Calcium channel blocking activity: Screening methods for plant derived compounds

Phytomedicine Vol. 4 (2), pp. 167-181, 1997 © 1997 by Gustav Fischer Verlag

Calcium channel blocking activity: Screening methods for plant derived compounds H. VUORELA1, P. VUORELA1, K. TORNQUIST2 and S. ALARANTA3 Division of Pharmacognosy, Department of Pharmacy, University of Helsinki, Finland. Division of Animal Physiology, Department of Biosciences, University of Helsinki, and Minerva Foundation Institute, Helsinki, Finland. 3 Leiras Oy, Turku, Finland. 1

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Summary Calcium channel blockers are a heterogenous group of substances that inhibit influx of Cal. into the cell. Their main therapeutical influence is on the function of heart and blood circulation. A vast number of pure natural compounds with calcium antagonistic activity, mainly coumarins, have been isolated from plants and identified. Screening natural products for calcium channel blockers has been done through classical in vitro aorta or ileum strip assays, but recently in vitro screens using animal cell lines also have been established. The number of in vivo assays on natural compounds is quite limited so far. Much more research is needed to clarify the basic pharmacology and to determine the possible clinical use of pure compounds discovered from plants. Key words: Calcium channel blocker, calcium antagonist, plant compounds, screening methods.

Introduction Calcium antagonistic drugs have received remarkable attention in the treatment of cardiovascular disorders such as angina pectoris, myocardial infarction, atherosclerosis and hypertension. This heterogenous group of drugs acts on calcium channels by inhibiting the Ca 2+ influx into the cell (Raeburn, 1987). Calcium antagonists influence many body functions such as muscle contraction, gland secretion, platelet cell activity, changes in gene expression and the release of transmitters (Davis, 1992). However, their main therapeutical influence is on the function of heart and circulation of the blood and their influence on other tissues is clinically less important. Calcium channel blockers are coronary dilators (Fleckenstein, 1975); they reduce peripheral vascular resistance and hence reduce the cardiac workload (Nayler, 1993). They inhibit Ca 2 + influx through voltage-sensitive Ca 2 + channels and may therefore slow the early rise in cytosolic Ca2 +. Furthermore they are energy sparing drugs that slow the rate of ATP exhaustion (Nayler and Szeto, 1972). They may also

protect against oxyradical-induced lipid peroxidation (Nayler, 1988, 1991, 1992, 1993). The calcium channel blockers that are in use can be divided into three main groups that differ clinically from each other: dihydropyridines, benzodiazepines and phenylalkylamines. They include common compounds used in therapy such as nifedipine, diltiazem and verapamil (Fig. 2). It is known that congestive heart failure, peripheral artery diseases, renal insufficiency, stroke and coronary artery disease occur more often in hypertensive than in normotensive patients (Collins et aI., 1990; Mancia, 1991). There has been a persistent attempt to develop pharmacological agents that not only have antihypertensive efficacy but also reduce the incidence of cardiovascular morbidity and mortality.

Mode of action The widespread term calcium channel blocker or calcium antagonist is used for substances with different modes of

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action. Nifedipine, diltiazem and verapamil, for example, block the voltage-operated channels (VOCCs) in the membranes of smooth muscles. Substances like prenylamine and fendiline also block these channels but may also interact with receptor-operated Ca2 + channels (ROCCs), Na' channels or calmodulin and are therefore classified as "nonselective" calcium-antagonists by WHO (Bolton, 1979; Godfraind et aI., 1986; Raeburn, 1987; Nayler, 1992, 1993). An increase in intracellular calcium concentrations contracts heart muscle cells and the smooth muscle cells in blood vessels' walls. However, the calcium influx has a greater influence on the contraction of heart muscle cells than on the contraction of smooth muscle cells. In the blood vessels the release of calcium from intracellular stores has a great influence on the contraction of muscle cells. The intracellular concentration of free calcium is ten thousand times less than the extracellular. This concentration gradient is maintained by the Ca2+ pumps in the membranes and the membranes of intracellular Cal. stores. Depolarization, ligands and mechanical factors control the calcium influx by regulating how long the calcium channel is open. The calcium channels that are controlled by voltage can be divided into L-, N-, T- and P-types according to their electrophysiological and pharmacological characteristics. Those of the N-type are associated with the release of neurotransmitters. T-type (transient) channels are opened for a short time at the beginning of the action potential. P-channels are located in the Purkinje cells. L-type (slow) channels are found in heart and smooth muscle. Calcium channel blockers inhibit calcium influx from the extracellular space into the cell by binding at various sites on the L-type channels; exactly where depends on their structure. Calcium channel blockers are effective in treating heart and circulatory disorders because the heart and blood vessels contain a higher number of L-type channels than other parts of the body (Nayler, 1993). Calcium channel blockers mainly relax the smooth muscles of the arteries and arterioles, thereby decreasing periferic restriction and blood pressure. All the calcium channel blockers dilate coronary arteries and increase the coronary artery blood circulation. Some have been shown to reduce the extent of nerve cell death following a stroke (Kobayashi et aI., 1992). Calcium channel blockers modify the atrioventricular conductivity and the sinus rhythm maintained by the sinotrial node. The effect depends partly on their influence on the recovery of the channel, i. e. it makes the channel open again after the action potential, and therefore they can be used as antiarythmic drugs. The contraction of many arteries is biphasic, reflecting a two-phase calcium mobilization that involves a rapid release of intracellular calcium and a sustained transplasmalemmal influx of extracellular calcium through the slow Ltype calcium channels activated by membrane depolarization (Van Breemen, 1969; Steinsland et a!., 1973; Deth and van Breemen, 1974; Godfraind, 1976; Droogmans et a!.,

1977; Casteels et aI., 1981; Somlyo and Somlyo, 1968; Droogmans et aI., 1977; Bolton, 1979; Karaki and Weiss, 1981). While the ROCCs remain unidentified, the effects of different ROCC blockers are being investigated; new ones are to be discovered. Among them is the slime mold differentiating factor DIF-1, which potently inhibits receptor-operated calcium entry and might represent a useful lead for new compounds. It is hoped that potent ROCC inhibitors will help to better characterize these channels, isradipine has done this for the L-type VOCCs (Riiegg and Hof, 1995). Prototype compounds

