Intracellular localization of the calcium antagonist propyl-methylenedioxyindene in cardiac tissue

Gen. Pharmac. Vol. 14, No. 6, pp. 571-578, 1983

0306-3623/8353.00 + 0.00 Copyright © 1983 Pergamon Press Ltd

Printed in Great Britain. All tights reserved

INTRACELLULAR LOCALIZATION OF THE CALCIUM ANTAGONIST PROPYL-METHYLENEDIOXYINDENE IN CARDIAC TISSUE JOSEPH J. LYNCHt, RALF G. RAHWAN.1, DONALD T. WITIAK2 and FREDERICK D. CAZER3 Divisions of ~Pharmacology and 2Medicinal Chemistry, Lloyd M. Parks Hall, and 3Radiochemistry Laboratory, Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210, U.S.A. (Tel: (614) 422-6636) (Received 8 March 1983)

Abstract--l. Propyl-methylenedioxyindene (pr-MDI) is a coronary dilating and antiarrhythmic calcium antagonist with an intracellular site of action in cardiac tissue, probably involving inhibition of calcium mobilization from the sarcoplasmic reticulum and preservation of mitochondrial structural and functional integrity. 2. Perfusion of isolated gUinea-pig hearts with [t4C]pr-MDI resulted in negative inotropism, with the time-course of this effect paralleling the uptake of the drug in the whole heart and the time-course of its accumulation in atrial and ventricular tissue. 3. Tissue-to-medium ratios ofpr-MDI for both atria and ventricles were high, and washout of the drug from the heart was biexponential. 4. Pr-MDI associated poorly with a cardiac subcellular fraction exhibiting the highest specific activity of sarcolemmal marker enzyme. 5. The highest specific activity of pr-MDI occurred in the cardiac mitochondrial fraction and in a fraction enriched with a putative sarcoplasmic reticular marker, and the accumulation of drug in these fractions was temporally correlated with its negative inotropic action. 6. The results support an intracellular site of action of pr-MDI in cardiac tissue.

INTRODUCTION The methylenedioxyindenes (MDIs) are tertiary amines with calcium antagonistic properties (Rahwan and Witiak, 1982), that were originally reported from our laboratory (Rahwan et al., 1977). They differ from membrane calcium channel blockers in that they exert their calcium antagonistic action intracellularly, primarily (though not necessarily exclusively) through inhibition of calcium mobilization from the endoplasmic reticulum, and possibly through preservation of mitochondrial structural and functional integrity (Rahwan and Witiak, 1982). Support for the intracellular calcium antagonistic action of the tertiary 2-propyl and 2-n-butyl MDIs drives from the following findings: (1) their ability to interfere with barium-induced nonvascular smooth muscle contraction (Rahwan et al., 1977) which involves mobilization of intracellular calcium; (2) the reversibility of their vascular and non-vascular smooth muscle relaxant properties by increasing the extracellular calcium concentration (Rahwan et al., 1977; Piascik et al., 1979b); (3) their ability to inhibit calcium-dependent (but not calcium-independent) evoked adrenomedullary catecholamine secretion without interfering with cellular calcium uptake (Piascik et al., 1978); (4) their ability to reduce the quantity of calcium released from the sarcoplasmic reticulum upon stimulation of skeletal muscle, as evidenced by depression of activation heat *Author to whom all correspondence should be addressed.

