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`THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
`Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics
`JPET 294:179–186, 2000 /2393/
`
`Vol. 294, No. 1
`Printed in U.S.A.
`
`Differential Inhibition of the Prejunctional Actions of
`Angiotensin II in Rat Atria by Valsartan, Irbesartan, Eprosartan,
`and Losartan
`
`SURAJ S. SHETTY and DOMINICK DELGRANDE
`Novartis Institute for Biomedical Research, Metabolic and Cardiovascular Diseases, Summit, New Jersey
`Accepted for publication March 15, 2000
`This paper is available online at http://www.jpet.org
`
`ABSTRACT
`The effects of valsartan and other nonpeptide angiotensin II
`type 1 (AT1) receptor blockers on the prejunctional actions of
`angiotensin II were investigated in the isolated left atria of rat.
`Norepinephrine stores in rat atria were loaded with [3H]norepi-
`nephrine, and neuronal norepinephrine release was deduced
`from the radioactivity efflux. Angiotensin II (1029 to 1026 M)
`produced concentration-dependent enhancement of the elec-
`trical stimulation-induced efflux of [3H]norepinephrine from the
`preparation. Pretreatment of tissues with valsartan, irbesartan,
`eprosartan, or losartan (1028 to 1026 M) produced concentra-
`tion-dependent inhibitions of the stimulation-induced efflux of
`radioactivity observed in the presence of angiotensin II (1027
`M). The AT1 receptor blockers did not decrease the “basal”
`stimulation-induced overflow of radioactivity but rather selec-
`tively inhibited the angiotensin II-mediated augmentation of the
`response. Regression analyses of the inhibition of the angio-
`
`tensin II-mediated response by valsartan, irbesartan, eprosar-
`tan, and losartan revealed corresponding log IC50 values (log
`M, with 95% confidence intervals) of 27.78 (28.19, 27.51),
`27.65 (28.02, 27.40), 27.12 (27.37, 26.86), and 26.75
`(27.00, 26.40), indicating that the IC50 values for valsartan and
`irbesartan are significantly lower than those for eprosartan and
`losartan. Thus, valsartan is a potent inhibitor of the prejunc-
`tional facilitatory effect of angiotensin II on the release of nor-
`epinephrine from peripheral sympathetic nerves. This implies
`that the therapeutic domain of valsartan may be extended to
`include pathophysiological conditions such as congestive heart
`failure wherein prejunctional angiotensin II receptors apparently
`play a significant role. Whether the high potency of valsartan
`translates into a significant clinical advantage relative to the
`other agents tested remains to be ascertained.
`
`The renin-angiotensin system (RAS) and the sympathetic
`nervous system (SNS) are important regulators of cardiovas-
`cular function. Angiotensin II (Ang II), the effector peptide of
`RAS, elicits potent vasoconstrictor effects on interacting with
`specific Ang II receptors in vascular smooth muscle (Mendel-
`sohn, 1985). Experimental data indicate that it also modu-
`lates peripheral sympathetic neurotransmission in vitro and
`in vivo by enhancing the release of the adrenergic transmit-
`ter in several tissues (Rand et al., 1990) and augmenting the
`effects of the transmitter at the postjunctional sites (Nicho-
`las, 1970), thereby exerting a facilitatory effect at the adren-
`ergic neuroeffector junction. These observations have been
`substantiated in studies with pithed rats wherein endoge-
`nous Ang II was shown to facilitate sympathetic neurotrans-
`mission after spinal cord stimulation (Wong et al., 1992). Ang
`II-induced facilitation of peripheral adrenergic transmission
`has also been demonstrated in hand veins and resistance
`
`Received for publication December 13, 1999.
`
`vessels of humans (Benjamin et al., 1988; Seidelin et al.,
`1991). Consistent with these findings, treatment with angio-
`tensin-converting enzyme inhibitors has been reported to
`decrease circulating norepinephrine (NE) concentrations
`(Wenting et al., 1983).