An agent which blocks one of the key steps in the glutamate-activated cascade, such as glutamate receptor activation or calcium influx through voltate-dependent calcium channels, is supposed to reduce the extent of nerve cell death following a stroke. This hypothesis has been tested in animal models using several compounds among them Ca2+ channel blockers. One of the compounds of particular interest is CNS 2103. It blocks a broad spectrum of VOCCs in neuronal cell bodies, including calcium channels which are resistant to the actions of dihydropyridine compounds such as nimodipine (Kobayashi et a!., 1992). The smooth muslce relaxant papaverine was one of the first compounds known to exhibit calcium channel blocking activity. Cinnarizine and verapamil-like molecules were the first Ca2+ entry blockers identified independently by Godfraind and Fleckenstein in 1968-1969 (Godfraind et a!., 1968; Fleckenstein, 1975), followed soon after by the identification of 1,4-dihydropyridine nifedipine. These prototype molecules have altered in industrial and university laboratories to improve their therapeutic properties. Because of their potential use as anti-vasoconstrictor agents in hypertension and angina pectoris, the discovery groups at e. g. Sandoz AG, Basel Switzerland in the 1970s and 1980s synthesized and tested a new series of dihydropyridines (Hof and Hof, 1981; Hof, 1983; Hof and Vuorela, 1983; Hof et aI., 1984 a; Riiegg et a!., 1985 a, b; PerezVizcaino et a!., 1993). The key selection criterion was vascular selectivity;i. e. the ratio between vascular and smooth muscle cell lines and electrically stimulated contractions of single rat cardiac myocytes. The result was the discovery of the highly selective compound isradipine that has a great affinity on the smooth muscle L-type VOCe. According to these studies SDZ 202-791, a nitro analog of isradipine, has a dual activity in smooth muscles: its (+ )enantiomer behaves as a calcium channel activator, whereas its (-)enantiomer is a calcium entry blocker (Riiegg and Hof, 1995). It has been shown that many plant-derived compounds have a possible calcium blocking activity. For example, dihydropyranocoumarin visnadin has been the subject of several investigations to evaluate its toxicity, frequency of side effects, and its potential use as a coronary vasodilator in

Calcium channel blocking activit y: Screening methods for plant derived compounds humans (von Bargheer et al., 1967; Eyrand and Aurou sseau, 1973). Various other dihydrop yranocoumarins isolated from Umbelliferous plants are also known to exert spasmolytic and coronary vasodilatory activity in isolated organs (Thastrup et al., 1983 ). Etbn opbarmacological background for screening

The rapid progress in molecular biology over the last decade has led to the ident ification of the molecular substrates of many diseases, thereby providing an abundance of novel targets for therapeutic intervention. The resulting demand for novel lead compounds can currently best be met by high throughput screening. Recent rapid developments in the field of combinatorial chemistry permit the creation of combinatorial libraries of thousands or even millions of compounds for lead discovery. The resources and manpower being applied to the human genome project will identify an increasing number of molecular target opportunities, increasing the demand for such compounds. Combin atorial libraries will most likely continue to develop and provide opportunities for lead discovery. Traditionally, proprietary compound libraries ha ve provided the largest source of chemicals for screening, but the diversity of chemical templates with in these collections has been said to be restricted. Therefore , natural product librar ies will continue to provide a rich source of novel and often structur ally complex compounds with exploitable biological activity (Harris et al., 1992; Sutter and Wang, 1993; Bevan, 1995 ). Since calcium antagonists are in wide clinical use as therapeutic agents for the treatment of cardiovascular disorders, some screening programs for plant extracts with calcium-antagonistic activity have been established (Yamahara et al., 1985; Vuorela, 1988 a; Ichikawa et al., 1989; Ko et al., 1991 a, b, 1992 a, b, 1993; Wagner, 1993; Rauwald et al., 1994 a). As a result of such investigations, naturally occurr ing substances with possible calcium channel blocking activity have been found in various groups of secondar y products such as alkaloid s (Yano et al., 1991; Martin et al., 1993), coumarins (Vuorela et al., 1988 b; Harrnala et al., 1992 a; Rauwald et al., 1991 , 1994 b), lignans (Ichikawa et al., 1986 ), phen ylpropanes (Hwang et al., 1987; NeuhausCarlisle et al., 1993, 1996; Sensch et al., 1993) and flavonoids (Morales and Lozoya, 1994). An editorial in The Lancet (1994) stated recently, that natural product screening is a viable source of new drugs and the world-wide investment in drug discovery sho uld be encouraged, especially regarding heart and blood circulation diseases. Structure-based rational design is a very powerful tool in the discovery of new drugs. Howev er, there currently are only a limited number of therap eutic targets, since the structure of only a few receptors and their proteins are known with high accuracy at this time. In the case of calcium channel blockers, the channel s are not dearly defined

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structurally. Therefore, dru g discovery relying on assays and screening of natural produ cts and compounds is still valid. The following approaches can be used in the selection of plants for calcium blocking activity: - chemoraxonorny-guided chemical and pharmacological screening - screening of natural comp ound s structurally related to known calcium-antagonists - idiosynchrony-guided screening, e. g. nonspecific spasmolytic activity in which the mode of action is not clearly shown - screening of plants used to treat cardiovascular diseases in traditional medicine. Selecting the solvent to ext ract the active component from the traditional medicine is very important (Yamahara et al., 1985). Usually, polar or moder ately polar organic solvents are used. Trad itional healers usually prepare their remedies by extraction with water, either by boiling the coarsely powdered plant parts or by soaking them in cold water (Samuelsson et al., 1985). In a few cases the plant powde r is added to food. Harrnala et al, 1992 a, b showed an integrated strategy for the extraction, chromatographic separation and purification of compounds tested in vitro for their calcium antagonistic activity. An integrated strategy is necessary to avoid ending up using a laborous trial and error optimization method when testing solvents with various chemical natures in the choice of extra ctant s or mobile phases in liquid chromatography. Pharmacological assays have been used to choose solvents for the extraction of biological material (e. g. Verpoort e et al., 1982; Vuorela et al., 1987; Glinski et al., 1990) . This method uses the activity shown in the test system as the basis for choosing the extraction solvent. It can involve either a specific assay for a certain activity or a general screening. It can also follow activity through the isolation procedure, i. e. directed fractionation of most active extracts (Samuelsson et al., 1985; Orjala, 1993; Andersson Dunstan, 1995 ). To be useful, a pharmacological screening assay must be rapid , inexpensive and easy to perform. The techniques available for the evaluation of pharmacologically active compounds are becoming rapidl y more sensitive and many easy in vitro test procedures are now available to do primary screening of extra cts without having to resort to whole animal experimentation (Vuorela 1988 a; Harrnala, 1991 ). When a strategy for isolating biologically active compounds has been created, and the optimization system carefully studied and the strate gy tested, new active principles can be found for further pharm acological investigations, and the challenge of the ethnopharmacological approach to new drug development becomes worthwhile.

