), and Newman-Keuls Multiple Range Test, when three or more groups were compared. The minimum level of significance was considered p < .05.

Drugs

Amlodipine was a gift from Pfizer (Groton, CT); 1- and d-NOR were obtained from Aldrich (Milwaukee, WI), and d,I-NOR was from Arynco (Carlstadt, NJ). PPA and the individual enantiomers d- and 1-NOR were tested for their ability to rotate polarized light on a Perkin-Elmer polarimeter (model number 241MC), and additionally they were melted in previous studies from this laboratory to confirm their identity. All other drugs were obtained from Sigma (St. Louis, MO).

RESULTS Physical Properties

The optical rotations for 1- and d-NOR were -32.2 and +33.0, respectively, which are consistent with published values for these species. The racemic d,l-NOR mixture, as expected, failed to induce optical rotation when exposed to polarized light.

Relative Potency

Phenylpropanolamine and its enantiomers were relatively weak in their vasoconstrictor activity when compared to NE (Fig. 1; Table 1). The endogenous neurotransmitter had a -log ED,, value of 5.92 2 0.35, compared to 3.53 2 0.01 for d,l-NOR, 3.83 +. 0.04 for 1-NOR, and 3.02 2 1.90 for d-NOR, indicating a potency for NE of about 250 times that for d,l-NOR. Moreover, the slope of the linear regression for the dose-response curve of NE was significantly greater than the slopes for d,l, 1-, and d-NOR (p < .05). While NE was a full agonist when compared to the maximal contractions induced by methoxamine, d,l-NOR and 1-NOR


100

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Fig. 1. norepinephrine.

The relative potency and intrinsic activity of dJ-, d-, and I-norephedrine compared to 1-

TABLE 1. Intrinisic Activity of d,b, d-, I-NOR, I-NE, and d-NORP Compared to Maximal Contraction Induced bv Methoxamine (50 ul: 1 X lo-’ M)t

Re 1 at i v e -Log ED,, Enlax potency

1-NE 5.92 & 0.35 101.12 & 3.13* 245 * * d,l-NOR 3.53 * 0.01 42.24 5 4.89 1-NOR 3.83 * 0.04 48.63 k 6.69 2.31** d-NOR 3.02 * 1.90 40.43 k 5.31 0.059** d-NORP 14.12 * 7.83*

+The Em,, was expressed as percent of the maximum contraction induced by methoxamine. Relative potency is compared to d,l-NOR. Because there was no intrinsic activity seen with d-NORP, relative potency could not be determined. Each value repre- sents the mean 5 S.E.M. for 5 or 6 tissues. *Significant at p < .01. **Significant at p < .05.

-

-

were able to induce only 42% and 49%, respectively, and d-NOR only 40%, of the maximal contractions of NE or methoxamine. When the vasoconstrictor activity of d,I-NOR was compared to its enantiomers, I-NOR was 2.3 times more potent than d,l-NOR, and d,l-NOR 16.8 times more potent than d-NOR in inducing contraction of rat caudal artery (p < .05). The slopes for linear regressions generated from the dose-response curves indicated that the slope for I-NOR was significantly greater than that for d-NOR (p < .05). Slopes for d,l-NOR did not significantly differ from those of d-NOR or I-NOR. The diasteriomer d-NORP demonstrated no significant direct vasoconstrictor activity in isolated caudal artery exposed to doses ranging from 1 X lop4 M to 1 X lo-’ M. Analysis of the regression line indicated no significant difference from zero.

Sensitivity of Adrenoceptor Subtypes

Dose response-curves were performed for d,l-NOR and its enantiomers in the presence of antagonists for specific adrenoceptor subtypes. To examine the effect of alpha-adrenoceptor


TABLE 2. The Effect of Alpha-Adrenoceptor Blockade on Vasoconstriction Induced by d,l, d-, and I-NOR?

Relative -Log ED,, Emax potency

d,I-NOR Control 3.50 t 0.05 209 t 16 -

Yohimbine 3.44 * 0.04 175 t 21 0.60 Corynanthine 2.65 * 0.54 64 t 6* 0.002* *

Corynanthine & yohimbine 2.95 t 0.07 71 t 5* 0.002** I-NOR

Control 3.69 t 0.09 233 2 16 -

Yohimbine 3.59 f 0.11 211 t 11 0.64 Corynanthine & yohimbine 3.37 & 0.13 90 ? 10* 0.005* *

Control 1.95 k 0.11 98 & 10 -

Cory nanthine 2.01 t 0.17 51 t 4" 0.032** Yohimbine 1.46 ? 0.15 78 t 19 0.52

Corynanthine & yohimbine 1.68 & 0.11 41 ? 7* 0.005*

?Each value is the mean t S.E.M. of 5 or 6 tissues. *Significant at p < .01. **Significant at p < .05.