(Burchfield et al., 1982); (5) their inhibitory effect on caffeine-induced contractures of skeletal muscle (which result from mobilization of sarcoplasmic reticular calcium) in the presence and in the absence of extracellular calcium (Rahwan and Gerald, 1981); (6) their inhibitory effect on thrombin-induced platelet secretion (Miirer et al., 1981) which involves intracellular calcium mobilization; (7) their inhibitory effect on swelling and uncoupling of oxidativephosphorylation induced by inorganic phosphate in isolated cardiac mitochondria (Matlib et al., 1981); (8) their ability to inhibit the contractile effect of U44069 (which mobilizes intracellular calcium) on the isolated aorta in a calcium-free medium (Heaslip and Rahwan, 1982a); (9) their ability to block the two intracellular-calcium-dependent phases (Heaslip and Rahwan, 1982b) of norepinephrine-induced contractions of the isolated aorta in a calcium-free medium (Heaslip and Rahwan, 1983); (10) their inability to block myocardial membrane slow calcium channels or other presumptive membrane calcium channels at concentrations which depress cardiac inotropism (Lynch and Rahwan, 1982); (11) their ability to uncouple excitation from contraction in superfused papillary muscle preparations at low concentrations which do not reduce action potential characteristics (Rahwan et al., 1981); and (12) the relatively much weaker in-vitro activity of the quaternary ammonium derivatives of the MDIs on inotropy of electrically-driven left atria (Lynch et al., 1982a) and on aortic contractions induced by norepinephrine or potassium (Witiak et al., 1982) or by

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JOSEPH J. LYNCH et al.

U44069 (Heaslip and Rahwan, 1982a). In the perfused intact heart, the tertiary MDIs do not interfere

with the mitochondrial respiratory electron transport chain and do not uncouple oxidativephosphorylation (Lynch and Rahwan, 1983). Contrary to a preliminary report (Piascik et al., 1981), the predominant evidence indicates that the tertiary MDIs do not bind to calmodulin and do not inhibit calmodulin-dependent processes (personal communications from W. G. Nayler, F. Siegel, and F. Butcher). The objective of the present investigation was to determine the cellular uptake and subcellular distribution of radiolabeled propyl-MDI ([14C]pr-MDI), in order to provide direct evidence in support for the proposed intracellular site of action of the tertiary MDIs. Cardiac tissue was selected since the major pharmacological actions of the MDIs so far described are coronary dilation (Piascik et al., 1979b) and antiarrhythmic effects (Piascik et al., 1979a; Lynch et al., 1981, 1982b). MATERIALS AND METHODS Synthesis of [14C]pr-MDI The 14C label was introduced in the 3-position of the indene ring, using a slight modification of the original procedure for the synthesis of nonlabeled pr-MDI (Witiak et al., 1974), adapted for the micromolar scale synthesis of [14C]pr-MDI. 14C-labeled dimethylformamide (DMF, 22/~1; 0.28 mmol, 54 mCi/mmol) was allowed to warm to room temperature. POC13 (14/~l; 0.15mmol) was added to the DMF and the solution stirred for 30min at room temperature. To this solution was added 20 #l (0.095)mmol) of the olefin (E)-l-(3,4-methylenedioxyphenyl)-pent-l-ene,and the stirred reaction mixture was heated at 89°C for 2 hr in a sand bath. The resultant dark reaction mixture was quenched by the addition of 2 ml of ice-cold water, and the unreacted olefin was extracted into three 2 ml aliquots of ether, while the hydrochloride salt of the MDI remained in the aqueous layer. The aqueous layer was made basic by the dropwise addition of 10% NaOH until the solution became cloudy. The basic solution was extracted with five 2 ml aliquots of ether which extracted the MDI base into the organic phase. The combined ether phases containing the MDI were dried with Na2SO4 then acidified with HCI gas. Some crystals of pr-MDI formed while standing in the freezer; however, the mother liquor still contained over 90% of the MDI as visualized by u.v. and by a Baird Beta Camera. The [14C]pr-MDI hydrochloride product appeared to be greater than 95% radiochemically pure by TLC; however, absolute quantification of purity was difficult due to the slight tailing of the compound (silica GF eluted with isopropanol:acetronitrile, 1:1). The specific activity of [t4C]pr-MDI hydrochloride was 52 mCi/mmol.