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Test methods in use to detect calcium channel blocking activity Test models in which response (increase in force, uptake of calcium or increase in [Ca2+li) is elicited by depolarization (increase in potassium concentration) can only be a first step in determining a possible calcium channel blocking effect. The effect of depolarization is not restricted to the opening of calcium channels but may be complicated by intracellular calcium release. Additionally, calcium fluxes which are independent from calcium channels may playa role. The only method for determining a calcium channel blocking effect with certainty is the measurement of calcium inward current through calcium channels. Since calcium channel blockers are predominantly indicated in cardiovascular diseases, test models, in which vascular smooth muscle or heart muscle preparations are used, should be preferred.

1. Experiments with animals Measurement of blood pressure and blood flow

An elegant way to monitor the vasodilator effects of VOCCs is to use flow probes placed arount blood vessels of individual vascular beds in anesthetized open-chest cats or dogs (Hof and Hof, 1981; Hof et al., 1982 b; Hof et al., 1984 b). The microsphere method allows regional blood flow to many vascular beds to be measured at the same time by using radioactively labeled spheres (Hof et al., 1980). Although these techniques are very usefull in monitoring effects in vivo, they are both laborous and time-consuming and need special skill to be performed successfully. However, Takeuchi et al. (1991) and Chang et al. (1994) have used them to show the cardiohemodynamic effects of 3-angeloyloxy-4'-acetoxy-3',4'-dihydroseselin (= praeruptorin A) isolated from the Chinese medicinal plant Peucedanum praeruptorum Dunn. in anesthetized open-chest dogs. A dose of 0.3 mg/kg of the compound increased coronary blood flow and significantly decreased the mean aortic pressure and the rate pressure product while increasing the heart rate. These results suggest that praeruptorin A is a Ca 2+ channel blocker. In these experiments it is crucial to dissolve the sample in a proper way as will be discussed later. An easier but less specific approach is to study the in vivo effects of calcium channel blockers on the high blood pressure of spontaneously hypertensive rats. The measurement of the blood pressure of unanesthetized rats by a tail-cuff method is easy and relatively inexpensive to perform (Summanen et al., 1994; Vuorela et al., unpublished results). Gilani et aI. (1994 b) measured the pressure of the carotid artery of anesthetized rats via an arterial canula connected to a pressure transducer and reported that Artemisia scoparia may contain calcium channel blocker-like constituent(s) that may explain its hypotensive effect observed in vivo. Po-

li et al. (1992) tested the crude extracts of the whole plant of Elephantopus scaber with a similar experimental setup but their results revealed only low cardiovascular activity. Pang et al. (1996) used a. o. this method to describe the calcium antagonistic activity of tetramethylpyrazine.

2. Experiments with isolated muscle preparations 2.1. Vascular smooth muscle preparations

Smooth muscle preparations of the rabbit or rat aorta can be used as testing models. Contractions of the aorta are caused by a K+ depolarization that opens VOCCs to allow extracellular Ca 2+ into the cytosol; calcium channel blockers inhibit these contractions noncompetitively (Spedding and Cavero, 1984). However, positive results from this model do not necessarily indicate a calcium channel blocking mode of action; compounds that interfere with Ca 2+ release from intracellular stores might also give positive results (Kobayashi et al., 1989). Only substances that inhibit contractions in this test model are worth further characterization of their mode of action. To characterize the effect of the compounds it is preferable to replace K+ by noradrenaline because it causes Ca 2+ to enter through ROCCs. Calcium channel blockers like nitrandipine or nifedipine only inhibit K+ induced contractions, since they have only weak affinity to ROCCs (Karaki and Weiss, 1988; Godfraind and Miller, 1983; Hof et al., 1982 a). Therefore, a comparison of these two substances can give information about the selectivity of the tested compounds. The inhibition may, however, be influenced by differences in species and the type of vessel studied (Golenhofen and Hermstein, 1975; Kazda and Towart, 1981). Rat and rabbit aortic preparations seem to be suitable as an in vitro screening model for calcium antagonists. For further functional evidence that a compound is a blocker of VOCCs, a calcium channel modulator such as Bay K 8644 can be used to induce contractions in the vascular preparation due to an increased Ca 2+ influx through VOCCs. There are three basic methods for assessing calcium blocking activity on vascular smooth muscle (Hof and Vuorela, 1983). They all rely on stimulation with high external potassium (40-400 mM KCI) (Weiss, 1975). In the first method, potassium chloride is added cumulatively to the organ bath to cause a graded depolarization (Towart, 1981). The second method is based on inducing contractions by adding calcium to a calcium-free depolarizing solution (Godfraind et al., 1968). The rings are pretreated with the test compounds before the calcium is added. In the third method the calcium antagonist is added to a preparation

that has been contracted by high external potassium (Spedding, 1982). 1. In the fist method, mM KCI can be added either to the

Calcium channel blocking act ivity: Screening methods for plant derived compounds

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Fig. 1. Tracing of the ill vitro test calcium cha nnel blocking activity. Rabbit aortic rings were twice contracted with a single dose of 55 mM KCl, whereas the third dosing of KC! was perform ed cumulatively up to 55 mM after the addition of an ant agonist (Ho f et a!', 1983; Vuorela, 1988 a).