`The nexus between the SNS and the RAS could have seri-
`ous implications in the pathogenesis of various cardiovascu-
`lar disorders. An increase in sympathetic neural activity is
`believed to be important in the pathogenesis of hypertension
`in spontaneously hypertensive rats (Judy et al., 1976). Con-
`sistent with this view, an increased transmitter turnover was
`detected in some vascular beds and in the heart during the
`development of hypertension (Adams et al., 1989). The activ-
`ity of the SNS was also found to be augmented in congestive
`heart failure (CHF) (Rector et al., 1987; Francis, 1989). The
`ensuing increase in cardiac NE spillover has been associated
`with malignant ventricular arrhythmia (Meredith et al.,
`1991), presumably accounting for the positive correlation
`noted between plasma NE concentrations and mortality
`
`ABBREVIATIONS: RAS, renin angiotensin system; SNS, sympathetic nervous system; AT1, angiotensin II type 1; Ang II, angiotensin II; NE,
`norepinephrine; CHF, congestive heart failure; SI, stimulation-induced; PSS, physiological salt solution; CI, confidence interval; FR, fractional
`release.
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`180
`
`Shetty and DelGrande
`
`rates in this condition (Rector et al., 1987). Furthermore, Ang
`II levels are also reportedly elevated in CHF (Francis, 1989),
`suggesting that the augmentation of the activity of the SNS
`in this condition is secondary to an upsurge in the levels of
`the peptide.
`Ang II elicits its vast array of pharmacological actions by
`binding to specific receptors located on the membranes of its
`target cells. Based on the differential binding affinities of
`selective ligands, losartan, CGP42112, and PD 123319, two
`receptor subtypes were identified and subsequently catego-
`rized as Ang II type 1 (AT1) and type 2 (AT2), respectively
`(Chiu et al., 1989; Whitebread et al., 1989; Bumpus et al.,
`1991). The AT1 subtype appears to be the principal mediator
`of all the known physiological actions of Ang II, whereas the
`function of the AT2 subtype is poorly defined at present. The
`receptor mediating the prejunctional facilitatory effects of
`Ang II on sympathetic neurotransmission was proposed to be
`of the AT1 subtype based on the antagonistic effect of losar-
`tan, the prototypical AT1 receptor blocker (Tofovic et al.,
`1991; Wong et al., 1992; Foucart et al., 1996). Given the
`prognostic and pathophysiological significance of the inter-
`action between the RAS and the SNS regarding cardiovascu-
`lar disorders, it was considered desirable to ascertain and
`quantify the inhibitory effects of valsartan, a potent Ang II
`receptor blocker (Criscione et al., 1995), on the prejunctional
`actions of Ang II. An ancillary goal of the present investiga-
`tion was to compare the potency of valsartan with those of
`three other AT1 receptor blockers (losartan, eprosartan, and
`irbesartan) for effecting this inhibitory action. The isolated
`rat left atrial preparation was used as a model system in this
`investigation because it is amenable to direct measurements
`of the parameters of interest without being encumbered by
`confounding physiological mechanisms operative in more
`complex in vivo systems.
`
`Materials and Methods
`Animal care and experimental procedures were in accordance with
`the National Institutes of Health Guide for the Care and Use of
`Laboratory Animals and were approved by the Animal Care Com-
`mittee of Novartis Institute for Biomedical Research. Male Sprague-
`Dawley rats (300–400 g) were anesthetized with sodium pentobar-
`bital (65 mg/kg i.p.). After opening the chest, the hearts were
`removed, cannulated at the aorta, and immediately suspended in a
`Langendorf apparatus for retrograde perfusion (4 ml/min) of the
`coronary system with warm (37.5°C) oxygenated (95% O2, 5% CO2)
`physiological salt solution (PSS). The PSS contained 118.0 mM NaCl,
`4.7 mM KCl, 1.03 mM KH2PO4, 0.45 mM MgSO4, 2.5 mM CaCl2, 25.0
`mM NaHCO3, 11.1 mM dextrose, 0.14 mM ascorbic acid, and 0.067
`mM disodium EDTA. The procedure described by Foucart et al.
`(1996) was adopted with some modifications for the assessment of
`agents affecting the overflow of the radiolabeled neurotransmitter.