Cardiac effects of pr-MDl The time course of pr-MDI effects on cardiac inotropy and chronotropy was investigated in short-haired guinea-pigs of either sex (Carr Animal Supply, Powell, Ohio), weighing 600-1000 g. The animals were killed by a blow on the head, and the hearts were rapidly excised and prepared for Langendorff perfusion. A peristaltic perfusion pump (Model 375A, 2.5 mm tubing diameter; Sage Instruments, Cambridge, Mass.) delivered the perfusion fluid retrogradely into the coronary vasculature through the aortic stump at a constant rate of 6 ml/min. A 3-way stopcock allowed near-instantaneous changes in perfusion fluid. The composition of the physiological Krebs-Henseleit solution was (g/I): NaCI 6.896, KC1 0.358, CaCI2.2H20 0.367,

NaHCO 3 2.285, MgSO4.7H20 0.296, KH2PO4 0.136, and dextrose 2.0. The MDI was also dissolved in Krebs-Henseleit medium. All perfusion fluids were aerated with 95% 02/5 % CO 2 and delivered at 37°C. Heart rate and isometric force of contraction were recorded with Narco Biosystems force transducers (E and M Instrument Co., Houston, Texas) connected to a Physiograph DMP-4B recorder (E and M Instrument Co.). A resting diastolic tension of 2 g was applied to each heart. The hearts were equilibrated for 15-20 min by perfusion with Krebs-Henseliet solution, in order to establish baseline force and rate of contraction. Subsequently, the inotropic and chronotropic responses of the hearts were monitored for up to 45 mins during perfusion with 3 x 10 -5 M pr-MDI (with or without tracer amounts of I4C-pr-MDI). Control hearts were perfused for equivalent periods of time with drug-free physiological solution.

Tissue uptake and washout of [14Clpr-MDI Determination of the washout characteristics of [14C]pr-MDI in the isolated perfused guinea-pig heart was made by measurement of the effluent content of ~4C during the course of the 45 min perfusion with 3 x 10 ~M pr-MDI (with a mean 0.13~o _ 0.03% of drug present as [14C]pr-MDI tracer) and the subsequent 45 min washout with drug-free Krebs-Henseleit solution. The effluent samples were collected at l, 2, 4, 8, 16, 30 and 45 min of drug perfusion, and at l, 2, 4, 8, 16, 30 and 44mins of drug-free washout. Aliquots of effluent were added to Thrift-Solve scintillation solution (Kew Scientific, Columbus, Ohio), and were counted in a Beckman LS-6800 liquid scintillation counter (Beckman Instruments, Palo Alto, California) using Hnumber to monitor and correct for quench. Counting efficiency was > 90%. From the DPMs of ~4C in the effluent samples, the total effluent concentration of pr-MDI was calculated.

Subcellular fractionation protocol Guinea-pig ventricles were excised from hearts perfused with Krebs-Henseleit solution containing 3 x l0 -~ M prMDI and from non-perfuscd hearts and analyzed separately. The individual ventricles were placed in ice-cold solution containing sucrose (250mM), Tris HCI (10mM) and EDTA (2 mM), pH 7.4, at 4°C. All subsequent separation steps were carried out at 4°C. The ventricles were minced and homogenized in a Polytron tissue homogenizer (Brinkman Instruments, Westbury, New York) at low speed for 5 sec, followed by three 5 sec periods at maximum speed. The resulting homogenate was passed three times through an all glass homogenizer, and then filtered through surgical gauze. The filtered homogenate was then diluted with sucrose-Tris-EDTA buffer (4°C) to an approximately 25% suspension (8-9 ml of whole homogenate diluted to a final volume of 35 ml). The diluted homogenate was centrifuged at I085g (avg.) for 15 mins (repeated once for l0 min) in a Sorvall RC-5B centrifuge (Dupont Industries, Newton, Conn.). The pellet from the 1085g centrifugations (designated the nuclear/cell debris fraction) was resuspended in 3-4 ml of sucrose-Tris-EDTA buffer in an all glass homogenizer. The combined 1085g supernatants were centrifuged at 14,500 g (avg.) for 20 mins (reapeated once). The 14,500 g pellet (designated the mitochondrial fraction) was resuspended in 4-5 ml sucrose-Tris-EDTA buffer in an all glass homogenizer. The combined 14,500g supernatants were centrifuged at 109,000g (avg.) in a Beckman L5-75 preparative ultracentrifuge for 60 min. The 109,000 g supernatant (designated as the supernatant or cytosolic fraction) was separated from the pellet. The 109,000g pellet was resuspended in approximately 2 ml of sucrose-Tris-EDTA buffer in a Teflon-glass homogenizer, and was layered onto a discontinuous sucrose density gradient made up of (bottom to top) 4 ml of 45% sucrose, 3 ml of 33% sucrose, and 3 ml of 28% sucrose, as described by Wei et al. (1976). The

