Pharmacology & Toxicology 1992, 70, 152-156.

Comparison of the Effects of Thapsigargin and BAY K 8644 on SpontaneousMechanical Activity in Rat Portal Vein and Contractile Responses of Rat Cardiac Muscle E. 0. Mikkelsen', S. H. Poulsen' and S. Bregger Christensen*

'Institute of Pharmacology, University of Aarhus, DK-8000 Aarhus C, and 2PharmaBiotec,Department of Organic Chemistry, Royal Danish School of Pharmacy, Universitetsparken 2, DK-2100 Copenhagen, Denmark (Received March 15, 1991; Accepted September 2, 1991) Abstract: The effect of thapsigargin, 10-9-10-6 M, and Bay K 8644, 10-9-10-7 M, was studied on isolated portal veins and cardiac muscles from rats. In rat portal veins thapsigargin induced a concentration dependent increase in the amplitude of the spontaneous mechanical activity without increasing the frequency of spontaneous activity. Thapsigargin was less effective than Bay K 8644 in increasing the amplitude of the mechanical activity. In contrast to thapsigargin Bay K 8644, M, and phentolamine, M, had no effect M increases the frequency of the mechanical activity. Atropine, M, totally abolished the on the thapsigargin and Bay K 8644 induced increase in mechanical activity. Nitrendipine, mechanical response in preparations stimulated by thapsigargin and Bay K 8644. In rat atrial and papillary muscles Bay K 8644 increases the frequency in right atrium and tension in both atrial and papillary muscles. Thapsigargin was without effect on the frequency and tension in the cardial preparations. In conclusion, thapsigargin increases the amplitude of spontaneous activity in rat portal veins. In contrast to Bay K 8644 thapsigargin was less effective in increasing the amplitude and had no effect on the frequency of spontaneous activity; furthermore, thapsigargin was without effect on cardiac muscles. The results support the view that an endoplasmaticCa2+-pumpsensitive to thapsigargin is of importance for spontaneous activity in portal veins while such pump is of minor importance for contractile activity in cardiac muscles.

The sesquiterpene lactone, thapsigargin, was isolated from the Mediterranean plant Thapsia garganica L in order to characterize the skin irritant principles (Christensen 1988). The skin irritation is probably caused by the ability of thapsigargin to stimulate different active cellular processes (Ali et al. 1985; Brayden et al. 1989). Studies on a molecular basis have indicated that the mechanism behind the cell activation involves an inhibition of the ATP dependent Ca2+-pumpof the endoplasmic reticulum (Thastrup et al. 1990). A consequence of the arrest of the Ca2+-pumpis that Ca2+ leaking from the microsomal Ca2+ pools increases the cytoplasmic concentration of Ca2+. In most cells the sequestration of microsomal stored Ca2+is followed by a Ca2+ influx from the extracellular milieu (Thastrup et al. 1990). The link between the sequestration of the microsomal Ca2+pools and the opening of the plasma membrane channels is still a subject of debate (Taylor 1990; Kwan et al. 1990). The inhibition of the endoplasmic reticulum Ca2+ATPases is highly specific, as thapsigargin has little or no effect on the Ca2+-ATPasesof the plasma membrane or the sarcoplasmic reticulum (Thastrup et al. 1989 & 1990). Previous studies indicate that thapsigargin partly acts by stimulating the influx of extracellular calcium (Ali et al. 1985; Mikkelsen et al. 1988). The calcium agonist Bay K 8644 is a Ca2+mobilizer, which in contrast to thapsigargin acts by directly affecting membrane channels (Schramm et al. 1985). Thapsigargin as well as Bay K 8644 induces a rise in the cytoplasmic Ca2+-concentrationin pituitary cell line GH&, only the latter drug, however, promotes a prolactin release. This difference probably reflects the different mechanisms of Ca2+mobilization (Law et al. 1990). The rat