Corynanthine 2.65 t 0.54 64 2 6* 0.002* *

d-NOR

blockade, propranolol (lop6 M) was added to buffer solutions to block possible beta-adrenoceptor activity. Additionally, the buffer contained either 1) yohimbine to block alpha,-adrenoceptors; 2) corynanthine to block alpha,-adrenoceptors; or 3) yohimbine and corynanthine to block both alpha-adrenoceptor subtypes (Table 2).

The presence of yohimbine ( lop7 M) in the buffer caused a small non-significant shift to the right in the dose-response curves for the d- and 1-NOR enantiomers and the racemic d,l-NOR, with relative potencies of 0.52, 0.64, and 0.60, respectively. Furthermore, there were no significant changes in the slopes of the linear regressions, or in the maximal response of the tissues, to stimulation by d-,I-NOR, or d,l-NOR in the presence of yohimbine-mediated blockade of alpha,-adrenoceptors. However, blockade of alpha,-adrenoceptors with corynanthine M) caused significant decreases of 31, 212, and 489-fold in the relative potencies d-,I-NOR, and d,l-NOR, respectively, when compared to control (p < .05), as well as significant decreases in the maximal contractions induced by each drug. The combination of yohimbine and corynanthine in the perfusion buffer produced results which were nearly iden- tical with the effects of blockade with corynanthine alone. The addition of corynanthine or corynanthine and yohimbine to the buffer did not significantly alter the slopes of the regression lines for d-&NOR, or d,l-NOR, indicating that these antagonists were acting competitively at their respective adrenoceptor sites.

The beta,-selective adrenoceptor antagonist metoprolol did not demonstrate any significant effects on the potency, maximum contractions, or slopes of the regression lines for vasoconstriction induced by d,l-NOR or its individual component enantiomers (Table 3). The relative potency of d-, I-NOR, and d,l-NOR in the presence of metoprolol ( M) were 0.92, 1.07, and 1.24, respectively, p > .05. There were more pronounced shifts to the left observed in the dose-response curves for d,l-NOR and its enantiomers in the presence of the beta- adrenoceptor antagonist propranolol ( M) in the buffer (Table 3). With 1-NOR, the relative potency of the responses of tissues perfused with the propranolol-containing buffer was 2.89 compared to control tissues, p < .05. In caudal arteries constricted with d,l-NOR and d-NOR the relative potencies were 3.34 and 2.74, respectively. There was no significant change in


TABLE 3. The Effect of Beta-Adrenoceptor Blockade on Vasoconstriction Induced by d.1, d-. and I-NOR*


d,l-NOR Control 3.01 + 0.05 139 ? 20 -

Metoprolol 3.01 * 0.01 136 f 9 1.07 Propranolol 3.37 * 0.06 166 f 21 3.34

1-NOR Control 3.21 * 0.05 170 & 16 - Metoprolol 3.42 f 0.13 167 f 11 1.24 Propranolol 3.55 & 0.07 198 + 14 2.89

Control 1.79 * 0.18 69 f 7 Metoprolol 1.88 * 0.15 85 & 7 0.92 Prouranolol 1.99 f 0.20 56 & 3 2.74

d-NOR -

*Each value was the mean + S.E.M. of 6 tissues

either maximum response, or slopes of linear regression in the presence of propranolol. These resulted suggest that d,l-NOR and its enantiomers mediate the contraction of rat caudal artery primarily via activation of alpha,-adrenoceptors, and that alpha,- and beta-adrenoceptors exert only a limited influence on vasoconstriction induced by these agonists.