`Briefly, the left atrial walls were dissected from the suspended and
`perfused hearts and incubated for 25 min in 5 ml of PSS to which
`[3H]NE (5.7 mCi/ml) was added. The radiolabeled incubation solution
`was maintained at 37.5°C and continually oxygenated as before. At
`the end of the incubation period, the tissues were lightly blotted on
`a filter paper to remove superficially bound [3H]NE and transferred
`to 0.5-ml perfusion chambers (one atrium per chamber). The perfu-
`sion system used was a Brandel suprafusion system (SF-06; Brandel
`Inc., Gaithersburg, MD). The perfusion rate was set at 0.4 ml/min,
`and the temperature of the perfusion solutions was kept constant at
`37.5°C with the help of a water bath and an environment cage.
`Electrical stimulation of the preparations was performed by a mul-
`tichannel electrical stimulator (ES2-069-55; Brandel Inc.) and plat-
`
`Vol. 294
`
`inum screened electrical probes. Effluents were collected into 8-ml
`vials placed in vial trays. From reagent to effluent, each channel was
`completely isolated from the others.
`The atria were washed for 65 min with PSS during which a
`priming stimulus (PS; 3 Hz, 50 mA, 1 ms, 60 s) was given at 50 min
`to eliminate the superficial or loosely bound [3H]NE. The effluent
`was subsequently collected once every 5 min for a total of 70 min (14
`sampling periods). During this period, the atria were field stimulated
`twice (S1 and S2; 3 Hz, 50 mA, 1 ms, 60 s) at 20 and 55 min as
`described by Foucart et al. (1996). Thus, initiations of PS, S1, and S2
`were each separated by 35 min. Ang II was included in the perfusing
`solution 20 min before the second stimulation (S2) in select experi-
`ments. Test compounds (or vehicle) were typically included in the
`perfusing solution 20 min before S1. Thus, the tissues were initially
`treated with each of the test compounds (or vehicle) for 35 min and
`then exposed to Ang II for 20 min in the continued presence of the
`agents before being subjected to S2. The effects of test compounds on
`the control (or basal) stimulation-induced (SI) efflux were also as-
`certained by including the compounds in the perfusion solution 20
`min before either S1 or S2.
`At the end of the experiment, the atria were lightly blotted,
`weighed, and placed in 7-ml vials, each containing 1 ml of Soluene-
`350 (Packard Instrument Co., Meriden, CT). The vials were shaken
`at 50°C for 2 h to solubilize the tissues. The radioactivity present in
`the solutions (effluents, solubilized tissues) was determined by liquid
`scintillation counting (Beckman LS6500; Beckman Instruments, Ir-
`vine, CA) after the solutions were mixed with 5 ml of Pico-Fluor 40
`(Packard Instrument Co., Meriden, CT). The spontaneous (resting)
`radioactive outflow during the 5-min period before the stimulation
`was measured, and the SI component of the outflow of radioactivity
`was determined by subtracting the resting radioactivity from the
`total radioactivity content of the 5-min sample collected during the
`stimulation period. The SI outflow of radioactivity measured during
`the second period of stimulation (S2) was expressed as the percent-
`age of the first period of stimulation (S1). The values were initially
`standardized for the total radioactivity in the tissue at that time
`point by expressing them as fractional releases (FR) of radioactivity,
`and the ratio % FR2/FR1 was then used to indicate the effects of
`pharmacological interventions.
`Drugs. Desipramine hydrochloride, oxymetazoline hydrochloride,
`fenoterol hydrobromide, and Ang II (synthetic, human sequence)
`were obtained from Sigma Chemical Co. (St. Louis, MO). Valsartan
`was synthesized in-house at Novartis (Summit, NJ). Losartan was a
`gift from DuPont Merck Pharmaceuticals (Wilmington, DE). Epro-
`sartan and irbesartan were synthesized in Novartis (Basel, Switzer-
`land). Stock solutions (1022 M) of desipramine, oxymetazoline, and
`fenoterol were prepared fresh each day in PSS. Stock solutions (1023
`M) of Ang II prepared in deionized water were stored in 100-ml
`aliquots at 280°C. All other compounds were prepared fresh each
`day in DMSO to a concentration of 1022 M. Further dilutions were
`made in PSS. Tritiated NE (NE, levo-[ring-2,5,6-3H]) was purchased
`from NEN Life Science Products, Inc. (Boston, MA) with a specific
`activity of 62.3 Ci/mmol and a radioactive concentration of 1 mCi/ml.