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organ bath solution to give a final concentration that produces the maximal contraction, or KCI can be exchanged for NaCI to keep the bath iso-osmotic, Noncompetitive antagonism can usually be observed and the results obtained from cumul ative concentration response cur ves appear to be primarily based on the inhibition of the slow tonic contraction, as is shown in Fig. 1. The osmolarity of the organ bath does not influence the inhibitin g effect of an antagonist. 2. In the second method the organ rings are suspended in a calcium-free solution (Krebs-H enseleit or Tyrode ) or TRIS bath solution which also contains KCI at the concentration that produces the maximal contraction. Calcium is added as an agon ist. Competitive inhibitio n of the calcium effects can be seen with calcium-bl ockers such as nifedipine and verapamil. However, very high concentrations of calcium have relaxant effects and distort the results. 3. In the third method, the organ rings are contracted by the concentration of KCI that produces the maximal contraction and then relaxed by adding the calcium antagonist to the bath. By comparing an iso-osmotic solution to a solution made hyperosmotic with sucrose it can be concluded that hyperosmolarity itself can con tribute to the tension development. The calcium antagonists are less active in hyperosmolar solution, so such bath conditions can considerably bias the results. Relaxation is measured 15-30 min after addition of the antagonist. Cumulative dose response curves for the agonist are usually obtained according to the method of Van Rossum and Van den Brink (1963). Rabb its, rat s or guinea-pigs are killed with a sharp blow to the base of the skull. The descending thorac ic aorta is excised immediately, cleaned from conne ctive tissue, and placed in a Krebs-Henseleit or similar solution. Th e aorta is cut into 2, 3 and 5 mm wide rings or spirally cut into aortic strips. The tension of the aortic rings or strips can be record ed isometr ically with force-displ acement transducers, electromechanical transducers and a potentiometric recorder. The rings are stretched to an initial tension of 800 mg-2 g, and then equilibrated for 1-2 hours until they reach a resting

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tension (VuoreIa, 1988 b; Rauwald et al., 1994 a; Gilani, 1994 ). Two control contractions for each preparation are induced, and the antagonists can then be added 15-30 minutes prior to cumulative dosing with KCl. To eliminate the effect of acetylcholine or noradrenaline release, the endothelium of the artery sho uld bc remo ved by rub bing with a cotton-wool ball; the absence of acetylcholine-induced relaxation can be taken as an indication that the endothelium has been successfully removed. The contr actile effect of Ca 2+ can then further be studied in rings stabilized in high K+ solution without Ca 2+ (Yu et aI., 1994 ). Howe ver, the method can easily lead to art ifacts in results.

2.2. Oth er smooth muscle preparations Guinea-pig trachea has been used by Yu et aI., 1994 (see also Teng et aI., 1990). Male Dunk in-Hartley guinea-pigs (450-550 g) are killed and the tracheas are dissected out , tr ansferred to Krebs solution and cut transversely between the segments of cartilage. Several segments, usually about 3-5 mm, can be tied togethe r to form a chain . Arteche et al. (1995) have used estroge n-primed rat myometrium (40 0 llg/kg 17 ~-oestradi ol benzoate i. p. 24 h before the experiments). Longitud inal strips of uterine smooth muscle (approx imately 1 em in length and 2 mm in width) are prepared and mounted in tissue bath s containing Sund's solution. Similarly the calcium blocking activity of compounds on smoo th muscle preparations have been tested using prep arations of rabb it jejunum and rat uterus (Calixro and Santa na, 1990 ) rat du odenum (Sanchez de Roja s et aI., 1995) and mou se hemidiaphragm (Singh and Dryden, 1990).

2.3. Heart muscle Dichtl and Vierling (1991 ) have used papillary muscles isolated from the right ventr icles of guinea-pig hearts. For isomet ric force measurements, they are mounted vertically in Reiter's medium (Reiter et aI., 1984 ). The muscles are stimul ated through punctate electrodes at their mural end

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by square-wave pulses. Compounds of further interest can then be investigated using electrophysiological methods. Transmembrane action potentials are obtained from horizontally mounted muscles by means of conventional glass micro-electrodes filled with KCl.

3. Experiments with cell lines Cell lines have increased their importance as tools for preliminary screening in vitro since they are easily obtainable, quite stable and do not have the difficulties associated with the breeding of laboratory animals. Measurements in whole tissue are complicated by the presence of extracellular matrix and of other cell types. Furthermore the advantage of cultured cells is that a relatively homogenous population can be studied directly. One of the major pathways leading to enhanced levels of cytoplasmic calcium is the VOCCs present in isolated excitable cells (Bolton, 1979). Mechanisms controlling the gating of this channel can be studied directly by electrophysiological means, especiallyby the patch clamp technique (Reuter, 1983; Hess et al., 1984). Dichtl and Vierling (1991) have studied the effect of magnesium on the inward current using the whole cell voltage clamp technique (Hamill et al., 1981). The micropipettes are filled with a buffer solution. DC resistance ranges from 1-2 mn. An Ag-AgCI electrode is connected via an agar bridge to the bath solution serving as reference. The micropipettes are connected to a patch clamp amplifier. The current and the voltage signals are digitized. This method gives much useful information but is quite laborous. In studies to delineate the calcium blocking activity, the cells are subjected to high levels of K+ or ATP, thyrotropin releasing hormone (TRH) or some other stimulant. This is followed by the measurement of Ca-influx, Ca-efflux or the release of intracellular calcium (Law et al., 1990; Tornquist and Vuorela, 1990). In calcium uptake studies 45Ca2+ is often measured by liquid scintillation counting. The development of Ca-complexing fluorescent agents such as Fura-2 and Fluo-3 (Minta et al., 1989) has made it possible to relatively easily measure the changes in intracellular calcium concentration by fluorimetry. 3.1. Vascular smooth muscle cells Vascular smooth muscle cells are obtained by enzymatic digestion from e. g. Sprague Dawley rat tail artery and the dispersed cells can be used in a whole cell version patch clamp technique. In this technique calcium channel activity is determined using patch micropipettes with the aid of a hydraulic micromanipulator to break the patch membrane. A holding potential can be set and a current-voltage plot constructed using peak current values (Pang et al., 1996). The isolated tail artery cells can also be used for determina-

