103
`
`Desensitization of Adenylate Cyclase and
`Increase of Gia in Cardiac Hypertrophy Due
`To Acquired Hypertension
`
`Michael Bohm, Peter Gierschik, Andreas Knorr, Katharina Larisch,
`Korinna Weismann, and Erland Erdmann
`
`The present study investigated whether reduced adenylate cyclase activity and an increase in inhibitory
`guanine nucleotide binding proteins (G^), which have been observed in the failing human heart, already
`occur in myocardial hypertrophy before the stage of heart failure. In membranes of hypertrophic hearts
`from rats with different forms of experimentally induced hypertension without heart failure (one-kidney,
`one clip rats, deoxycorticosterone-treated rats, and rats with reduced renal mass), basal as well as
`isoprenaline-, 5'-guanylylimidodiphosphate-, and forskolin-stimulated adenylate cyclase activity was
`reduced. The activity of the catalyst was depressed in deoxycorticosterone but unchanged in one-kidney,
`one clip and reduced renal mass compared with controls. The number of 0-adrenergic receptors was
`similar in all groups. Radioimmunological quantification of G^, proteins revealed an increase by 73% in
`one-kidney, one clip, 67% in reduced renal mass, but only 20% in deoxycorticosterone compared with
`sham-operated, age-matched control rats. The increase of G^, was accompanied by smaller changes of
`pertussis toxin-induced [uP]ADP-ribosylation of a 40-lid membrane protein. It is concluded that Gu,
`contributes to the reduced adenylate cyclase activity in cardiac hypertrophy in one-kidney, one clip and
`reduced renal mass and to a smaller extent in deoxycorticosterone. It is suggested that an enhanced
`expression of Gt, could occur not only in severe heart failure but also in cardiac hypertrophy and could,
`therefore, contribute to myocardial depression and progression of disease in heart failure. In addition, G u
`might represent an important regulatory mechanism for cardiac adenylate cyclase activity and thus, might
`play an important role in various cardiac diseases. (Hypertension 1992^20:103-112)
`KEY WORDS • experimental hypertension • hypertrophy • adenyl cyclase • guanine nucleotide
`regulatory protein • adrenergic receptors • cardiomyopathy • heart failure
`
`I n the failing human heart desensitization of adenyl-
`
`ate cyclase occurs, which leads to blunted effects
`of cyclic AMP (cAMP)-dependent positive ino-
`tropic agents and endogenous catecholamines.1-4 This
`desensitization, which also impairs the effects of non-
`/3-sympathomimetic cAMP-dependent positive inotro-
`pic agents,2-4'6 has been attributed to a decline in the
`number of /3-adrenergic receptors1"6 and to an increase
`of pertussis toxin substrates.3-4-7 However, these data
`were obtained in human myocardium from terminally
`failing hearts obtained at heart transplantation. Hith-
`erto, it has been unknown whether an increase of
`inhibitory guanine nucleotide binding proteins (G^)
`also occurs earlier during the development of cardiomy-
`opathy, namely, in cardiac hypertrophy.
`Myocardial hypertrophy has been regarded as an
`adaptive process to reduce wall stress when an in-
`
`From the Medizinische Klinilc I der Universitat Munchen
`(MB., K.L., K.W., E.E.), Klinikum Groflhadern, Munich; Phar-
`maiologisches Institut (P.G.), Universitat Heidelberg; and Bayer
`AG (A.K.), Leverkusen, Germany.
`Supported by the Deutsche Forschungsgemeinschaft (MB).
`Address for reprints: Michael Bohm, MD, Medizinische Klinilc
`I der Universitat Munchen, Klinikum GroBhadern, Marchionin-
`istr. 15, 8000 Munchen 70, Germany.
`Received October 14, 1991; accepted in revised form February
`7, 1992.
`
`creased pressure load is imposed on the myocardium.8
`Hypertrophy has been suggested to be one of the initial
`steps that reduces the contractility of myocardium in
`the development of heart failure.910 However, it is
`unclear whether changes of /3-adrenergic receptors or
`GJO proteins occur in the hypertrophied human heart
`before the onset of heart failure. Since viable human
`myocardial tissue with hypertrophy is not available, we
`investigated different forms of experimentally induced
`hypertensive cardiomyopathy in rats. In renal hyperten-
`sive rats and in deoxycorticosterone (DOCA) rats, a
`reduction of /3-adrenergic receptor-stimulated adenyl-
`ate cyclase has been reported.11-13 The data on the
`density of ^-adrenergic receptors are controversial. In
`DOCA rats, some investigators observed a decline of
`the number of 0-adrenergic receptors,11 whereas others
`observed no changes.14 In renal hypertensive rats, in-
`creases1516 as well as decreases12-13 or no changes14-17 of
`/3-adrenergic receptors have been observed. Some in-
`vestigators hypothesized that defects of G protein-
`effector coupling mechanisms (e.g., changes of G,,,
`proteins or the catalyst) could occur in cardiac hyper-
`trophy due to acquired forms of hypertension and could
`be independent from /3-adrenergic receptors.14-19 How-
`ever, experimental data on GL, changes in support of
`this suggestion are lacking.