`Statistical Analysis. All data are expressed as mean 6 S.E.
`Student’s t test (two-tailed, unpaired) was used to determine statis-
`tical significance of differences between means of control and treat-
`ment groups. An ANOVA followed by Dunnett’s test was used for
`multiple comparisons with a control group. Differences with P , .05
`were considered significant.
`For estimation of potency differences among the four drugs, the
`“proportion inhibition” effected by each of the drugs was determined.
`The proportion inhibition of the Ang II response in each individual
`tissue exposed to the receptor blocker was determined by subtracting
`the individual response from the average response seen in the pres-
`ence of Ang II alone and by dividing that value by the average net
`increase effected by Ang II relative to the average basal response.
`The concentration-response relationships for the four Ang II receptor
`blockers were linearized by log transformation of the data. The
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`AT1 Receptor Blockers and Prejunctional Actions of Ang II
`
`181
`
`regression lines were tested for equality of their slopes, and the IC50
`values with 95% confidence intervals (CIs) for each of the four drugs
`were computed (Grieve, 1996). Differences among the IC50 values were
`considered statistically significant when the CIs did not overlap.
`First, a linear regression model was fitted in which all four re-
`gression lines retained their individual slopes and intercepts:
`Model 1: proportion inhibition 5 b0 1 b1 (dose) 1 b2 (L) 1 b3 (E)
`1 b4 (I) 1 b12 (dose 3 L) 1 b13 (dose 3 E) 1 b14 (dose 3 I), where L,
`E, and I are indicators for losartan, eprosartan, and irbesartan,
`respectively.
`In model 1, b0 and b1 correspond to the intercept and slope of the
`regression line for valsartan, b0 1 b2 and b1 1 b12 are the intercept
`and slope for losartan, b0 1 b3 and b1 1 b13 are the intercept and
`slope for eprosartan, whereas b0 1 b4 and b1 1 b14 are the intercept
`and slope for irbesartan.
`A likelihood ratio test was applied for testing the equality of slopes
`by fitting the following model (2), which is nested in model 1:
`Model 2: proportion inhibition 5 b0 1 b1 (dose) 1 b2 (L) 1 b3 (E)
`1 b4 (I).
`
`Results
`Rat left atrial preparations loaded with [3H]NE and sub-
`jected to electrical field stimulation produced an increased
`efflux of the radioisotope. The efflux of radioactivity from the
`tissue into the superfusion solution in control experiments is
`
`shown in Fig. 1. The SI fractional release of radioactivity
`during S2 (FR2, 0.532) when expressed as a percentage of
`that released during S1 (FR1, 0.524) yielded a value of 101.5.
`The ability of the in vitro assay system to detect alterations
`in the overflow of the released NE was ascertained by expos-
`ing the tissues to agents known to modulate the reuptake or
`release of the neurotransmitter. Exposure of the rat atrial
`preparation preloaded with the [3H]NE to desipramine (1026
`M), an inhibitor of the neuronal uptake of NE, 20 min before
`S2 resulted in a significant augmentation of the radioactivity
`efflux on electrical stimulation (Table 1). Desipramine, how-
`ever, did not cause any discernible augmentation of the rest-
`ing efflux of radioactivity. Similar applications of the a2
`agonist oxymetazoline (1026 M) or the b2 agonist fenoterol
`(1026 M) to the rat atrial preparation resulted in correspond-
`ing inhibitory or facilitatory effects on the SI overflow of
`[3H]NE (Table 1).
`Exposure to Ang II (1029 to 1026 M) 20 min before S2 did
`not significantly alter the resting efflux of [3H]NE but pro-
`duced a significant increase in the SI efflux (Fig. 2). A max-
`imal augmentation of about 60% was observed with 1028 M
`Ang II. No diminution of the response was evident on increas-
`ing the concentration of the peptide to 1027 or 1026 M.