Cellular uptake of methylenedioxyindene gradient was centrifuged at l12,000g (avg.) for 120 min in a swinging bucket SW-36 rotor (Beckman instruments). Protein bands were then harvested from the three interfaces using Pasteur pipettes, and the resultant three fractions were diluted with Tris HC1 (10 mM)-EDTA (2 raM) buffer and centrifuged at 122,000g (avg.) for 60min to yield three pellets. Each 122,000g pellet was resuspended in 1.0-1.5 ml of sucrose-Tris-EDTA buffer in a Teflon-glass homogenizer, and these final three fractions were designated as interfaces 1, 2 and 3 corresponding to the top, middle, and lower protein bands of the density gradient, respectively.

573

and ventricular tissues, and its intraceUular localization in ventricular cells, was made by measurement of the content of [14C]pr-MDI tracer in tissue samples, subcellular fractions, and effluent aliquots from isolated guinea-pig hearts which had been perfused with 3 x 10 -5 M pr-MDI (with a mean 0.37% + 0.03% of drug present as [14C]pr-MDI tracer) for 8, 15, 30 or 45 mins. Subsequent to drug perfusion, the hearts were immediately perfused with drug-free Krebs-Henseleit medium for a 10-min washout period, which was sumcient to remove excess MDI from the intercellular spaces (see results). Tissue samples were then taken from the right and left atria and ventricles (15-50 mg), and the remainder of the ventricles were homogenized and subjected to differential and density gradient centrifugation as described above to obtain the various subcellular fractions. The atrial and ventricular whole tissue samples and aliquots of the ventrieular subcellular fractions were incubated in Protosol (New England Nuclear, Boston, Mass.) for 24hr at 50°C for protein solubilization, and then neutralized with glacial acetic acid. The digested samples were then added to Thrift-Solv emulsion-type scintillation solution, and counted in a Beckman LS-6800 liquid scintillation counter using H-number to monitor and correct for quench. From the dpms of ~4C in each sample, the total content of pr-MDl was calculated for whole tissue and for each subeellular fraction. Effluent samples from the perfused hearts were also counted for ~4C as described above, and the data obtained used for calculating the tissue/medium ratios for each duration of drug exposure. All values are reported as the means _ SEM. Paired or unpaired t-tests were used for statistical evaluation of the data.

Characterization of the subcellular fractions The relative purity of each subcellular fraction was determined by assays of marker enzymes. Protein content in each subcellular fraction was determined by the method of Lowry et al. (1951). A Gilford model 250 spectrophotometer attached to a Gilford Model 6050 recorder (Gilford Instruments, Oberlin, Ohio) was utilized in the measurement of enzyme activities and protein concentrations. ATPase activities were determined by a modification of the method of Kidwai et al. (1971). Total ATPase activity ( N a + + Mg 2+ + K +) was determined in a reaction mixture of 1 ml containing 10 mM Tris HCI (pH 7.4 at 25°C) with 50 mM histidine, 5 m M MgCI2, 100mM NaC1, 10mM KC1, 25-100/~g of fraction protein, and 3 mM ATP (Mg salt). The reaction was started by adding the ATP, and the mixture was incubated at 37°C for 10 rain. The reaction was stopped by adding 1 ml of 12~o trichloroacetic acid. After centrifugation at 12,000g (avg.), the liberated inorganic phosphate was determined by the method of Fiske and Subbarow (1925). The fraction of total ATPase activity designated as Na+/K÷-ATPase activity was calculated as the difference between activities in the presence and in the absence of 1 mM ouabain in the reaction mixture. NADPH cytochrome c-reductase activity was measured by the spectrophotometric method of Phillips and Langdon (1962) in 25-200/~g of fraction protein. Succinate cytochrome creductase activity was determined by the spectrophotometric method of Tisdale (1967) in 2/~ g of fraction protein.