`
`Downloaded from http://ahajournals.org by on September 22, 2020
`
`NOVARTIS EXHIBIT 2002
`Biocon v. Novartis, IPR2020-01263
`Page 1 of 10
`
`

`

`104
`
`Hypertension Vol 20, No 1 July 1992
`
`The present study investigated whether changes of
`adenylate cyclase similar to those observed in the failing
`human heart, namely, heterologous adenylate cyclase
`desensitization involving G^ proteins, occur in the hy-
`pertrophied myocardium of rats with different forms of
`acquired hypertension before the development of heart
`failure. Thus, we studied one-kidney, one clip (1K1C)
`renal hypertensive rats, DOCA-treated one-kidney rats,
`and rats with reduced renal mass (RRM). Changes of
`cardiac adenylate cyclase activity were characterized and
`related to the amount of a 40-kd pertussis toxin substrate
`and the amount of expressed G^ proteins. G^ proteins
`were studied using a novel radioimmunochemical
`method with the iodine-125-radiolabeled synthetic
`C-terminal decapeptide of retinal transducin a (KEN-
`LKDCGLF), which has been described recently.20 Car-
`diac /3-adrenergic receptors were studied for comparison.
`
`Methods
`Wistar-Hagemann rats (n=62) were purchased from
`Hagemann (Miinster, Germany) and were divided into
`four groups. Rats in the 1K1C group underwent clip-
`ping of the right renal artery with a clip 0.18 mm in
`diameter. One week later nephrectomy of the left
`kidney was performed. In the second group (DOCA
`rats), DOCA pellets (50 mg) were implanted subcuta-
`neoush/ and the left kidney was extirpated. These
`animals were given 1% NaCl for drinking water. In the
`third group (RRM rats), the upper and lower pole of
`the left kidney was dissected. One week later, the
`animals underwent nephrectomy on the right side.
`These procedures resulted in a reduction of renal mass
`by about 68-70%. RRM rats were given 0.5% NaCl for
`drinking water. All control animals underwent sham
`operations. Experiments were conducted 4-7 weeks
`after the operations were performed. In all experimen-
`tal animals, hypertension was established for 2 weeks.
`Blood pressure was measured with the tail-cuff method
`according to Pfeffer et al.21 The last measurement was
`taken immediately before the rats were killed.
`
`Adenylate Cyclase Determination
`Paniculate washed membrane fractions (10,000g sed-
`iment) were prepared according to the method of Kruse
`and Scholz22 from homogenates of rat hearts. The
`activity of adenylate cyclase was determined in a reac-
`tion mixture containing 50 jtmol/1 f^Ja-ATP (approx-
`imately 0.3 jiCi/100 /il), 50 mmol/1 triethanolamine-
`HC1, 5 mmol/1 MgCl2, 100 /xmol/1 EGTA, 1 mmol/1
`3-isobutyl-l-methylxanthine, 5 mmol/1 creatine phos-
`phate, 0.4 mg/ml creatine kinase, and 0.1 mmol/1 cAMP
`at pH 7.4 in a final volume of 100 fi\. The mixture was
`preincubated for 5 minutes at 37°C. The reaction was
`started by addition of the membrane suspension (30 yjg
`per tube) and was continued for another 20 minutes at
`the same temperature. Reactions were stopped by the
`addition of 500 /tl of 120 mmol/1 zinc acetate. cAMP
`was purified by chromatography on neutral alumina.
`After centrifugation for 5 minutes at 10,000g, 0.8 ml of
`the supernatant was applied on neutral alumina col-
`umns equilibrated with 0.1 mmol/1 Tris/HQ, pH 7.5.
`The effluent was collected, and the [^JcAMP was
`determined by measuring radioactivity in a liquid scin-
`tillation spectrometer.
`
`Radioligand Binding Experiments
`Rat hearts were chilled in 30 ml ice-cold homogeni-
`zation buffer (10 mmol/1 Tris-HCl, 1 mmol/1 EDTA,
`dithiothreitol 1 mmol/1, pH 7.4). Connective tissue was
`trimmed away, myocardial tissue was minced with scis-
`sors, and membranes were prepared with a motor-
`driven glass-Teflon homogenizer for 1 minute. After-
`ward, the membrane preparation was homogenized by
`hand for 1 minute with a glass-glass homogenizer. The
`homogenate was spun at 484g (rotor, Beckman JA 20)
`for 10 minutes. The supernatant was filtered through
`two layers of cheesecloth, diluted with an equal volume
`of ice-cold 1 mol/1 KC1, and stored on ice for 10
`minutes. This suspension then was centrifuged at
`100,000g for 30 minutes. For radioligand binding exper-
`iments, the pellet was resuspended in 50 vol incubation
`buffer (50 mmol/1 Tris-HCl, 10 mmol/1 MgCl2, pH 7.4)
`and homogenized for 1 minute with a glass-glass homog-
`enizer. This suspension then was recentrifuged at
`100,OOQt» for 45 minutes. The final pellet was resus-
`pended in incubation buffer (50 vol) and was stored at
`-70°C. Storage did not alter the results.