`Pretreatment of tissues with each of the four AT1 receptor
`
`Fig. 1. Mean effluxes of radioactivity into
`5-min collections of the PSS bathing the
`atria in control experiments (n 5 15). Elec-
`trical stimulations (3 Hz, 50 mA, 1 ms,
`60 s) are indicated by S1 and S2. In these
`experiments, the mean content of radioac-
`tivity remaining in the atria at the end of
`the experiment was 2.36 3 106 dpm.
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`The likelihood ratio test for testing regression model 2
`versus model 1 (i.e., testing for equality of the slopes of the
`four concentration-response lines as indicated in Statistical
`Analysis) yielded an F value of 0.0882. Comparison with an F
`distribution with 3 and 91 df yielded a P value of .97,
`indicating that there is no evidence that the slopes are dif-
`ferent, thereby necessitating estimation of only one slope.
`The concentration-response lines thus obtained for each of
`the four agents is shown in Fig. 4. The log IC50 values (log M,
`with the 95% CIs in parentheses) accordingly computed for
`the drugs were as follows: valsartan, 27.78 (28.19, 27.51);
`irbesartan, 27.65 (28.02, 27.40); eprosartan, 27.12 (27.37,
`26.86); and losartan, 26.75 (27.00, 26.40). Thus, the log
`IC50 values obtained with valsartan and irbesartan were
`significantly lower than those obtained with eprosartan and
`losartan. Furthermore, on translation into IC50 values (16.6
`nM, valsartan; 22.4 nM, irbesartan; 75.9 nM, eprosartan;
`177.8 nM, losartan) these values reveal that valsartan is 4.6
`and 10.7 times as potent as eprosartan and losartan, respec-
`tively, in inhibiting the prejunctional actions of Ang II (1027
`M).
`
`Discussion
`Previous studies with atrial and other sympathetically in-
`nervated tissues incubated with [3H]NE have shown that
`[3H]metabolites mainly constitute the spontaneous (resting)
`outflow of radioactivity, whereas intact [3H]NE accounts al-
`most entirely for the SI outflow of radioactivity (Angus et al.,
`1984; Rump et al., 1994). Thus, SI outflow of radioactivity
`from tissues preincubated with [3H]NE is frequently used as
`an index of NE release from sympathetic nerves (Fuder and
`Muscholl, 1995). The observation in this study that the frac-
`tional release of radioactivity during the second period of
`stimulation (FR2) is essentially equivalent to that detected
`during the initial stimulus (FR1) is consistent with the ob-
`servations of Chulak et al. (1995) with atrial preparations
`obtained from Wistar rats and subjected to similar treat-
`ment. Thus, the fractional release of radioactivity from the
`tissue remains basically unchanged during two periods of
`stimulations spaced 35 min apart even though the resting
`outflow of radioactivity from the tissue declines somewhat,
`thereby emphasizing the stability of the preparation. The
`expression of the SI efflux as a fraction of the radioactivity in
`the tissue clearly adds to the precision of the index and
`facilitates comparison of releases during consecutive periods
`of stimulation.
`The observation that desipramine, a tricyclic antidepres-
`sant known to inhibit the neuronal uptake of NE (Franco et
`al., 1976), caused a significant increase in the radioactive
`content of the effluent from electrically stimulated tissues
`demonstrates the ability of the in vitro assay system to detect
`alterations in the overflow of the released neurotransmitter
`after pharmacological interventions. The use of the prepara-
`tion for assessing the effects of agents on sympathetic neu-
`rotransmission was further affirmed by using substances
`known to modulate release of NE. There is compelling evi-
`dence indicating that the release of NE from sympathetic
`nerve terminals is modulated by endogenous or exogenous
`substances acting at receptor sites associated with the nerve
`terminals (Westfall, 1977; Fuder and Muscholl, 1995). The
`prejunctional receptors at peripheral neuroeffector sites,
`
`182
`
`Shetty and DelGrande
`
`TABLE 1
`Effect of a 20-min treatment with the indicated agent (1026 M) on
`stimulation-induced outflow of radioactivity from rat atria
`FR1 and FR2 are the fractional releases of radioactivity evoked by the two electrical
`stimuli and are calculated by subtracting the resting radioactivity from the total
`radioactivity content of the 5-min samples collected during each of the two stimula-
`tion periods (S1, S2) and expressed as a percentage of the total tissue radioactivity at
`that time. The exposure of the tissue to the indicated agent (dissolved in PSS)
`commenced 20 min before S2 and continued for the remainder of the experiment.