RESULTS

Cardiac effects o f p r - M D ! The effects of 3 × 10 -5 M p r - M D I o n cardiac force a n d rate of c o n t r a c t i o n o f the isolated perfused guinea-pig hearts is plotted as a function o f time in Fig. 1. P r - M D I p r o d u c e d a negative inotropic effect t h a t was immediate in onset, reaching a peak o f 45~o below control values at 35 m i n o f perfusion, with the m a g n i t u d e of this inotropic difference between

Tissue concentration and subcellular distribution of [14C]pr-MD1 The determination of pr-MDI uptake into cardiac atrial

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Fig. 1. Time course of the effects of 3 x 10 -5 M pr-MDI on inotropy and chronotropy of the isolated perfused guinea-pig heart. Each point represents the mean +__SEM of 8 preparations. The broken line in the left panel represents the normalized negative inotropic effect of 3 x 10 -5 M pr-MDI, corrected at each time point for deviations of the control (0.0 M pr-MDI) force of contraction from 100%.

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Fig. 2. Time course for the uptake and washout of 3 x 10-5 M pr-MDI (30 nmol/ml, with [t4C]pr-MDI) from isolated perfused guinea-pig hearts. Each point represents the mean + SEM of 4 preparations. treated and untreated hearts being maintained for the remaining 10 min of perfusion time. Cardiac chronotropy, on the other hand, was not affected by pr-MDI. Tissue uptake and washout o f [14C]pr-MDI The effluent concentrations of pr-MDI, calculated from the 'ac content of effluent aliquots for various time periods during the 45-min cardiac perfusion with ['4C]pr-MDI and subsequent 45-min washout of radioactivity with drug-free medium, are plotted as a function of time in Fig. 2. During the 45 min of perfusion with 3 x 10-SM pr-MDI (30 nMol/ml), a steady state is essentially achieved by 35-45 mins, at which time the hearts are extracting approximately 4 5 ~ of the perfusing drug with 55~o of the drug appearing in the effluent. On the other hand, an equilibrium condition (with equality between the infusate concentration of 30 nMol/ml of pr-MDI and the steady state effluent concentration of the drug) is not established by the end of the 45-min drug perfusion period. During the 45-min washout period, MDI concentrations in the effluent were fitted to both a monoexponential and a biexponential model with the aid of the N O N L I N computer program (Metzler et al., 1974). Visual inspection of the distribution of experimental points about the theoretical lines and comparison of the sum of squared deviation suggest that these drug concentrations decline in a biphasic rather than a monophasic manner. The first washout phase is a rapid, nonlinear exponential phase occurring at the onset of the washout and lasting approximately 5 min. This is followed by a second, log-linear washout phase lasting throughout the remainder of the 45 min washout period. In general, the washout portion of Fig. 2 tends to indicate a rather extensive loss of MDI from the heart over the 45min of washout. The results obtained in this section of the study were used to establish the parameters of drug perfusion and washout for the subsequent examination of tissue uptake and subcellular distribution of pr-MDI.