`The radioligand binding assays were performed in a
`total volume of 250 fi\ incubation buffer. The incubation
`was carried out at 37°C for 60 minutes. These conditions
`allowed for complete equilibration of the receptors with
`the radioligand. The reaction was terminated by rapid
`vacuum filtration through Whatman GF/C filters, and
`filters were immediately washed three times with 6 ml
`each of ice-cold incubation buffer. All experiments were
`performed in triplicate. Myocardial /3-adrenergic recep-
`tors were studied using l25I-cyanopindolol as radiola-
`beled ligand. Specific activity was 2,000 Ci/mmol; 3
`^.mol/1 (-)-propranolol was used to determine nonspe-
`cific binding.
`
`Pertussis Toxin-induced [32P]ADP Ribosylation
`[^PJADP-ribosylation of G^ by pertussis toxin was
`performed for 1 hour at 37°C in a volume of 50 fi\
`containing 100 mmol/1 Tris/HCl, pH 8.0 at 20°C, 25
`mmol/1 dithiothreitol, 2 mmol/1 ATP, 1 mmol/1 GTP, 50
`nmol/1 ["PJNAD (800 Ci/mmol), and 20 Mg/ml pertussis
`toxin that had been activated by incubation with 50
`mmol/1 dithiothreitol for 1 hour at 20°C before the
`labeling reaction. Optimal labeling conditions were
`achieved when 0.5% (vol/vol) Lubrol PX was present in
`the assay medium. Samples were subjected to sodium
`dodecyl sulfate-polyacrylamide gel electrophoresis
`(SDS-PAGE) (10% [wt/vol] acrylamide, 16 cm total gel
`length). Gels were stained with Coomassie blue and
`dried before autoradiography was performed.
`
`Immunoblotting Techniques
`Immunoblotting techniques were performed according
`to Gierschik et al.23 The retinal transducin a-subunits
`were purified from bovine rod outer segments as described
`elsewhere.2*24 The polyclonal antiserum (DS 4) was raised
`in rabbits against the C-terminal decapeptide of retinal
`transducin a KENLKDCGLF coupled to keyhole limpet
`hemocyanine as described by Goldsmith et al.25. Under
`the conditions used, this serum was strongly reactive
`against a,, a,u and eta but weakly reactive against OQ and
`a,,. The electrophoretic transfer and immunostaining
`methods have been described previously.20
`
`Downloaded from http://ahajournals.org by on September 22, 2020
`
`NOVARTIS EXHIBIT 2002
`Biocon v. Novartis, IPR2020-01263
`Page 2 of 10
`
`

`

`Bohrn et al Adenylate Cydase in Acquired Hypertension
`
`105
`
`TABLE 1. Systolic Blood Pressure, Bod; Weight, and Cardiac Hypertrophy Expressed in Heart Weight and Heart
`Weight-to-Body Weight Ratio in One-Kidney, One Clip Rats, Deoxycorticosterone-Treated Rats, Rats With
`Reduced Renal Mass, and Control Rats
`Control
`(» = H)
`96.4±3.6
`260.9±10.8
`604.8+19.0
`
`Measurements
`Systolic BP (mm Hg)
`Body weight (g)
`Heart weight (mg)
`Heart weight/body weight
`3.6±0.2*
`4.4±03'
`4.4±0.1*
`2.3±0.1
`ratio (mg/g)
`1K1C, one-kidney, one clip rats; DOCA, deoxycorticosterone rats; RRM, reduced renal mass rats; BP, blood
`pressure.
`*p<0.01,1p<0.05, significantly different from control.
`
`1K1C
`("=6)
`222±6.1'
`235 ±9.7
`l,032±21.0*
`
`DOCA
`(«-6)
`204.8±6.8*
`213.3±11.0*
`925.2±43.9*
`
`RRM
`(«=«)
`207.8±4.5*
`218.3±9.0*
`770.0±44.0t
`
`Iodination of C-Terminal Synthetic Peptide and
`Radioimmunoassay
`The C-terminal synthetic peptide was iodinated by
`conjugation to the iodine-125-labeled acylating agent
`N-succinimidyl 3-(4-hydroxy, 5-[125I]-iodophenyl) propi-
`onate according to Bolton and Hunter26 and modified as
`described recently.20 The assay was performed in a final
`volume of 80 y\, containing 20 y\ radiolabeled peptide
`diluted to 10,000 cpm/tube in 50 mmol/1 Na-phosphate
`buffer with 0.25% gelatine at pH 7.5, 20 yi antiserum
`dilution (1:100), and 40 yX solubilized membranes,
`transducin a in 50 mmol/1 Na-phosphate buffer at pH
`7.5 or solubilization buffer instead of membranes. Stan-
`dard curves were constructed by using 0.25-25 y.glm\
`retinal transducin OL The assay was performed at 4°C for
`18 hours. For precipitation of immune complexes, sam-
`ples were supplemented with 30 yX staphylococcal pro-
`tein A suspension and incubated for 30 minutes on ice.