`Results are expressed as mean 6 S.E.
`
`Treatment
`
`% FR2/FR1
`
`% Control
`
`n
`
`104.13 6 4.97
`3
`Control
`233.57 6 19.43a
`Desipramine
`3
`113.29 6 8.26
`6
`Control
`68.14 6 4.88a
`6
`Oxymetazoline
`96.01 6 8.36
`6
`Control
`138.82 6 16.14a
`6
`144.6
`Fenoterol
`a Significantly different from the corresponding value obtained with control group
`(t test, P , .05).
`
`224.3
`
`60.1
`
`blockers (1026 M) for 20 min before S1 inhibited the subse-
`quent Ang II (1027 M)-induced augmentation of the SI re-
`lease of [3H]NE (Fig. 3A). The percent inhibition of the Ang II
`response ranged between 73.2% (losartan) and 92.7% (val-
`sartan). Inclusion of lower concentrations of the AT1 receptor
`blockers (1027 and 1028 M) in the perfusing solution 20 min
`before S1 also inhibited the Ang II response. Although all four
`compounds attenuated the Ang II-mediated facilitatory ef-
`fects, the inhibition produced by eprosartan and losartan did
`not attain statistical significance at these lower concentra-
`tions (Fig. 3, B and C). Furthermore, the percent inhibition of
`the Ang II response by the four compounds at each of the
`three concentrations tested (1026, 1027, and 1028 M) re-
`tained the same rank order of activity: valsartan . irbesar-
`tan . eprosartan . losartan. Valsartan, the principal focus
`of this study, was further tested in tissues challenged with a
`10-fold higher concentration of the agonist. Under these con-
`ditions, valsartan (1026 M) effected a 40.3% inhibition (68.5
`versus 40.9%; augmentation of the SI outflow of radioactivity
`by Ang II in the absence and presence of valsartan, respec-
`tively) of the response elicited by Ang II (1026 M, n 5 6).
`The effects of the Ang II receptor blockers on the control
`responses were ascertained by including the agents (1026 M
`each) in the perfusion solution before S1 as before while
`omitting the subsequent application of Ang II before S2. The
`fractional release of radioactivity during S2 relative to that
`during S1 (% FR2/FR1) remained unaltered (104.4 6 12.1,
`DMSO, n 5 5; 100.4 6 3.3, losartan, n 5 6; 104.6 6 2.9,
`eprosartan, n 5 6; 102.1 6 2.4, irbesartan, n 5 6; 104.6 6 1.4,
`valsartan, n 5 6) despite exposure of the tissues to each of the
`four agents for an additional 35 min. The fractional releases
`of radioactivity (FR1) during S1 were also unaffected by the
`20-min pretreatment with the drugs (Table 2). The effects of
`the AT1 receptor blockers on SI overflow of radioactivity were
`further explored by including a 10-fold higher concentration
`(1025 M) of the agents in the perfusion solution 20 min before
`S2. This protocol enabled comparison of the SI efflux in the
`absence and presence of the agent in the same tissue. There
`was again no statistically significant difference between com-
`pound-treated and vehicle-treated tissues regarding the ratio
`of the fractional releases of radioactivity (Table 3), indicating
`that the Ang II receptor blockers do not alter either the
`resting or the basal SI efflux (in the absence of added Ang II)
`of radioactivity.
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`AT1 Receptor Blockers and Prejunctional Actions of Ang II
`
`183
`
`Fig. 2. Effect of Ang II (1029 to 1026 M) on
`the stimulation-induced efflux of radioactive
`NE from [3H]NE-labeled isolated left atria of
`rat. The tissues were electrically stimulated
`twice at 35-min intervals. Ang II was intro-
`duced into the perfusing solution 20 min be-
`fore the second period of stimulation. Col-
`umns represent the mean 6 S.E. from 6 to 12
`determinations. *P , .05, compared with con-
`trol response (ANOVA followed by Dunnett’s
`test).