portal vein is suitable for studies of the effect of Ca2+ mobilizers as especially the spontaneous mechanical activity in these vessels is highly dependent on extracellular calcium. Therefore, drugs which act on the transmembranal influx of calcium would influence the spontaneous mechanical activity in these vessels (Sutter & Ljung 1977; Pegram & Ljung 1981; Vanhoutte 1982; Mikkelsen 1985). It is well known that activation of heart muscles is also highly dependent on the influx of extracellular Ca2+,and that drugs which act on the cellular Ca2+transport influence the contractile force in these muscles (see e.g. Braunwald 1982). The present paper reports the different contractile responses obtained by incubation of portal veins, atrial and papillary muscles of rats with thapsigargin and Bay K 8644. Materials and Methods Studies on portal veins. Male Wistar rats (own breed) aged 4 5 months were used. After the animals were killed by a blow to the neck, the portal veins were isolated and removed. In order to obtain optimal mechanical activity, and as it is known that the longitudinal muscle layer in portal veins is more sensitive to agonist than the circular muscle layer (Sutter 1990) the veins were cut longitudinally and preparations of about 4 mm in length were mounted vertically in 30 ml organ baths containing physiological salt solution (PSS) maintained at 37" and bubbled with a mixture of 5% CO, and 95% 0, (pH 7.4). Isometric tension was recorded by a Grass Ft03 transducer connected to a Beckmann polygraph (model R611). The resting tension was adjusted to about 10 mN, which was the optimal tension for recording spontaneous mechanical activity in the preparations. When the mechanical activity was stable the effect of thapsigargin, Bay K 8644, acetylcholine,atropine, phentolamine and nitrendipine was studied.

THAPSIGARGIN AND BAY K 8644 I N PORTAL VEINS AND CARDIAC MUSCLES Studies on atrial and papillary muscle preparations. The auricles and papillary muscles of the left ventricle were dissected free from the heart. The atria were separated by incision through the interatrial septum and the spontaneously beating right atrium was separated from the left atrium. Special precautions were taken to select papillary muscle preparations almost of cylindrical uniformity with a diameter < 1.O mm to ensure adequate oxygenation of the central part of the preparations (Gibbs 1978). The preparations were then mounted in organ baths containing 15 ml PSS (composition see below). The organ baths were continuously bubbled with a mixture of 95% 0, and 5% COz,pH 7.4, Tp 32”. The right atria and left papillary muscles were mounted on platinum electrodes and the papillary muscles electrically stimulated by punctate electrodes at a rate of 1 Hz, duration 5 ms (Blinks 1966). The papillary muscle was held and stimulated at its ventricular end, the corda tendinae was tied with silk to a wire connected to a force transducer. The intensity of stimulation was about 20% above threshold, (10-20 mA). The resting tension was adjusted to a level that achieved maximal force of contraction, corresponding to 5 mN and 2.5 mN for the papillary and atrial preparations, respectively. In order to evaluate a possible stimulatory effect of thapsigargin on agonist induced contractions in the cardiac muscles, the papillary muscles were pretreated for 30 min. with lo-’ M thapsigargin whereafter concentration response curves were constructed by cumulative addition of either calcium or isoprenaline. The isometric tension of the preparations was recorded by a Hypart twin-press isometric force transducer connected to a Graptec recorder WER7600. Drugs and solutions. Thapsigargin was isolated as previously reported (Rasmussen et al. 1978), (*)-Bay K 8644 (Bayer AG, Germany), ( f )-nitrendipine (Bayer AG, Germany), acetylcholine chloride (Sigma, U.S.A.), atropin (DAK, Denmark). Phentolamine methansulphonate (Rigitin@, Ciba-Geigy, A/S, Switzerland), (*)isoprenaline chloride (Sigma, U.S.A.). Bay K 8644 and nitrendipine were dissolved in ethanol 95% and thapsigargin in DMSO. In 5 control experiments the maximal final concentrations of ethanol and DMSO did not affect the basal tone or mechanical activity of the vena porta or the contractile force of the cardiac muscles. The solution of substances was added to the organ bath in values of 0.03-0.3 ml to give required concentrations. The physiological salt solution (PSS) had the following composition (mM): NaCl 119, NaHCO, 25, KCI 4.7, MgSO, 1.2, CaCI, 2.5, EDTA 0.027 and

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glucose 11. K-PSS was PSS in which NaCl was exchanged for KCI on an equimolar basis. Statistics. Results are given as mean S.E.M., differences between means have been tested using Student’s t-test. Level of significance was set at Pc0.05.

Results Thapsigargin ( 10-9-10-6 M) induced a concentration dependent increase in spontaneous mechanical activity in rat portal veins without changing the frequency of the spontaneous activity. Thapsigargin was less effective than Bay K 8644 in increasing the amplitude of the mechanical activM) Bay K 8644 ity (fig. 1). In high concentrations in contrast to thapsigargin increased the frequency of the mechanical activity from average 6& 1 to 1 1 f 1 contractiondmin. (n = 5) (PO.Ol, *** = P

Comparison of the effects of thapsigargin and BAY K 8644 on spontaneous mechanical activity in rat portal vein and contractile responses of rat cardiac muscle.

The effect of thapsigargin, 10(-9)-10(-6) M, and Bay K 8644, 10(-9)-10(-7) M, was studied on isolated portal veins and cardiac muscles from rats. In r...
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