`Details have been described elsewhere.20
`
`Miscellaneous
`Protein was determined according to Lowry et al27
`using bovine serum albumin as standard. SDS-PAGE
`was performed as described by Lammli.28 5'-Nucleotid-
`ase activity was investigated with the method of Dixon
`and Purdom.29 Human leukemia (HL-60) cells were
`grown in suspension culture and were induced to dif-
`ferentiate into mature myeloid forms by cultivation in
`the presence of 1.25% (vol/vol) dimethyl sulfoxide for 5
`days. Details of cultivation and membrane preparations
`are described elsewhere.30
`
`Materials
`Forskolin was donated by Dr. Metzger (Hoechst AG,
`Frankfurt, Germany). Carbachol was from Sigma Chem-
`ical Co., Deisenhofen, Germany. Guanosine 5'-triphos-
`phate, 5'-guanyrylimidodiphosphate [Gpp(NH)p], aden-
`osine 5'-triphosphate, creatine phosphate, and creatine
`kinase were purchased from Boehringer-Mannheim
`(Germany), and isobutylmethylxanthine was purchased
`from EGA-Chemie, Steinheim, Germany. The ligand
`l25I-Cyp was from Amersham-Buchler, Braunschweig,
`Germany. Dithiothreitol was from Serva, Heidelberg,
`Germany. Pertussis toxin was from List Biological Labo-
`ratories, Campbell, Calif.). Antiserum (DS 4) was raised
`against the C-terminal decapeptide of bovine retinal
`transducin a. Bolton and Hunter reagent for peptide
`iodination (Af-succinimidyl 3-(4-hydroxy, S-^IJiodophe-
`nyl) propionate, specific activity 2,000 Ci/mmol) was
`purchased from Amersham-Buchler. The C-terminal
`
`decapeptide of transducin a was kindly provided by Prof.
`Dr. U. Weber, Tubingen, Germany. Immunoprecipitin
`(staphylococcal protein A adsorbant) suspension was
`from Gibco/BRL, Eggenstein, Germany. All other com-
`pounds used were of analytical or best grade commer-
`cially available. Only deionized and twice-distilled water
`was used throughout.
`
`Statistics
`The data shown are mean±SEM. Statistical signifi-
`cance was estimated with Student's / test for unpaired
`observations and analysis of variance according to Wal-
`lenstein et al.31 A value of p<0.05 was considered
`significant. Ko values and the drug concentration pro-
`ducing 50% of the maximal effect (EQo) were deter-
`mined graphically in each individual experiment.
`
`Results
`Blood Pressure and Myocardial Hypertrophy
`As shown in Table 1, systolic blood pressure was
`elevated similarly above 200 mm Hg in all experimental
`groups of acquired hypertension. Consistently, heart
`weight and heart weight-to-body weight ratio were
`increased in all groups with hypertension compared
`with controls. In all groups, there was a decrease of
`body weight. The decline in weight was significant and
`stronger in DOCA and RRM than in 1K1C rats.
`
`Adenylate Cyclase Activity
`Concentration-response curves for isoprenaline sum-
`marize the effects of ^-adrenergic receptor stimulation
`on cardiac adenylate cyclase activity (Figure 1, left
`panel). In either group with experimentally acquired
`hypertension, the ability of isoprenaline to stimulate
`adenylate cyclase activity was reduced. This reduction
`was more pronounced in DOCA than in 1K1C or RRM
`rats. To investigate whether the effects of agents bypass-
`ing the /J-adrenergic receptor-mediated stimulation of
`adenylate cyclase are sustained, we investigated the
`poorly hydrolyzable guanine nucleotide analogue Gpp-
`(NH)p and the diterpene derivative forskolin. The
`effects of both Gpp(NH)p (Figure 1, middle panel) and
`forskolin (Figure 1, right panel) were reduced com-
`pared with control rats. As with isoprenaline, the de-
`cline of the effectiveness of Gpp(NH)p and forskolin
`was strongest in DOCA rats as compared with 1K1C
`and RRM rats. One general problem for the determi-
`nation of adenylate cyclase activity in hypertrophied
`myocardium was to control the preparation for the
`amount of membranes. To overcome this potential
`
`Downloaded from http://ahajournals.org by on September 22, 2020
`
`NOVARTIS EXHIBIT 2002
`Biocon v. Novartis, IPR2020-01263
`Page 3 of 10
`
`

`

`106
`
`Hypertension Vol 20, No 1 July 1992
`
`I 200
`
`So
`
`• CM
`
`"S• £
`a o
`
`£100si
`
`6 0 0
`
`4 0 0
`
`200
`
`800
`
`600
`
`4 0 0
`
`200
`
`o-o
`
`CONTROL
`(n»5>
`
`1C-1K
`(n-5/8)
`
`DOCA
`(n-5)
`
`RRM
`(n»6/6)
`
`100
`10
`1
`0 0.01 0.1
`Concentration of Isoprenaline (pmol/l)
`
`100
`10
`1
`0 0.01 0.1
`Concentration of Qpp(NH)p (pmol/l)
`
`100
`10
`1
`0 0.01 0.1
`Concentration of forskolin (jjmol/l)
`
`FIGURE 1. Line plots show concentration-dependent effects of isoprenaline (left panel, 0.01-100 fimol/l), 5'-guanytylimido-
`diphosphate [Gpp(NH)p] (middle panel, 0.01-100 fimol/l), and forskolin ("right panel, 0.01-100 fimol/l) on cardiac adenylate
`cyclase activity in one-kidney, one clip (1C-1K) rats, deoxycorticosterone-treated rats (DOCA), and rats with reduced renal mass
`(RRM). Experiments in the control rats (control) are shown in all panels for comparison. cAMP, cyclic AMP.