Direct vs. Indirect Activity

Reserpine, a drug which depletes catecholamine neuronal storage vesicles, was administered to a group of rats to determine whether d,l-NOR and its individual component enantiomers induced vasoconstriction indirectly, via the release of NE from neuronal stores. To confirm the effectiveness of the treatment, caudal arteries from animals which had been reserpine pretreated were tested with the indirect adrenergic agonist tyramine and found to be insensitive to this agent. When dose-response curves for d,l-NOR were compared among the tissues from animals pretreated with reserpine, control tissues perfused with buffer containing cocaine (lop5 M), and control tissues perfused without cocaine, there was no significant differences in relative potency, maximal response developed, or the slopes of the regression lines between the groups (Table 4). The relative potency between the control group without cocaine and the reserpine group was 0.77, and between the cocaine and control groups was 0.78 (p > .05). These results indicate that the vasoconstriction of caudal arteries, induced by d,l-NOR, is not mediated via the displacement of NE from neuronal storage vesicles and supports a direct receptor activation in this tissue.

Calcium Channel Antagonists

M) or verapamil (lop7 M) in the perfusion solution resulted in a non-significant shift to the right of the dose-response curve for d,l-NOR, with no significant differences in the slopes of the regression lines. There was, however, a significant (p < .01) decrease in the maximal response of 45 and 53 mm Hg for amlodipine and verapamil, respectively (Table 5).

The presence of the calcium channel antagonists amlodipine

DISCUSSION

The results of these experiments demonstrate that PPA and its individual component enantiomers 1- and d-NOR are relatively weak adrenergic agonists, with the most active species, 1-NOR, being 124 times less potent, and having only half the intrinsic activity of the


TABLE 4. The Effects of Reserpine Pretreatment and Cocaine on Vasoconstriction Induced by d,l-NOR*

Relative -Log EDSO Emax potency

Control 3.06 t 0.19 148 t 21 -

Cocaine 3.01 t 0.05 139 * 20 0.77 Resemine 3.09 t_ 0.09 121 t 19 0.78

*Each value was the mean t S.E.M. of 6 tissues

TABLE 5. The Effect of Calcium Channel Antagonists on Vasoconstriction Induced by d,l-NORt


Control 3.31 t 0.07 162 2 17 -

Amlodipine 3.47 t 0.25 106 f 8* 0.34 Verapamil 3.43 t 0.07 96 t l l * 0.25

?Each value was the mean t_ S.E.M. of 6 tissues. *Significant at p < .01, when compared to control.

endogenous neurotransmitter NE. Studies by Ruffolo et al. [ 19821, demonstrated that alpha- methylnorepinephrine with the 1R,2S( -)-erythro configuration was more potent than the three other stereoisomers of alpha-methyl-NE in field-stimulated guinea pig ilium and central anti- hypertensive activity. The results of this investigation are similar in that the lR,2S(-)-erythro configuration, 1-NOR, was also the most active enantiomer of norephedrine. This finding is consistent with the Easson-Stedman hypothesis which states that the beta-hydroxy carbon must be in the R configuration for most favorable binding to adrenergic receptors [Easson and Stedman, 19331.

The results of this investigation are also consistent with the results of previous investigations, both in whole animals and in other isolated tissues. Research by Minneman [1983], utilizing isolated rat vas deferens, reported the intrinsic activity of PPA to be 47% of that for NE, which compares favorably with the 42% maximal response observed in the present study. Additionally, Minneman found PPA to be 1,000 times less potent than NE, more than a 4-fold greater difference than the 245-fold relative potency observed in this investigation. The difference in the potency of PPA between caudal artery and vas deferens could be related to differences in adrenoceptor density and/or type, as well as the different methodologies employed. Minneman observed that the adrenoceptor population mediating contraction of rat vas deferens was homogeneous for the alpha, subtype, while in rat caudal artery, populations of both alpha, - and alpha,-adrenoceptors have been identified [Medgett and Langer 19841. In studies of isolated human digital arteries removed postmortem, Stevens et al. [1981] observed a relative potency ratio of 76 between NE and PPA; however, they reported no significant differences in E,,,. The variability in the relative potency between NE and PPA was further demonstrated by Persson et al. [1973] who found the potency of NE in isolated strips from rabbit aorta to be only 50 to 80 times that of PPA. As a consequence of the relatively weak vasoconstrictor activity of d-NOR, the racemate PPA was half as potent as its I-enantiomer at inducing vasoconstriction of the rat caudal artery. The results of the current investigation are therefore consistent with previous findings from this laboratory utilizing whole animal and pithed rat preparations, which found 1-NOR and PPA to be potent pressor agents when administered IV, and d-NOR only marginally effective in raising blood pressure [Moya-Huff and Maher 1987; Moya-Huff et al., 19871.