`
`which have been most thoroughly studied, are the inhibitory
`a2- and the facilitatory b2-adrenoceptors. Application of the
`a2 agonist oxymetazoline or the b2 agonist fenoterol to rat
`atria loaded with [3H]NE was found in this study to decrease
`or increase, respectively, the release of NE on electrical stim-
`ulation. These results are consistent with those reported by
`Abadie et al. (1996) with human atrial appendages subjected
`to similar treatment. Thus superfused rat atrial prepara-
`tions, when used as indicated in this report, provide a stable
`and reliable in vitro model system for studying modulations
`of sympathetic neurotransmission.
`The maximal augmentation (60%) in the SI efflux observed
`in this study with Ang II is consistent with the values re-
`ported with the peptide in other sympathetically innervated
`tissues (Brasch et al., 1993; Cox et al., 1995). The observed
`lack of any attenuation of the response on increasing the
`concentration of the peptide to 1027 or 1026 M, however, is in
`contrast to studies with other preparations wherein a de-
`creased augmentation of the response was seen with supra-
`maximal concentrations of Ang II (Cox et al., 1996; Guima-
`raes et al., 1998). The apparent resistance of the isolated rat
`left atrial preparation to any tachyphylaxis or desensitiza-
`tion on exposure to Ang II under these experimental condi-
`tions makes the preparation especially suitable for delinea-
`tion of
`the effects of AT1
`receptor blockers on the
`prejunctional actions of Ang II.
`
`The observed inhibition of the Ang II responses by all four
`Ang II receptor blockers tested suggests that the enhance-
`ment of sympathetic neuroeffector transmission in the rat
`heart by the peptide entails activation of AT1 receptors. Sim-
`ilar inferences have also been drawn from studies using
`human atrial tissues (Munch et al., 1996; Rump et al., 1998).
`Thus, facilitation of neuronal NE release by Ang II acting via
`prejunctional AT1 receptors apparently is a phenomenon ev-
`ident across diverse species. The observation that the frac-
`tional releases of radioactivity during the two consecutive
`
`TABLE 2
`Fractional release of radioactivity on electrical stimulation of tissues
`pretreated for 20 min with DMSO (0.01%, v/v) or the indicated AT1
`angiotensin II receptor blocker (1026 M)
`FR1 is the fractional release of radioactivity evoked by the electrical stimulus and is
`calculated by subtracting the resting radioactivity from the total radioactivity con-
`tent of the 5-min sample collected during the stimulation period and expressed as a
`percentage of the total tissue radioactivity at that time. The exposure of the tissues
`to the vehicle (DMSO) or the indicated agent commenced 20 min before S1 and
`continued for the rest of the experiment. Results are expressed as mean 6 S.E. The
`differences in the values obtained with tissues treated with DMSO and losartan,
`eprosartan, irbesartan, or valsartan were not statistically significant (Dunnett’s test).
`
`Treatment
`
`DMSO
`Losartan
`Eprosartan
`Irbesartan
`Valsartan
`
`FR1
`0.54 6 0.05
`0.64 6 0.06
`0.61 6 0.05
`0.54 6 0.03
`0.53 6 0.05
`
`n
`
`10
`11
`11
`11
`11
`
`BIOCON PHARMA LTD (IPR2020-01263) Ex. 1004, p. 005
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`184
`
`Shetty and DelGrande
`
`Vol. 294
`
`Fig. 3. Effects of losartan, eprosartan, irbe-
`sartan, and valsartan (1026 M, A; 1027 M, B;
`1028 M, C) on Ang II (1027 M)-evoked aug-
`mentation of the stimulation-induced efflux
`of radioactive NE from [3H]NE-labeled iso-
`lated rat left atrial preparation. The atria
`were electrically stimulated twice at 35-min
`intervals. The AT1 receptor blockers or vehi-
`cle (DMSO) was included in the perfusing
`solution 20 min before the first period of stim-
`ulation. Ang II was included in the perfusing
`solution 20 min before the second period of
`stimulation in the continued presence of the
`drugs or DMSO. Data represent the mean 6
`S.E. from 5 to 11 determinations. *P , .05,
`compared with control Ang II response
`(ANOVA followed by Dunnett’s test).