Characterization o f subcellular fractions Ventricular subcellular fractions obtained by the differential and density gradient centrifugation protocol described under Methods were examined for relative purity by assays of marker enzymes. Six non-perfused guinea-pig hearts were individually homogenized and the subcellular fractions characterized enzymatically. Additionally five guinea-pig hearts were perfused for 45 min with 3 x 10 -5 M pr-MDI, and then immediately homogenized (without a washout period), and the subceilular fractions characterized enzymatically. The purpose of omitting the washout of pr-MDI prior to homogenization was to ensure the maximum presence of the drug during the subsequent subcellular fractionation steps, and thus maximize the detection of any drug-induced changes in enzyme distribution or activity that would confound the interpretation of subsequent results involving the subcellular distribution of radiolabeled drug. The mean enzyme activities and protein contents of the subcellu!ar fractions and the overall enzyme activity profiles for the non-perfused and MDI-perfused hearts were found to be remarkably similar, indicating that the 45-min perfusion with 3 x 10-SM pr-MDI had minimal effects upon the enzyme activities monitored or the fractionation protocol itself. Figure 3 depicts the pooled enzyme activity profiles for the subcellular fractions from the 11 guinea-pig hearts, for each of the following marker enzymes: succinate cytochrome c-reductase, a constituent of mitochondrial particles (Green and Burkhardt, 1961); N A D P H cytochrome c-reductase, a putative marker for sarcoplasmic reticulum (Kidwai et al., 1971; Tibbits et al., 1981); total (Mg 2÷ + Na ÷ + K ÷) ATPase, and N A + / K + ATPase, a commonly used marker enzyme for sarcolemma (Kidwai et al., 1971; Hui et al., 1976; Collins and Cook, 1981). As evident from the enzyme distribution pattern in Fig. 3, the mitochondrial fraction shows the highest specific activity of succinate cytochrome c-reductase. The specific activity of the putative sarcoplasmic reticulum marker, N A D P H cytochrome c-reductase,

Cellular uptake of methylenedioxyindene

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Fig. 3. Distribution of marker enzyme activities in subcellular fractions of guinea-pig ventricles. Each bar represents the mean _ SEM of 11 preparations. Numbers within the bars represent purification indices (ratio of enzyme activity in a given fraction to that of the homogenate); the indices were omitted if they were 0.13 or less. H = homogenate; N = nuclei/cell debris; M = mitochondria; S = supernatant/cytosol; I1, 12 and 13 = sucrose gradient interfaces 1, 2 and 3; Pi = inorganic phosphate.

is highest in the three interface fractions harvested from the discontinuous sucrose density gradient (see Methods), with a suggestive higher relative enrichment in interface 1. The total ATPase activity, as expected, was moderate in the mitochondrial fraction and high in the three interface fractions harvested from the sucrose gradient. The sarcolemmal marker enzyme, N a + / K ÷ ATPase shows a significantly higher specific activity (P < 0.01) in interface 2 than in interfaces 1 or 3 harvested from the sucrose gradient.

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Figure 4 shows the p r - M D I content of atrial and ventricular tissues after perfusion of isolated guineapig hearts for various periods of time with 3 x 10-SM p r - M D I containing [14C]pr-MDI tracer. It is evident that marked quantitative increases in the tissue retention of p r - M D I in both the atria and ventricles occur throughout the drug perfusion period. Ventricular tissue consistently displayed a greater retention of drug than atrial tissue at all time periods tested. Simple tissue/medium ratios (shown on Fig. 4 in parentheses for each time point), as well as tissue/medium ratios calculated according to the assumptions of Lorenzo and Spector (1973) (shown in the legend to Fig. 4), are quite high. This is consistent with a steady state uptake well below equilibrium conditions. The tissue/medium values for ventricular tissue are slightly greater than the corresponding values for atrial tissue, reflecting greater accumulation of p r - M D I in ventricular tissue.