tion of intracellular calcium using Fura-2 for fluorometric detection in a Sykes-Moore chamber on the stand of an inverted microscope (Pang et al., 1996). 3.2. Myocardial cells The myocardial cells are usually used as primary cultures. In the whole cell voltage clamp technique the cells for the experiments are enzymatically dispersed: ventricular myocytes can be isolated from the hearts of adult guinea pigs by enzymatic digestion. Perfusion with nominal Ca 2+ free ]oklik solution at 35°C at a flow rate of approximately 10 rnl/min for 4 min is followed by perfusion with ]oklik solution for 10 min. After mincing the ventricle with scissors, the tissue fragments are incubated for 5 min at 35°C. After filtering, the myocytes are spun down for 5 min, then resuspended in ]oklik solution with 250 11M CaCl 2 and an additional 10 gil albumine. All solutions are vigorously gassed with 5% CO 2 in O 2 ; the pH is 7.4 (Hamill et al., 1981). On the average, 50% of the cells recovered are viable and rod-shaped with clear cross-striation. The resting membrane potential and holding current is measured. The cell suspension is placed in a perfusion chamber (volume 1 ml) mounted on an inverse microscope. Explanted cardiac cells have been used to study drug actions (Schanne and Bkaily, 1981). Several animal species have been utilized: studies have used the hearts of 6 to 20day-old chicken embryos, 1 to -l-day-old rats and mouse embryos about 14-days-old. Namba et al. (1988) have established a myocardial cell culture of hearts taken from 14 to 16-day-old mouse embryos that are digested with a mixed solution of trypsin and collagenase in buffer solution. The fibroblast-like cells are removed by a method based on the different rate of adhesion between myocardial cells and fibroblast-like cells. The myocardial cells are seeded onto a glass coverslip which is coated with fibronectin. To obtain the large cell sheets, the cell-suspension medium (2-5 X 10 6 cells/ml) must be shaken gyratorally to gather the cells in the center of the coverslip. The cell-attached glass slip is mounted upside down on a small chamber in a stainless plate. To obtain the control value for measuring the cell beating the cell sheet attached to the glass coverslip is stabilized under the microscope. HEPES buffered medium in the chamber is displaced with the same medium supplemented with the tested compounds and the same point of the cell sheet is observed in the course of a measurement. For the Ca-uptake experiments, cardiac cell sheets cultured on plastic dishes are used. The seeding medium is removed and cells are washed with washing solution. Then the cells are treated with the medium supplemented with 45CaCl2 (2.011Ci/ml). After incubation, the cells are suspended in NaOH and equal amounts are removed and a scintillation solution added for scintillation counting.

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with the test. The test substances and reference compounds are added to the cells (10f..ll/990f..ll of the cell suspension). 4.1. Vascular smooth muscle cells The mixture is incubated for 2 hours to measure basal proRuegg et a1. (1985 b) has used a smooth muscle cell line lactin (PRL) release, and for 30 min for TRH- or Kt-depoderived from the thoracic aorta of fetal rats to study cal- larization induced PRL release. TRH or K+ is added to give cium entry through VOCCs. The A7rs cells are plated at a a final concentration of 0.1 u.M or 55 mM, respectively. Afdensity of 7,000 cells/well in Dulbecco modified Eagles me- ter incubation, the tubes are centrifuged and the supernadium containing 7.5% fetal calf serum. At confluency (after tant used for PRL analysis, done by a rat PRL kit. 4-5 days) each well contained a monolayer of 30,000 to For the studies, the GH 3 and GH 4C1 cells are grown on 50,000 cells (0.06 to 0.10 mg of protein). About 15 passag- 35-mm dishes. The cells are then preincubated in buffer at es of a single batch of cells are possible without morpholog- room temperature or at 3rC (basal and K+ stimulated upical changes. The depolarizing buffer used contains 95 mM take). Fresh buffer containing 0.5-1.5 f..lCi 4sCaz+/ml and NaCl and 55 mM KC1. The experiments to measure cal- the appropriate agonist is added. At the end of the incubacium uptake are carried out on confluent monolayers of tion period, the cells are washed three times with Ca z+ free cells. The A 7rS is prepared on plates with 12,000 cells/well buffer containing La3+ (Tan and Tashjian, 1984; Tornquist and the cells are used for flux studies from day 4 to day 6. and Tashjian, 1989). The cells are solubilized with 0.1 N Cells are preincubated with buffer that contains no CaCl z NaOH, and the cell-associated radioactivity is measured by but does contain 0.1 mM EGTA to remove extracellular liquid scintillation. Ca z+. Uptake measurements are performed in plain buffer Further studies on the mode of action of the possible calor 55 mM KCI buffer containing 0,25 f..lCi of 4sCaClz. The cium channel blocker can be carried out as Kummala et a1. cells are then washed with sodium dodecyl sulfate. Radio- (1996) has done using rat thyroid FRTL-5 cells. The cells activity is measured by scintillation counter. are grown in Coon's modified Ham F 12 medium, supplePrimary smooth muscle cells (SMC) of rat aorta are treat- mented with 5% calf serum and hormones (insulin, transed in the same way, except plate cultures are prepared on ferrin, hydrocortisone, the tripeptide Gly-L-His-L-Lys, plates with 20,000 cells/well and the cells are used at days 8 TSH, somatostatin) (Arnbesi-Irnbiomato et al., 1980). Beand 9. fore an experiment, cells from one donor culture flask are harvested with a 0.25% trypsin solution and plated onto 4.2. Other cultured cells: pituitary cells, thyroidea cells 35-mm culture dishes (for 4SCa2+-experiments) or 100-mm Cell cultures that have also been used to screen out pos- culture dishes (for [Caz+]i-experiments) (Tornquist, 1991). sible calcium antagonistic activity in plant extracts and The cells are grown for 7-8 days before an experiment. compounds have been rat pituitary tumor cell culture lines To measure [Caz+L, FRTL-5 cells are harvested with GH 3 and GH 4C1• The cells of the pituitary gland have been HBSS that lack CaCl z but contain 0.02 % EDTA and 0.1 % found to possess VOCCs (Ozawa and Kimura, 1982) that trypsin. The cells are incubated with 1.5 mM Fluo-3-AM can be stimulated by thyrotropin-releasing hormone (TRH) (Minta et al., 1989). The cells are washed with HBSS and and by depolarization with high external potassium. In the added to a quartz cuvette. Fluorescence is measured with a presence of calcium, both TRH and high potassium induce, fluorometer (the excitation wavelength is 506 nm and emisthe release of prolactin (PRL) from the pituitary cells (Tan sion 526 nm). Maximum fluorescence values (Fm.xl are oband Tashjian, 1984). The influx of calcium can be blocked tained by adding CaCl z, digitonin and Tris-base to mainby calcium channel blockers such as verapamil. Channel tain pH above 8.3. Minimum values (Fmin ) are obtained by blockade results in a decrease in the secretion of PRL chelating extracellular calcium with EGTA. [Caz+l is calcu(Ozawa and Kimura, 1982; Enyeart et al., 1985). This lated as described by Grynkiewicz et a1. (1985), using a Kd means that the pituitary cells can be used as a model for of 0.45 nM for Fluo-3-AM. To measure 4SCaZ+ uptake the cells are preincubated in studying compounds that interact with calcium channels. The properties of the GH 3 and GH 4C1 cell lines and the HBSS-buffer. HBSS with or without the blocker containing methods of cell culturing have been described by Tashjian ATP and 1 f..lCi 4sCaz+/ml is added. After the incubation, the et a1. (1968). The cells can be grown in monolayer cultures cells are washed with Ca z+ free HBSS containing 0.1 mM in Ham's F-10 medium supplemented with 15% horse ser- LaC1 3• The cells are solubilized with NaOH, and the cell-asum and 2.5% fetal calf serum at 37°C in a humidified at- sociated radioactivity is measured by liquid scintillation mosphere of 5% COz and 95% air. Prior to use, the cells counting (Tornquist, 1991). are harvested using 0.5% trypsin for 5 min at 37°C. For 4SCaZ+ efflux studies the cells are preloaded with bufThe tests with GH 3 or GH 4C1 cells can be performed in fer containing 1 f..lCi 4sCaz+/ml and NaHC0 3 • They are plastic test tubes or in monolayer cultures containing washed with buffer. The antagonist is added with and with0.1-0.2 X 106 cells/ml in a final volume of 1 m1. The test out ATP, the cultures are incubated and the cell-associated samples and reference compounds such as verapamil or ni- radioactivity is measured by liquid scintillation counting fedipine are dissolved in a vehicle that does not interfere (Tornquist, 1991).