`
`TABLE 3
`Stimulation-induced outflow of radioactivity from tissues treated for 20
`min with DMSO (0.1%, v/v) or the indicated AT1 angiotensin II
`receptor blocker (1025 M)
`FR1 and FR2 are the fractional releases of radioactivity evoked by the two electrical
`stimuli and are calculated by subtracting the resting radioactivity from the total
`radioactivity content of the 5-min samples collected during each of the two stimula-
`tion periods (S1, S2) and expressed as a percentage of the total tissue radioactivity at
`that time. The exposure of the tissues to DMSO (vehicle) or the indicated agent
`commenced 20 min before S2 and continued for the rest of the experiment. Results
`(mean 6 S.E.) obtained with two sets of experiments (DMSO/valsartan and DMSO/
`losartan/eprosartan/irbesartan) conducted separately are shown. The differences in
`the values obtained with tissues treated with DMSO or valsartan were not statisti-
`cally significant (Student’s t test). Similarly, the differences in the values obtained
`with tissues treated with DMSO and losartan, eprosartan, or irbesartan were also
`not statistically significant (Dunnett’s test).
`
`Treatment
`
`DMSO
`Valsartan
`DMSO
`Losartan
`Eprosartan
`Irbesartan
`
`% FR2/FR1
`
`118.11 6 5.84
`121.15 6 3.38
`105.73 6 1.37
`119.25 6 6.12
`95.77 6 3.57
`107.51 6 2.98
`
`n
`
`6
`5
`3
`4
`4
`4
`
`periods of stimulation spaced 35 min apart remained con-
`stant despite continued presence of the drugs indicates that
`the observed inhibition of the Ang II response by the drugs is
`not a consequence of any time-dependent attenuation of the
`basal SI efflux by the agents masking the Ang II response.
`This inference was reinforced by observations that inclusion
`of high concentrations of the agents (1025 M) between the
`two periods of stimulation (S1, S2) also does not alter the
`basal SI efflux. Furthermore, the observation that fractional
`releases (FR1) during S1 are similar across all groups treated
`with vehicle or drug for 20 min reiterates that the agents per
`se do not alter the basal SI efflux but that they selectively
`inhibit facilitation of the response by Ang II. These conclu-
`sions are in consonance with those drawn by Foucart et al.
`(1996) after their examination of the effects of losartan in the
`isolated rat atria.
`Although all four Ang II receptor blockers that we tested
`inhibited the prejunctional actions of Ang II in the rat atria,
`significant differences were noted in their relative potencies
`to effect this action. The log IC50 values computed from the
`
`BIOCON PHARMA LTD (IPR2020-01263) Ex. 1004, p. 006
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`Downloaded from
`
`jpet.aspetjournals.org
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` at ASPET Journals on April 28, 2017
`
`2000
`
`AT1 Receptor Blockers and Prejunctional Actions of Ang II
`
`185
`
`Fig. 4. Regression analysis of the data pre-
`sented in Fig. 3. The proportion inhibition of the
`Ang II response in each individual tissue ex-
`posed to the receptor blocker was determined by
`subtracting the individual response from the
`average response seen in the presence of Ang II
`alone and by dividing that value by the average
`increase effected by Ang II relative to the aver-
`age basal response. Figure represents the pro-
`portion inhibition of the Ang II-mediated re-
`sponse by each of the four AT1 receptor blockers
`across the indicated concentration range. The
`regression lines were plotted and rendered par-
`allel as described in Results. The dotted line
`indicates a proportion inhibition level of 0.5.
`The symbols for valsartan, eprosartan, and lo-
`sartan at each of the three concentrations tested
`have been shifted slightly from their precise x
`coordinates to prevent their overlap and to help
`discern all data points.
`
`concentration-response relationships of the individual agents
`indicated that valsartan and irbesartan are significantly
`more potent than eprosartan and losartan in inhibiting the
`prejunctional facilitatory actions of Ang II. The potency dif-
`ference between valsartan and irbesartan, however, did not
`attain statistical significanc