Attempts were made to determine the relative activity of PPA and its enantiomers at


specific adrenoceptor subtypes. For alpha-adrenoceptors which mediate vasoconstriction of blood vessels, blockade of the alpha,-subtype with yohimbine resulted in only small non- significant shifts to the right of the dose-response curves for PPA, d-, and 1-NOR. Blockade of alpha, - adrenoceptors with corynanthine, however, resulted not only in dramatic decreases in the potency of the individual norephedrines, but in significant decreases in the maximum response as well. It must be recognized, however, that these investigations were undertaken with the realization that differences in the number and distribution of the adrenoceptor subtypes, as well as differences in how these subtypes are eventually coupled to the actin-myosin protein complex, could affect the degree of smooth muscle contraction. Thus, full activation of one adrenoceptor subtype may not result in the same degree of vasoconstriction as full activation of another, and, therefore, one cannot assume that a greater response necessarily means greater binding to, or activation of, a particular adrenoceptor subtype. As an example, research by Cheung and Triggle [ 19881 reported equal numbers of alpha,- and alpha,-adrenoceptor binding sites present in the caudal arteries of SD rats. However, studies by Medgett and Langer [ 19841 revealed that vasoconstriction of SD caudal arteries initiated by stimulation of sympathetic nerve terminals are mediated primarily by activation of alpha,-adrenoceptors, and that even with the application of exogenous adrenergic agonists, alpha,-adrenoceptors play only a minor part in the constriction of this blood vessel. Other investigators, utilizing caudal arteries from spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) demonstrated that alpha,-adrenoceptor activation by specific adrenergic agonists accounted for only 20% and 5% of the contraction in tissues from WKY and SHR, respectively [Aqel et al., 19861. Therefore, rat caudal artery may be a less suitable tissue to study alpha,-adrenoceptor activation than other tissues such as the marginal ear vein of the rabbit, or the middle cerebral artery of the cat, in which constriction is mediated predominately via the alpha,-adrenoceptor subtype [Medgett and Langer, 1983; Daly et al., 1988a,b]. The above results, therefore, should not be misconstrued as suggesting that PPA and its enantiomers do not bind to or activate alpha,-adrenoceptors. It may be concluded, however, that the vasoconstriction induced by the norephednnes in this study was mediated primarily via alpha,-adrenoceptors. Other investigators have demonstrated the activity of PPA at alpha,-adrenoceptors. Results of experiments of the pressor activity of PPA, d-, and 1-NOR, in rats by Moya-Huff and Maher [1987], found greater decreases in BP with the alpha, antagonist prazosin than with yohimbine. Utilizing competitive binding studies with the radiolabeled alpha, antagonist l2'IBE 2254, Minneman et al. (19831 observed that PPA bound to alpha,-adrenoceptors with low affinity (Kd 220 f 41). An unexpected result of the current study revealed decreased maximal responses for the norephedrines in the presence of corynanthine, a competitive alpha, antagonist. This effect could be related to the relatively low affinity of d- and 1-NOR for this receptor subtype, which might possibly prevent a truly competitive displacement of corynanthine from the receptor site by d- and I-NOR.

There has been general acceptance over the years that PPA has only minor activity at beta-adrenoceptors [Weiner, 19801. Classic experiments by Trendelenburg et al. [ 1962al and Liebman [1961] demonstrated that PPA increased heart rate in cat and isolated dog heart, respectively, primarily by indirect actions, and with low potency. However, in pithed rats, Moya-Huff and Maher [1987], found that PPA, I-, and d-NOR, increased heart rate primarily by direct activation of cardiac beta-adrenoceptors, with some minor indirect activity observed with I-NOR. Furthermore, Moya-Huff and Maher demonstrated a modest depressor activity for the norephedrines, attributed to beta,-adrenoceptor activation, when alpha- and beta,- adrenoceptors were blocked and the rats were infused with the potent pressor agent angiotensin 11. In the present study, blockade of beta,-adrenoceptors with metoprolol failed to shift the dose-response curves for d-, I-NOR, or PPA, suggesting that this adrenoceptor subtype does not significantly influence vasoconstriction in the isolated rat caudal artery. This result is not unexpected since beta, -adrenoceptors are not commonly associated with vasoconstrictor or dilator actions in vascular smooth muscle. The presence of the non-selective beta adrenergic


antagonist propranolol did modestly shift the dose-response curves for all three compounds to the left (significantly in the case of I-NOR), suggesting that the vasoconstrictor activity of PPA and its enantiomers may be moderated, to some degree, by stimulation of beta-adrenoceptors.