4. Cultured cells

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5. Principles of preparation of plant extracts; problems with solvents

Preparations of a sample from natural products is sometimes difficult since the compounds are seldom water soluble. Solvents must be able to totally dissolve the extracts. The solvents used should not have any influence or synergistic effect on the test method in the terms of the response measured. The solvents most frequently used to dissolve the compounds into the vehicle have been ethanol and polyethyleneglycol400, dimethylsulphoxide (DMSO), pyrrolidone, pyrrolidone-chremophor® (6.8%) 9:1 (v/v) or water containing 20% v/v Cremophor and EGTA or EDTA (Kozawa et aI., 1981; Vuorela, 1988 b; Yamahara et aI., 1990; Rauwald et aI., 1994; Namba et aI., 1988). It has been shown that when the final concentration of the solvent in the organ bath is less than 1%, it has no measurable influence on the response of smooth muscle preparations or pituitary tumor cells (Vuorela, 1988 b; Vuorela et aI., 1988) or cell beating of cultured myocardial cells (Namba et a!., 1988). According to Rauwald et a!. (1994 a) DMSO influences the K+- and noradrenaline-induced contractions less strongly than the other solvents they tested, and therefore should be used as solvent whenever possible. Due to the vehicle (ethanol and PEG 400 mixture, 2+1) the compounds remain dissolved in the salt solutions. The addition of 0.8-1.6 flUml vehicle provides an acceptable concentration of the tested compound or extract, e. g. 10-4 M for coumarins (approximately 0.025 mg/ml depending on the structure of the compound) (Vuorela, 1988 b), at which concentration range the responses can be regarded as nonspecific. The extracts can be dissolved in dimethylsulphoxide (DMSO 4 ul/rnl in the organ bath) according to Rauwald et al. (1994 a) and added to the bath to give a final concentration of the extract 10-3 g/ml or 10-4g/ml; the latter concentration may be used in the case of solubility problems. Extracts that have no significant activity at these extremely high concentrations are not worth examining further. Calcium channel blockers detected in plants

Table 1 shows some plant extracts and compounds. Cachannel blocking active compounds have been found in the classes of furanoconmarins, furanochromones, stilbenes, coumarins, flavonoids, lignans, naphtoquinones, phenylpropanoids, monoterpenes, sesquiterpenoids and alkaloids. The measurement of their possible calcium channel blocking activity by different preparations and techniques is tabulated and the concentration range where activity could be shown is indicated. The table indicates that many compounds in nature have an activity comparable to compounds currently used in therapy and are thus worth examining further with regard to their pharmacological properties. Some attempts to determine structure-activity relationships between the calcium channel blocking activity and

molecules have been performed (Rampa et a!', 1995). Mannhold et al. (1982) investigated earlier derivatives of nifedipine and verapamil and reviewed their results. Vuorela et al. (1992) and Salo et al. (1996) have used regression, factor and cluster analysis as well as neural network to describe coumarins by 180 topological indices in order to explain their activity in inhibiting depolarization-induced Ca 2 + uptake. Vierling et a!. (1995) showed that the benzol ring as well as the aliphatic chain of phenylpropane derivatives is essential for the calcium channel blocking effect. Concluding remarks on drug development of calcium channel blockers from natural sources