`
`pitfall, we related adenylate cyclase activity to both
`membrane protein and 5'nucleotidase activity as mem-
`brane marker enzyme. As summarized in Table 2, basal
`adenylate cyclase activities were similarly reduced in the
`experimental hypertensive animals when the data are
`related to milligrams membrane protein or to 5'nucle-
`otidase activity (Table 2).
`To study whether the reduced adenylate cyclase
`activity is due to defective G protein-effector coupling,
`enzyme activity was studied in the presence of 5 mmol/1
`MnCl2. Manganese ions are known to uncouple the
`catalytic subunit from guanine nucleotide regulatory
`sites.32-33 Thus, under these conditions the activity of the
`catalyst itself can be predominantly studied. Figure 2
`summarizes the data. In 1K1C and in RRM rats,
`adenylate cyclase activity in the presence of MnCl2 was
`not different compared with controls. This holds true
`for basal adenylate cyclase activity, adenylate cyclase
`stimulated with forskolin, and forskolin plus Gpp-
`(NH)p. Addition of Gpp(NH)p did not further increase
`the effect of forskolin, indicating that in the presence of
`MnCl2, only the effect of forskolin on the catalyst is
`studied. In contrast, in DOCA rats adenylate cyclase
`activity assayed in the presence of MnCl2 was reduced
`compared with control rats and also compared with
`1K1C and RRM rats. Similar results were obtained
`when the data were related to milligrams membrane
`protein or to 5'nucleotidase activity (Table 2). In
`DOCA the adenylate cyclase activity in the presence of
`MnCl2 was also not further increased by forskolin plus
`Gpp(NH)p compared with forskolin alone. These data
`show that the catalytic subunit was completely uncou-
`pled from the influences of guanine nucleotide-acti-
`vated G proteins. It is depressed in DOCA but not in
`RRM and 1K1C rats.
`
`Cardiac (1-Adrenergic Receptors
`Saturation experiments for 125I-Cyp binding to cardiac
`membranes showed that binding of the radiolabeled
`ligand was monophasic and saturable (not shown). Table
`3 summarizes the data. There were no differences in the
`numbers of cardiac /3-adrenergic receptors between
`1K1C, DOCA, RRM, or control rats. The data were
`similar when the receptor density was related to milli-
`grams membrane protein or to 5'nucleotidase activity.
`
`Gia Proteins
`Figure 3 shows a typical autoradiogram for the per-
`tussis toxin-induced ["P]ADP ribosylation of a 40-kd
`protein in rat myocardial membranes. This protein
`comigrated with the 40-kd pertussis toxin substrate G^,
`G w from human leukemia (HL-60) cells and with Gi/Go
`purified from bovine brain. Pertussis toxin labeling was
`enhanced in myocardial membranes from 1K1C and
`RRM rats and only slightly increased in DOCA com-
`pared with control. Figure 4 summarizes the data for all
`groups studied. There was a significant increase of
`pertussis toxin substrates only in 1K1C and RRM rats,
`whereas no significant increase in DOCA rats was
`detected.
`To study whether the increase of pertussis toxin
`substrates in 1K1C and RRM rats is due to an enhanced
`expression of G^ proteins or whether it is due to other
`factors affecting the pertussis toxin-catalyzed ADP-
`ribosylation reaction, we developed a radioimmunoas-
`say to directly quantify the amounts of G^ proteins in
`rat cardiac membranes. DS 4 antiserum was raised in
`rabbits against the C-terminus of retinal transducin a.