The studies of Trendelenburg, cited above, demonstrated that the actions of PPA may be either direct (nictitating membrane) or indirect (heart rate) depending on the particular tissue being examined. In isolated caudal artery, the presence of the uptake blocker cocaine, or acute pretreatment of the rat with reserpine failed to shift the dose-response curve for PPA. The results of the current study thus are in agreement with the findings of Persson et al. [1973], who found no effect with reserpine pretreatment in isolated rabbit aorta. In whole animals, Moya-Huff et al. [ 19871, demonstrated no tachyphylaxis following repeated administration of d,l-NOR, futher supporting a direct action for this sympathomimetic. The current investigation supports the conclusion that the mediation of vasoconstriction by PPA is via the direct activation of post-junctional adrenoceptors.

The calcium channel antagonists amlodipine and verapamil, in effective concentrations, failed to shift the dose-response curve, but significantly reduced the maximal response of the rat caudal artery to PPA. Previous investigations have demonstrated that alpha,-adrenoceptors are uniformly sensitive to calcium channel blockade, since they utilize extracellular Ca” to initiate contraction. However, in some tissues, contraction of smooth muscle activated by alpha,-adrenoceptors is relatively insensitive to calcium channel antagonists [Cavero et al., 19831. The lack of effectiveness of calcium channel antagonists with contractions mediated by this adrenoceptor subtype may be related to the utilization of intracellular Ca2+ stores to signal a portion of the contraction initiated by alpha,-adrenoceptor activation [Timmermans and Thollen, 19871. Thus, in the current experiments, with lower doses of PPA, the small contractions resulting from activation of a limited number of alpha,-adrenoceptors may have required only intracellular Ca2 + stores to produce contraction and consequently, the calcium channel blockers were ineffective. Maximal contractions of the caudal artery, however, may have required the mobilization of Caz+ from extracellular sources, as well as from intracellular stores, and thus the response was more susceptible to calcium channel blockade.

In summary the sympathomimetic properties of PPA and its individual component enantiomers d- and 1-NOR were examined in isolated perfused/superfused caudal arteries from SD rats. The results of these investigations revealed that the norephedrines act as partial agonists primarily via direct stimulation of postjunctional alpha,-adrenoceptors with relatively low potency; however, 1-NOR did have significant activity at beta,-adrenoceptors as well. When the enantiomers of PPA were compared, d-NOR had only minor vasoconstrictor activity, relative to I-NOR, which was well over 100 times less potent than the endogenous neurotransmitter NE. The maximal contractions produced by PPA in the rat caudal artery were reduced in the presence of calcium channel antagonists.

ACKNOWLEDGMENTS

This work was supported in part by a grant from the Thompson Medical Co. Inc. The authors wish to thank Dr. David A. Williams for assistance in determining the optical activity of the enantiomers employed in this study.

REFERENCES

Aqel, M.B., Sharma, R.V., and Bhalla, R.C.: Increased Ca2 + sensitivity of alphal-adrenoceptor- stimulated contraction in SHR caudal artery. Am. J. Physiol. 250:C275-C282, 1986.

Cavero, I., Sepperson, N., Lefevre-Borg, F., and Langer, S . Z . : Differential inhibition of vascular smooth muscle responses to alpha1 -adrenoceptor agonists by diltiazem and verapamil. Circ. Res. 52: 69-76, 1983.


Cheung, Y.-D., and Triggle, C.R.: Alpha adrenoceptor sites in vascular smooth muscle differentiation by selective antagonist binding. Biochem. Pharmacol. 37:4055 -4061, 1988.

Daly, C.J., McGrath, J.C., and Wilson, V.G.: Evidence that the population of postjunctiond-adrenoceptors mediating contraction of smooth muscle in the rabbit isolated ear vein is predominantly alpha,. Br,. J. Pharmacol. 94:1085-1090, 1988a.