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ventricular subcellular fractions obtained from guinea-pig hearts perfused for various time periods with 3 x 10-SM pr-MDI (containing [~4C]pr-MDI tracer). The high content of pr-MDI (calculated as nmol drug/mg protein) in the 109,000g supernatant (cytosolic) fraction is an artifact of the very low protein content of this fraction. The pr-MDI content of the 1085 g nuclear/cell debris fraction is a reflection of the presence of sheared cells in this fraction, as evidenced by activities of succinate cytochrome creductase, N A D P H cytochrome c-reductase, and total ATPase that are essentially equal to those of the total homogenate (see Fig. 3). Likewise, the timedependent increase in pr-MDI content in the nuclear/cell debris fraction and in the total homogenate fraction (Fig. 5) parallels the time-dependent increase in drug retention in whole ventricular tissue (see Fig. 4). Of greater interest in Fig. 5 are the pr-MDI concentration profiles for the 14,500g mitochondrial fraction and the three 122,000g interface fractions harvested from the sucrose density gradient. While all four of these fractions display a timedependent increase in pr-MDI concentration, the mitochondrial fraction and the interface 1 fraction clearly accumulate more drug (P < 0.05 or better at the 15 and 30 min time periods) than the interface 2 and 3 fractions. The mean recovery of radioactivity calculated in all experiments was 95.3 __+1.4%, and the mean recovery of the protein in the same experiments was 87.2 + 0.9%. These recovery values correspond to those reported by Marzo et al. (1977). DISCUSSION

The negative inotropic action of pr-MDI, as well as the lack of effect of this drug on cardiac chronotropy observed in the present study (Fig. 1), are consistent with previously published observations (Piascik et al., 1979a; Lynch et al., 1982a). The time course of the negative inotropic action of pr-MDI

corresponds to the time course of myocardial tissue uptake and accumulation of this drug. Thus, the maximum magnitude of the negative inotropic action of pr-MDI occurred at about 35 min of drug perfusion (Fig. 1), which corresponds to the time of steady state uptake of the drug in whole hearts (Fig. 2) and to the high levels and tissue/medium ratios of pr-MDI in atrial and ventricular tissues (Fig. 4). The subcellular fractionation protocol used in the present investigation (Fig. 3) resulted in fractions with relative purity at least comparable to those obtained by the techniques of Hui et al. (1976) and of Collins and Cook (1981), and it is noteworthy that the procedure does not involve the use of high salt extraction which results in alterations in membrane protein properties (See Bers et al., 1979, for references). It is evident from the present results (Fig. 5) that pr-MDI associates significantly with the mitochondrial fraction, which is consistent with the previously demonstrated direct protective effect of this drug on mitochondrial functional integrity (Matlib et al., 1981). There is also significant accumulation of pr-MDl in the fraction designated as interface 1 (Fig. 5), which is relatively enriched in endoplasmic reticular marker enzyme activity (Fig. 3). This would suggest binding of the drug to the sarcoplasmic reticulum, although this interpretation is rendered difficult by the relatively low accumulation of prMDI in interface fraction 3 at the 15 and 30min perfusion time intervals (Fig. 5), since this fraction also exhibits a high (though somewhat lower than interface 1) specific activity for the sarcoplasmic reticular marker (Fig. 3). Nevertheless, the possible association of pr-MDI with the sarcoplasmic reticulum would be consistent with the inhibitory effect of this drug on calcium mobilization from this intracellular organelle (Rahwan and Gerald, 1981; Burchfield et al., 1982). It is noteworthy that the high levels of pr-MDI achieved in the mitochondrial and interface 1 fractions by 30 rain of drug perfusion (Fig.