The first step in designing a screening system is to consider whether the treatment for disease is either inadequate or even non-existent, and whether an unmet therapeutic need can be filled by providing a superior drug. Since any natural product screening program is a major investment in manpower, money and time, the need has to be clarified carefully. It must be borne in mind that the screening of pharmacologically active compounds is only one of the first steps in drug development. The initial screening must be followed by thorough in vitro and in vivo animal experiments as well as by human clinical studies. Even though ion channel modulators are important drugs, as exemplified by calcium antagonists, it appears that ion channel modulators are a neglected area as far as natural product screening is concerned. This is most likely due to the fact that high capacity in vitro screens, those created for ACE inhibitors by Wagner (Elbl and Wagner, 1991; Wagner and Elbl, 1992; Wagner, 1993), do not seem to be readily available. Progress in natural product screening has always been intimately linked to both theoretical concepts and technical achievements. Theoretical concepts have led to be formulation of target-directed screening approaches. Screening techniques designed on the basis of known sites of action will not always reveal the existence of compounds having novel modes of action which may well be present. In addition to the choice of target and the choice of method, there remains the choice of biological source material. Source materials range from microorganisms, algae and plants to amphibians, spiders, snakes and scorpions. Most importantly, it is clear that natural product screening, if conducted intelligently, can be highly productive and can provide a competitive advantage in drug discovery. After many years of disfavor, it now appears that the natural product screening is again regarded as an important source of new drugs by the pharmaceutical industry. References Ambesi-Impiombato, F.S., Parks, L. A. M. and Coon, H. G.: Proc. Nat/. Acad. Sci. USA 77: 3455-3459, 1980. Andersson Dunstan, c.: Isolation and structure elucidation of

Calcium channel blocking activity: Screening methods for plant derived compounds NH

H,C ---

o

o

/

175

CH,

0 ...... CH,

(J(

Nifedipin

s 0

yCH'

N

S

H,CO

0

H,C-N

\

CH,

H,CO

Diltiazem Verapamil

OCH, OCH,

o Archangelicin

Columbianadin

Osthol

Atherosperminine

o

Fig. 2. Molecular structures of some calcium channel blockers commonly used in therapy and some of the most active natural substances.

o

>


o Crychine

~)::( H,C

CH,

Tetramethylpyrazine

176

H. Vuorela et al.

Table 1. Compounds and plant extracts that have shown activity in test methods related to the measurement of the possible calcium channel blocking activity (- no measurable activity) (see also Fig. 2).

Extracts/Compounds

Concentration range

Preparation

Reference

Okadaic acid Jatrophone

rc., 48.3 11M

10, 20 11M

rat myometrium (uterine) rat uterus anaesthetized rats rabbit jejunum human red blood cells

Arteche et aI., 1995 Calixto and Santana, 1990 Gilani et aI., 1994 b

rat pituitary cells, GH 4C t

Hadacek et aI., 1991

rabbit aorta rat pituitary cells, GH 4C t

Henry and Yokoyama, 1980 Harmala et aI., 1992 b

rat pituitary cells, GH 4C t

Harmala et aI., 1992 a

rat thoracic aorta rat thoracic aorta single canine ventricle cell

Ko et aI., 1992 a Ko et aI., 1993 Meselhy et aI., 1992

rat aorta guinea-pig aorta rat pituitary cells, GH 4C t

Morales and Lozoya, 1994 Mousa et aI., 1994

rat ganglion neurons mouse myocardial cells

Nah and McCleskey, 1994 Namba et aI., 1988

Artemisia scoparia Thunb.

3-30mg/kg IC so :::: 0.3 mg/ml ic., 0.24mM

E-2,3,5,4'-tetrahydroxystilbene 2-0-~­ D-glucopyranoside cis-E-3-butylidene-4,5,6,7rc., 0.16 mM tetrahydro-6, 7-dihydroxy-I (3 H)-isobenzofuranone trans-E-3-butylidene-4,5,6,7 rc., 0.26 mM tetrahydro-6, 7-dihydroxy-l (3 H)-isobenzofuranone E-2,4,6,4'- tetrahydroxystilbene 2-0-~-D-glucopyranoside Xanthalin rc., 5.6 11M Peuarenarin ic., 5.0 11M Peuarenin rc., 6.6 11M Peucedanin rc., 12.8 11M Athamantin rc., 3.7 11M 2-Methylchromone derivative rc., 4.3 11M Ergonovine EDso 2.0 11M

Angelicaarchangelica L., root methanol extract '" n-propanol extract ,,, n-pentanol extract ", chloroform extract ", n-hexane extract Osthol Archangelicin Bergapten Isoimperatorin Oxypeucedanin Oxypeucedanin hydrate Ostruthol Xanthotoxin Imperatorin Isopimpinellin Phellopterin Osthol Crychine Tinctormine Safflor yellow B Quercetin

Ficus benjamina L., fruit R sycomorus L., fruit R bengalensis L., fruit R religiosa L., fruit Panax ginseng L., root Osthol Esculetin Umbelliferone Fraxidin Fraxin Esculin 13-Methylumbelliferone

Scoparone Decursinol Decursin 3' -Dehydrodecursinol

IC so 3.9 ug/ml rc., 4.2 ug/ml rc., 4.2 ug/ml rc., 4.3 ug/ml IC so 5.711g/ml rc., 16.4 11M rc., 2.8 11M 92.6 11M IC so 25.5 11M 69.9 11M 65.8 11M 51.8 11M 92.6 11M IC so 40.0 11M 81.3 11M rc., 39.3 11M rc., 12 11M IC so ::: 25 ug/ml rc., -5 11M RC so 17.8 11M nc., 100 11M

10011g/ml

rc., 2.2mM rc., 2.8mM ic., 3.1mM rc; 4.1mM

IC so > 10mM IC so > 10mM Ieso > 10mM IC so > 10mM rc., 0.92mM rc., 1.9mM rc., 2.1mM

Gresh et aI., 1994

Calcium channel blocking activity: Screening methods for plant derived compounds Decursidin Nodakenetin Xanthotoxol Isopimpinellin Isobergapten Sphondin Imp erator in Co lum bianadin Bergapten Xant ho toxin Glycero l Co umestro l Isoglycyrol Dicoumarol Apiole

IC so > lOmM rc., 0.93mM IC so 5.2 mM IC so > 10mM IC so > 10 mM IC so > lOmM IC so > 10mM IC so > 10mM IC so > lOmM IC so > 10 mM IC so > 10mM IC so > lO mM IC so > 10 mM ICso > 0.1 7 mM rc., 31l!M