Daly, C.J., McGrath, J.C., and Wilson, V.G.: Pharmacological analysis of postjunctional alpha-adrenoceptors mediating contractions to (-)-noradrenaline in the rabbit isolated lateral saphenous vein can be explained by interacting responses to simultaneous activation of alphal- and alpha2- adrenoceptors. Br. J . Pharmacol. 95:485-500, 1988b.

Easson, L.H., and Stedman, E.: CLXX. Studies on the relationship between chemical constitution and physiological action. V. Molecular dissymmetry and physiological activity. Biochem. J . 27:

Horowitz, J.D., McNeil, J.J., Sweet, B., Mendelsohn, F.A.O., and Louis, W.J.: Hypertension and postural hypotension induced by phenylpropanolamine (Trimolets). Med. J . Aust. 1: 175,176, 1979.

Larson, W.L., and Rogers, A.: Overdosage from phenylpropanolamine: Experience of the hennepin regional poison center. Vet. Hum. Toxicol. 28546-548, 1986.

Lasagna, L.: “Phenylpropanolamine: A Review.” New York: John Wiley, 1988. Liebman, J.: Modification of the chronotropic action of sympathomimetic amines by reserpine in the

hear-lung preparation of the dog. J . Pharmacol. Exp. Ther. 133:63-69, 1961. Medgett, I.C., and Langer, S.Z.: Characterisation of smooth muscle alpha-adrenoceptors and of re-

sponses to electrical stimulation in the cat isolated perfused middle cerebral artery. Naunyn Schmiedebergs Arch. Pharmacol. 323:24-32, 1983.

Medgett, I.C., and Langer, S.Z.: Heterogeneity of smooth muscle alpha adrenoceptors in rat tail artery in vitro. J. Pharmacol. Exp. Ther. 229:823-830, 1984.

Minneman, K.P., Fox, S.W., and Abel, P.W.: Occupancy of alphal-adrenergic receptors and contraction of rat vas deferens. Mol. Pharmacol. 23:359-368, 1983.

Moya-Huff, F.A., Kiritsy, P.J., and Maher, T.J.: Cardiovascular differences between phenylpropanolamine and its related norephedrine isomers in the rat. J. Pharm. Sci. 76:114-116, 1987.

Moya-Huff, F.A., and Maher, T.J.: Adrenergic receptor subtype activation by (+)-, (-)- and ( + / -)-norephedrine in the pithed rat. J. Pharm. Pharmacol. 39:108-112, 1987.

Persson, C.G.A., Erjefalt, I., and Keman, M.: Brompheniramine and the cardio-vascular effects of norephedrine and noradrenaline. Acta Allergol. 28:401-415, 1973.

Ruffolo, R.R., Yaden, E.L., and Waddell, J.E.: Stereochemical requirements of alpha-2 adrenergic receptors. J. Pharmacol. Exp. Ther. 222:645-651, 1982.

Stevens, M.J., Rittinghausen, R.E., and Moulds, R.F.W.: Heterogeneity of human vascular pre- and post-synaptic alpha-adrenoceptors. Clin. Sci. 61:203S-206S, 198 1.

Timmermans, P.B.M.W.M., and Thollen, M.J.M.C.: CA’+ utilization in signal transformation of alpha- 1 adrenergic receptors. In Ruffolo, R.R., Jr., (ed.): “The Alpha-] Adrenergic Receptors.” Clif- ton, NJ: Humana Press, 1987, pp. 113-187.

Trendelenburg, U . , Muskus, A,, Fleming, W.W., and Alonso de la Sierra, B.G.: Modification by reserpine of the action of sympathomimetic amines in spinal cats; a classification of sympathomimetic amines. J. Pharmacol. Exp. Ther. 138:170-180, 1962a.

Trendelenburg, U., Muskus, A., Fleming, W.W., and Alonso de la Sierra, B.G.: Effects of cocaine, denervation and decentralization on the response of the nictitating membrane to various sympathomimetic amines. J. Pharmacol. Exp. Ther. 138:181-193, 1962b.

Weiner, N.: Norepinephrine, epinephrine, and the sympathomimetic amines. In Gilman, A.G., Good- man, A, , and Gilman, A. (eds.): “Goodman and Gilman’s The Pharmacological Basis of Ther- apeutics.’’ New York: MacMillan Publishing, 1980, pp. 138-175.

1257-1266, 1933.

vasoactive properties of phenylpropanolamine (d, i-norephedrine) and its enantiomers in isolated rat...

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