`Figure 5 shows that the antiserum was capable to
`immunostain a 40-kd membrane protein in rat cardiac
`membranes. Immunostaining of the 40-kd band was
`concentration dependency inhibited by the synthetic
`
`Downloaded from http://ahajournals.org by on September 22, 2020
`
`NOVARTIS EXHIBIT 2002
`Biocon v. Novartis, IPR2020-01263
`Page 4 of 10
`
`

`

`Bohrn ex al Adenylate Cyclase in Acquired Hypertension
`
`107
`
`TABLE 2. Adenylate Cyclase Activity Under Basal and Stimulated Conditions in Cardiac Membranes From One-
`Kidney, One Clip Rats, Deoxycorticosterone-Treated Rats, Rats With Reduced Renal Mass, and Control Rats
`Control
`DOCA
`
`1K1C
`
`RRM
`
`Condition
`Related to mg protein
`Basal
`
`Iso (10 fiM)
`
`Gpp(NH)p (100 nM)
`
`Forskolin (30 /iM)
`
`Mnd2
`
`MnCl2+forskolin (30 /iM)
`
`Mnd2+forskolin+Gpp(NH)p
`
`Related to 5'nucleotidase
`(nmol Pi/mg protein • min)
`Basal
`
`Iso (10 jiM)
`
`Gpp(NH)p (100 AIM)
`
`Forskolin (30 MM)
`
`MnCl2
`
`MnCl2+forskolin (30 /iM)
`
`MnQ2+forskolin+Gpp(NH)p
`
`All experiments with MnCl2 were
`costerone rats; RRM, reduced renal
`*p<0.01, tp<0.05.
`
`47.0+4.7
`(8)
`795 ±99.9
`(5)
`723.4±863
`(5)
`914.6±128.1
`(5)
`96.6±19.8
`(6)
`l,025.4±61.3
`(6)
`1,086.0+71.6
`(6)
`
`30.8±6.8
`(9)
`367.1±313t
`(5)
`454.9 ±74.6f
`(6)
`618.8±48.5
`(5)
`87.0±24.6
`(6)
`%9.5±36.1
`(6)
`984.6±38.1
`(6)
`
`18.5+2.7*
`(8)
`226.2+22.2*
`(5)
`272.3+20.6*
`(5)
`277.2+53.1*
`(5)
`55.5+12.0
`(6)
`529.0±603*
`(6)
`594.8±64.9*
`(6)
`
`29.1 ±43t
`(6)
`323.3±6.0t
`(5)
`411.6±66.6t
`(6)
`570.2±77.4
`(5)
`91.6±26.4
`
`1,008.3±25.8
`(6)
`1,089.1 ±55.5
`(6)
`
`1.0±0.2t
`0.5+0.1*
`1.5±0.1
`0.9±0.2f
`(6)
`(9)
`(8)
`(8)
`6.2+0.9*
`25.9±4.0
`11.2±1.7t
`ll.l±0.7f
`(5)
`(5)
`(5)
`(5)
`7.2±0.6*
`23.4±1.9
`14.9±2.6t
`13.0±2.3t
`(5)
`(6)
`(6)
`(5)
`7.5 + 1.4*
`18.5±2.7
`29.0±3.3
`17.8±1.8t
`(5)
`(5)
`(5)
`(5)
`3.3±0.9
`2.6±0.7
`3.2±0.6
`1.4±0.3t
`(6)
`(6)
`(6)
`(6)
`13.6+1.4*
`33±1.6
`29.5 ±1.6
`34.1±3.1
`(6)
`(6)
`(6)
`(6)
`15.2±1.4*
`33.2±0.8
`29.7±1.2
`34.5±4.6
`(6)
`(5)
`(5)
`(6)
`performed without MgCl2; 1K1C, one-kidney, one clip rats; DOCA, deoxycorti-
`mass rats; Iso, isoprenaline; Gpp(NH)p, 5'guanylylimidodiphosphate.
`
`C-terminus of retinal transducin a (KENLKDCGLF).
`DS 4 antiserum was used to quantify G;,, in a radioim-
`munoassay with iodinated retinal transducin a (125I-
`KENLKDCGLF) and retinal transducin a as standard.
`Figure 6 shows competition curves of increasing con-
`centrations of retinal transducin a and membrane ex-
`tracts from human leukemia cells (HL-60) and rat
`myocardial membranes. Transducin a, human leukemia
`cells (HL-60), and rat myocardial membranes were
`capable to concentration dependentry displace DS 4
`antiserum binding to iodinated retinal transducin a
`(1HI-KENLKDCGLF). As can be seen from the stan-
`dard curve with retinal transducin a, the sensitivity of
`the radioimmunoassay was 0.25 ng/ml transducin a.
`The interassay variation was less than 10%. Figure 7
`summarizes the results in the different groups with
`experimental hypertension. In 1K1C and RRM rats,
`there was an increase of G^ proteins by 73% and 67%,
`respectively; in DOCA rats, there was a small albeit
`significant increase by 20% compared with controls.