Allyitetrameth oxybenz ene M yristicin

IC so 63l!M ICso 88 l!M ic., 75 l!M rc., 76 l!M 63 l!M ic., 25 8l!M IC so 58 l!M IC so 44 1l!M rc., 190 l!M ic., 29l!M rc., 138l!M rc., 876l!M IC so 5 7l!M IC so 123l!M IC so 4 8l!M rc., 67l! M 5.6 l!M ICso 56l!M IC so 89 l!M rc., 999 l!M 265l!M rc., 879l!M rc., 83l!M IC so 93l!M ICso 15l!M rc., 55 1 l!M 49l!M rc., 1 mM rc., 20 rng/kg

trans-Anethole a-Asaron e ~-Asaron e

Methylchavicol Safro le Menthofuran Artem isin Bisabololox ide A Isopetasin Santonin ]uglone Men adi one 7-M ethyljuglone Plumbagin Allicin

trans-Ajoene Cyclos tac hin A Khellin Syringar esinol Eudesmin 4-Methylene-miltirone Gossypol Sang uinarin e Xantho toxi n Visna din Tetrameth ylpyrazine

Elephantopus scaber L., water extrac t

guinea pig papillary muscle Neuhau s-Carlisle et al., 1996

rc.,

rc.,

"

rc.,

rc.,

"' aqueous-alcoholic ext ract Ammi visnaga (L.) Lam 1O-3g1ml Guaiacum officinale L. 10-4g/ml Ruta graveolens L. 10- 3 glml Cryptolepis sanguinolenta Lindle y (Sclechrer), roo t Leonurus cardiaca L. Passiflora incarnata L. Solidago gigantea Ait. Olea europaea L., leaf 10- 3mglml 3,4-dihydroxy phenylethano l lO- lmg/ml Peucedanum ostruthium (L.) Koch , rh izom es 1O-3g/ml Isoimperat o rin 37.0 l!M Imperator in 37.0 l!M Ostruthin 33,6 l!M Ostruthol 25,9l!M 2 "-O-acetyl-oxypeucedanin hydrate 27.5l!M Ox ypeuc edan in hydrate 32.9 l!M Poeniculum vulgare var. dulce, Alef. 0.0 02g/ml

177

" ran tail artery strips anesthetized ra ts ureth an -an esth etized ra ts

Pan g et al., 1996

ra bbit thoracic ao rta

Rau wald et al., 1994 a

ra t ileum

Saleh er aI., 1996

Poli et a l., 1992

178

H. Vuorela et al.

Cistus populifolius L., leaf Musa sapientum L. var paradisiaca, stem juice Praerupterin A Xanthyletin Xanthoxyletin Suberosin Dicumarol Visnadin Pteryxin Isosamidin Samidin Disenecioyl cis-khellactone Diacetyl cis-khellactone Lomatin acetate Athamantin Isopeucedanin Peucedanin Archangelicin Diacetyl-vaginidiol Senecioyl-dihydrooroselol Columbianadin Vaginidin Eugenol Apiol Matricaria chamomilla, (L.) flowerheads, methanol extract Apigenin Apigenin 7-glucoside a-Bisabolol Peucedanum palustre (L.) Moench., root, pentane extr. Columbianadin Isoimperatorin Isobyakangelicin angelate Ostruthol (+ )-Oxypeucedanin (±)-Oxypeucedanin hydrate Peucedanum palustre (L.) Moench, root, pentane extract Columbianadin (+ )-Oxypel,lcedanin Isoimperatorin Ostruthol Vexibinol Kurarinone

reference compounds Verapamil Verapamil Verapamil Verapamil Nifedipin Papaverin

0.002g1ml ICso :::: 10M

rat urinary bladder rat duodenum

4 mglml (augmentation in mouse hemidiaphragms contraction) pD'25.26 quinea pig ileum 0.3 mglkg open-chest dogs ICso 7 JlM (at 0.3 JlM Ca2+) rat thoracic aorta rc., 38 JlM (at 0.3 JlM Ca 2+)" rc., 38 JlM (at 0.3 JlM Ca 2+)" rc., 70 JlM (at 0.3 JlM Ca 2+)"

rc., 17 JlM rc., 13 JlM rc., 16 JlM rc., 5.7JlM rc., 14JlM rc., 200JlM rc., 140JlM ic., 9.1 JlM ic., 65 JlM

quinea pig ileum

Sanchez de Rojas et al., 1995 Singh de Rojas et al., 1995 Takeuchi et al., 1991 Teng et al., 1992

Thastrup et al., 1983

ICso 29JlM ICso 4.7JlM

rc., 160 JlM rc., 29 JlM ic., 55 JlM rc., 100 JlM rc., 224JlM rc., 29JlM

1 X lO-4g1ml

guinea pig papillary muscles Vierling et al., 1995 rabbit thoracic aorta

Vuorela et al., 1985

rabbit thoracic aorta

Vuorela, 1988

z: 2.5-0.1 X lO-s glml

rat pituitary cells, GH 3

Vuorela et al., 1988

10mM 10mM 10mM O.lmM rc., O.lmM O.lmM 0.01 mM

rabbit thoracic aorta rat thoracic aorta rabbit thoracic aorta rat thoracic aorta

Yamahara et al., 1990

rabbit thoracic aorta mouse myocardial cells rat pituitary cells, GH4 C j rabbit thoracic aorta guinea pig papillary muscle

Vuorela, 1988 Namba et al., 1988 Harmala et al., 1992 a Rauwald et al., 1994 a Neuhaus-Carlisle et al., 1996 Thastrup et al., 1983

rc., 31.6JlM ic., 6.3 JlM rc., 10 JlM

rc., 3.6JlM rc., 25.1JlM rc., 12.6 JlM rc., 12.6 JlM rc., 5.0JlM

rc.,

rc., 0.3 JlM rc., 0.05 JlM rc., 2.0JlM ICso :::: 2JlM

rc., 0.3 JlM

quinea pig ileum

RC so - the concentration that produces 50% relaxation; IC so - the concentration that produces 50% inhibition

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Address Vuorela, H., Division of Pharmacognosy, Department of Pharmacy, P.O. Box 56, FIN-00014, University of Helsinki, Finland. Tel.:+358-9-70859167; Fax: +358-9-70859172; e-mail: [email protected]