Cellular uptake of methylenedioxyindene

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indenes, on mechanics and energetics of skeletal muscle 5) immediately precede the time of maximum negacontraction. Am. J. Physiol. 242, C347-C352. tive inotropic action exerted by this drug (Fig. 1). Collins M. J. and Cook D. A. (1981) Isolation and properA low accumulation of pr-MDI was found in the ties of a plasma membrane fraction from guinea-pig fraction designated as interface 2 (Fig. 5), which is heart. Proc. West. Soc. Pharmac. 24, 217-220. highest in specific activity for sarcolemmal marker Fiske C. H. and Subharow Y. (1925) The colorimetric enzyme (Fig. 3), at the time (30 min) immediately determination of phosphorus. J. biol. Chem. 66, 375-400. preceding the maximum peak negative inotropic Green D. E. and Burkhardt R. K. (1961) Studies on the effect of the drug (Fig. 1). It may be argued that the electron transport system. XXXIII. Succinic-cytochrome MDI could have been removed from membrane c reductase. Archs Biochem. Biophys. 92, 312-320. binding sites during the drug-free washout period Heaslip R. J. and Rahwan R. G. (1982a) Evidence for mobilization of intracellular calcium during the confollowing drug perfusion of the hearts. However, this tractile response of the rat aorta to U44069. Can. J. finding of low MDI accumulation in the interface 2 Physiol. Pharmac. 60, 743-746. fraction, coupled with the previous demonstration of Heaslip R. J. and Rahwan R. G. (1982b) Evidence for the an absence of inhibitory effect of this drug on myoexistence of two distinct pools of intracellular calcium in cardial membrane slow calcium channels or other the rat aorta accessible to mobilization by norpresumptive membrane calcium channels (Lynch and epinephrine. J. Pharmac. exp. Ther. 221, 7-13. Rahwan, 1982) at the concentration used in the Heaslip R. J. and Rahwan R. G. (1983) Norepinephrineinduced contractions of the rat aorta in the absence of present experiment, provides convincing evidence for extracellular calcium. II. Effects of calcium antagonists. a lack of pharmacologically significant sarcolemmal Gen. Pharmac. 14, 505-512. binding of the drug. Hui C.-W., Drummond M. and Drummond G. I. (1976) The preferential accumulation of pr-MDI in some, Calcium accumulation and cyclic AMP-stimulated phosrather than in all, subceilular fractions following phorylation in plasma membrane-enriched preparations perfusion of the drug into the heart, is taken as of myocardium. Archs Biochem. Biophys. 173, 415-427. evidence of uptake of the MDI into cardiac cells. If Kidwai A. M., Radcliffe M. A., Duchon G. and Daniel the presence of drug in subcellular fractions was an E. E. (1971) Isolation of plasma membrane from cardiac artifact of the homogenization and fractionation muscle. Biochem. biophys. Res. Commun. 45, 901-910. Lorenzo A. V. and Spector R. (1973) Transport of salicyclic procedure, the distribution of drug in all subceilular acid by the choroid plexus in vitro. J. Pharmac. exp. Ther. fractions would have been expected to be homoge184, 465-471. nous (Rahwan et al., 1973) and not time dependent. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall Further evidence in favor of uptake of the MDI into R. J. (1951) Protein measurement with the Folin phenol cardiac cells derives from the biphasic nature of the reagent. J. biol. Chem. 193, 265-275. washout of the drug following its perfusion into the Lynch J. J. and Rahwan R. G. (1982) Absence of blocking heart (Fig. 2), with the initial rapid phase generally effects on cardiac slow calcium channels by the intrainterpreted as representing washout of the drug from cellular calcium antagonist 2-n-propyl-3-dimethylaminothe blood vessels and intercellular compartment, 5,6-methylenedioxyindene.Can. J. Physiol. Pharmac. 60, while the subsequent slow phase representing the 841-849. Lynch J. J. and Rahwan R. G. (1983) Comparison of the gradual loss of drug from the intracellular compartcharacteristics of the negative inotropic actions of dinitroment (Rahwan et al., 1972; Rubin et al., 1967). phenol, rotenone, antimycin A, and the intracellular In summary, the present investigation provides calcium antagonist, propyl-methylenedioxyindene. Gen. evidence for the uptake of pr-MDI into myocardial Pharmac. 14, 437-443. cells, consistent with the prior assignment of an Lynch J. J., Rahwan R. G. and Witiak D. T. (1981) Effects intracellular site of action for this calcium antagonist of 2-substituted 3-dimethylamino-5,6-methylenedioxy(Rahwan and Witiak, 1982). The pattern of subindenes on calcium-induced arrhythmias. J. Cardiovasc. cellular distribution of pr-MDI correlates with the Pharmac. 3, 49-60. Lynch J. J., Rahwan R. G. and Witiak D. T. 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