`Figure 8 shows the correlation of systolic blood pressure
`(left panel) and heart weight-to-body weight ratio
`
`(right panel) to the density of G,,, proteins in control
`and hypertensive animals. It can be seen that in DOCA
`rats, the increase of G^ was less than in 1K1C or RRM
`rats, although elevation of blood pressure and cardiac
`hypertrophy were similar. Taken together, there is a
`substantial increase of expressed G^ proteins in 1K1C
`and RRM rats and a more moderate but significant
`increase in DOCA rats. The increase in Gto proteins
`poorly corresponds to the increase in pertussis toxin
`substrates, which was less pronounced and failed to
`detect the small increase of G^ in DOCA rats.
`
`Discussion
`The results of the present study show that in the
`hypertrophied myocardium from 1K1C and RRM renal
`hypertensive rats as well as that from DOCA rats, there
`is a desensitization of adenylate cyclase activity. The
`desensitization not only affects 0-adrenergic receptor-
`stimulated adenylate cyclase but also Gpp(NH)p- and
`forskolin-stimulated activities. Desensitization of ade-
`nylate cyclase was not accompanied by significant
`changes of myocardial 0-adrenergic receptors. Uncou-
`
`Downloaded from http://ahajournals.org by on September 22, 2020
`
`NOVARTIS EXHIBIT 2002
`Biocon v. Novartis, IPR2020-01263
`Page 5 of 10
`
`

`

`Rat ventricular myocardium
`
`40 kDa
`
`\
`
`FIGURE 3. Sodium dodecyl sidfate-pofyacrylamide gel elec-
`trophoresis (SDS-PAGE) shows [nP]ADP-ribosylation of a
`40-kd membrane protein by pertussis toxin in cardiac mem-
`branes from one-kidney, one clip (1C-1K) rats, deoxycortico-
`sterone (DOCA)-treated rats, rats with reduced renal mass
`(RRM), and control rats. Membranes from human leukemia
`(HL-60) cells and GJGo purified from bovine brain are
`shown for comparison. An autoradiograph of a representative
`experiment is shown. Proteins were separated on a 10%
`SDS-PAGE before autoradiography. Pertussis
`toxin-cata-
`lyzed [^PJADP-ribosylation was performed as described in
`"Methods" with 0.5% (vol/vol) Lubrol PXpresent in the assay
`medium
`to obtain optimal labeling conditions. Note that
`phosphorus-32
`incorporation by pertussis
`toxin was more
`pronounced in 1C-1K and RRM than in DOCA or control
`rats.
`
`colemmal membranes of renal hypertensive rats. There-
`fore, it has been proposed that the decline in adenylate
`cyclase activity could be due to alterations in /3-adren-
`ergic receptor-G protein effector coupling alone1517 or
`in concert with a reduction of /J-adrenergic receptors.18
`Similar data have been obtained with DOCA rats.
`There was a marked reduction of adenylate cyclase
`activity11 but only a marginal decline of the number of
`/3-adrenergic receptors.14 In the present study, desensi-
`tization of receptor-dependent and receptor-indepen-
`dent stimulation of adenylate cyclase activity was ob-
`served in DOCA, 1K1C, and RRM groups. These
`changes were not accompanied by a decline of the
`number of ^-adrenergic receptors (present study). One
`possible explanation for the discrepant findings con-
`cerning the number of /3-adrenergic receptors is the age
`of the animals and the duration of clipping of the renal
`artery. In studies in which a decline in the number of
`
`108
`
`Hypertension Vol 20, No 1 July 1992
`
`1000
`
`• 500
`
`I—I CONTROL
`
`400
`
`3 0 0
`
`£
`
`200
`
`s u
`
`MflCI,
`
`FIGURE 2. Bar graphs show basal adenylate cyclase activity
`and adenylate cyclase activity after stimulation with forskolin
`(30 fimol/l) and forskolin plus 5'-guanyfylimidodiphosphate
`[Gpp(NH)pJ (5 fimol/l) in the presence ofMnCl2 (5 mmoljl)
`in cardiac membranes from one-kidney, one clip (1C-1K)
`rats, deoxycorticosterone (DOCA)-treated rats, and rats with
`reduced renal mass (RRM) as well as control rats. cAMP,
`cyclic AMP.
`
`pling of the catalyst from influences of G proteins by
`manganese ions snowed that the activity of the catalyst
`is unchanged in 1K1C and RRM rats but markedly
`depressed in DOCA rats. Gi,, proteins, measured with a
`novel radioimmunochemical method, were observed to
`be increased by 73% in 1K1C and 67% in RRM but only
`by 20% in DOCA rats. The pertussis toxin-catalyzed
`[*P]ADP-ribosylation of a 40-kd Gia-related membrane
`protein showed smaller differences among the groups
`and only a marginal, insignificant increase in the DOCA
`group.
`In renal hypertensive rats, reduced effects of /3-ad-
`renergic receptor stimulation on adenylate cyclase have
`been observed.11-13151619 Consistently, the positive ino-
`tropic effects of /3-adrenergic receptor agonists were
`observed to be reduced.12 These functional alterations
`have been suggested to be due to a decline of the
`number of 0-adrenergic receptors.12-13 On the other
`hand, some investigators observed no changes14'17 or
`even an increase1516 of £-adrenergic receptors in sar-
`
`Group
`Control
`
`TABLE 3. Density and Antagonist Affinity of Myocardlal 0-Adrenergfc Receptors in Membranes From One-Kidney,
`One Clip, Deozycorticosterone-Treated Rats, Rats With Reduced Renal Mass, and Control Rats
`125I-Cyp bound
`l25I-Cyp bound (finol/nmol
`(pmol/1)
`(fmol/mg protein)
`Pi/mg protein • min)
`31.6±3.4
`66.9
`l.l±0J
`(48.6-86.8)
`(6)
`45.4
`37.6±2.2
`(38.4-52.9)
`(5)
`54.8
`36.6+2.9
`(48.8-61.0)
`(5)
`62.3
`32.8±4.2
`(48.4-77.3)
`(6)
`KD values are given as mean with 95% confidence intervals. 1K1C, one-kidney, one clip rats; DOCA, deoxycorti-
`costerone-treated rats; RRM, reduced renal mass rats.
`
`1K1C
`
`DOCA
`
`RRM
`
`13±0.3
`
`l.l±0.1
`
`1.2±0.1
`
`Downloaded from http://ahajournals.org by on September 22, 2020
`
`NOVARTIS EXHIBIT 2002
`Biocon v. Novartis, IPR2020-01263
`Page 6 of 10
`
`

`

`Bohm et al Adenylate Cyclase in Acquired Hypertension
`
`109
`
`COMPETITION FOR "•I-KENLKDCGLF BINDING
`
`coo
`o 100
`
`so
`
`o aoam U
`
`I
`
`RAT HEART
`
`20000
`
`10000
`
`m
`RRM
`DOCA
`1C-1K
`CONTROL
`FIGURE 4. Scatterplot shows incorporation of radioactivity
`by pertussis
`toxin-catalyzed [^PJADP-ribosylation
`into a
`40-kd membrane protein in cardiac membranes from one-
`kidney, one clip (1C-1K) rats, deoxycorticosterone (DOCA)-
`treated rats, rats with reduced renal mass (RRM), and control
`rats (control). Myocardial membrane proteins were separated
`by 10% sodium dodecyl sidfate-polyacrylamide gel electro-
`phoresis. Labeled bands were cut out, and incorporation of
`radioactivity was measured by determination of Cerenkov
`radiation. [^PJADP-ribosylation was performed with 0.5%
`Lubrol PX (volfvol) to obtain optimal radiolabeling as de-
`scribed in "Methods."
`
`/3-adrenergic receptors has been observed, the age of
`the animals was higher. In addition, renal hypertension
`was established for a longer period of time11-13 than in
`those studies in which a /3-adrenergic receptor-inde-
`pendent desensitization of adenylate cyclase was ob-
`served (present study and References 17 and 19).
`The reduced basal adenylate cyclase activity and the
`impaired effects of receptor-independent adenylate cy-
`
`Competltlon of Antlserum Binding to G | a
`
`byKENLKDCGLF
`
`Rat heart
`
`o o o o o o
`o °o
`
`o°
`
`40 kDa
`
`PeptkJe concentration (pg/ml)
`FIGURE 5. Sodium dodecyl sutfate-pofyacrylamide gel electro-
`phoresis (SDS-PAGE) shows inhibition of Gu, immunostaining
`by the C-terminal decapeptide of retinal transducin a (KEN-
`LKDCGLF)
`(0.001-1.0 umol/l) in rat myocardial membranes
`from a control rat Myocardial membranes were separated by
`10% SDS-PAGE before electrophoretic transfer to nitrocellulose
`membranes. Note that KENLKDCGLF concentration depen-
`dendy inhibited immunostaining of the 40-kd band, whereas
`nonspecific bands (e.g., 52 kd) were unaffected.
`
`2500
`
`2S0
`
`25
`
`0.025 0.28 2.S
`Protein concentration (pg/ml)
`FIGURE 6. Line plot shows concentration-response curves for
`the competition of increasing concentrations of purified retinal
`transducin a(KENLKDCGLF),
`solubilized membranes from
`human leukemia (HL-60) cells, and rat myocardial mem-
`branes for DS 4 annserum binding to
`I25I-KENLKDCGLF.
`Radioimmunoassay was performed as described in "Methods."
`
`clase stimulators indicate that defects in the catalytic
`subunit or changes of the regulation by G proteins occur
`in the hypertrophied myocardium. To study the activity
`of the catalyst directly, we investigated adenylate cyclase
`activity in the presence of manganese ions, which
`uncouple the catalytic subunit from G proteins.32-33
`Since the effects of forskolin to stimulate the ad