J Mel Cell Cardiol

23, 617-625

( 1991)

Alpha-adrenoceptor-mediated Isolated

Increase Cardiac

in Cytosolic Myocytes

Jiirgen Eckel, Elisaheth Gerlach-Eskuchen

Calcium

in

and Hans Reinauer

Diabetes Research Institute, 4000 Diisseldorf (Received 18 April

Free

1, FRG

1990, accepted in revised form 28 December 1990)

J. ECKEL, E. GERLACH-ESKUCHEN AND H. REINAUER. Alpha-adrenoceptor-mediated Increase in Cytosolic Free Calcium in Isolated Cardiac Myocytes. Journal of Molecular and Cellular Cardiolou (1991) 23,617-625. The effect of alpha-adrenoceptor stimulation on the concentration of cytosolic free calcium (Ca,s+) was determined by measuring indo-l fluorescence in isolated ventricular cardiomyocytes from normal and streptozotocin-diabetic rat; 1.3 x lo5 alpha,-adrenoceptors per normal myocyte and an unaltered number ofthese receptors in cells from diabetic rats were detected using the alpha,-selective ligand W&4101. Under basal conditions, CaiZ+ was found to be 154 + 4 IIM (n = 34) reaching a value of 192 f 10 no (R = 15) after stimulation of myocytes with a maximal dose of methoxamine for 5 min. Under the same conditions the leakage of dye produced a significantly smaller increase of basal values to 169 + 5 no (a= 17). Ind+l loaded cells did not respond to beta-stimulation unless in the presence of KC1 (50 mu), demonstrating the specificity of methoxamine action. Treatment of cells with nifedipine or chelation of extracellular calcium by EGTA did not modify the alpha-adrenergic responsr. Experiments with cardiomyocytes from streptozotocin-diabetic rats showed an unaltered modulation of Cai2’ by both alpha- and beta-receptor stimulation. It is concluded that signalling by alpha,-adrenoceptors in ventricular cardiomyocytes results in mobilization of intracellular calcium stores. KEY

WORDS:

Alpha-adrenoceptor;

Isolated

cardiac

myocytes;

Methoxamine;

Indo-1;

Cytosolic

calcium;

Diabetes

Iutroduction Alphai-adrenoceptors have been shown to mediate positive inotropic effects in a variety of cardiac tissue preparations from different speciesbeing well pronounced in rabbit, rat and man (for review, see Benfey, 1980; Bruckner et al., 1985; Scholz et al., 1986). Recently, a direct alpha-adrenergic-mediated prolongation of the action potential duration in isolated ventricular myocytes from adult rat heart has been reported (Vogel and Terzic, 1989). However, the molecular basis of this hormonal effect remains incompletely understood. It has been partly attributed to an increase in slow calcium inward current (Bruckner and Scholz, 1984), but has also been shown to be unrelated to changes in calcium current (Hartmann et al., 1988; Hescheler et al., 1988). Alternatively, alterations in calcium sensitivity of myofilaments (Endoh Abbreviations

used: Ca,“;

Cytosolic

Please address all correspondence 65, D-4000 Dusseldorf 1, FRG. 0022-2828/91/050617

+ 09 $03.00/O

free calcium

to: Priv.-Doz.

and Blinks, 1988) or an increased breakdown of myocardial phosphoinositide (Brown et al., 1985) have been suggested to be of major importance for the positive inotropic effect elicited by alpha-adrenoceptor stimulation. The latter hypothesis is supported by more recent work on isolated rat ventricle (Poggioli et al., 1986) and ventricular myocytes (Buxton and Doggwiler, 1988) demonstrating an increase in IP, by alpha-adrenergic stimulation. Furthermore, the increase in IP, preceded the increase in force of contraction and was directly correlated to the positive inotropit effect of the alpha-adrenoceptor agonist phenylephrine (Scholz et al., 1988). However, conflicting resultsconcerning the role of IP, in mobilizing intracellular calcium in cardiac muscle have been reported (Movsesian et al., 1985; Nosek et al., 1986). The situation might be further complicated by the presence of

IPs; Inositol-1,4,5-trisphosphate

Dr. Jiirgen

Eckel, Diabetes

Research

c

Institutr.

Auf’m

1991 Academic

Hrnnrkamp

Press Limitrd

J. Eckel et al.

618

subtypes of alphat-receptors which may control either mobilization of intracellular calcium or gating of extracellular calcium influx, as recently shown in smooth muscle (Han et al., 1987). Direct determinations of Cai2+ would be helpful in elucidating the mechanisms of alpha,-adrenergic receptor signal transduction. However, it has been argued that quin2loaded cardiomyocytes exhibit no responseof Cai2 ’ after alpha-adrenergic stimulation of the celIs with phenyIephrine or methoxamine (Movsesian et al., 1985). Using indo-l as fluorescent probe and our preparation of ventricular cardiomyocytes (Eckel et al., 1983; Eckel and Reinauer, 1988; Eckel and Reinauer, 1990) the present investigation was initiated with three aims in mind: (1) to expIore an effect of methoxamine on Cai*+; (2) to differentiate between enhanced influx or Cai2 + mobilized intracellularly and (3) to study the effects of diabetes on adrenergic modulations of Cai* +. This latter issueneeds specific attention sinceboth a decreased(Heyliger et al., 1982; Horackova and Murphy, 1988) and an increased (Downing et al., 1983) inotropic responseto alpha-adrenergic stimulation has been observed in hearts from insulin deficient animals. The data suggestexclusive involvement of intracellular calcium storesin alpha-adrenergic action on ventricular myocardium.

Materials

and

Methods

Chemicals

[3H]WB-4101 (sp. radioactivity 17.6 Ci/ mmol) and [ r2 I)iodocyanopindolol (sp. radioactivity 2200 Ci/mmol) were purchased from New England Nuclear, Dreieich, Germany. QuinS/AM (quin2 acetoxymethylester) and indol/AM (indo-l acetoxymethylester) were from Calbiochem, Frankfurt, Germany. Collagenase (EC 3.4.24.3) was purchased from Biochrom, Berlin, Germany. Bovine serum albumin (fraction V and fraction V, fatty acid free) was supplied by Boehringer, Mannheim, Germany. Hyaluronidase, (EC 3.2.1.35), isoproterenol, EGTA, TPEN (N,N,N’,N’-tetrakis-(2-pyridylmethyl) -ethylenediamine) and nifedipine were obtained

from Sigma, Munchen, Germany. Methoxamine was a gift from Deutsche Wellcome, Burgwedel, Germany. All other chemicals were analytical grade and supplied by Merck, Darmstadt, Germany.

Isolation of heart cells

MaleWistar rats fed ad libitum and weighing 280-320 g were used in all experiments. Calcium-tolerant myocytes were isolated by perfusion of the heart with collagenase as described in detail previously (Eckel et al., 1983). The final cell suspensionwas washed three times with Hepes buffer (composition: NaCl 130 mM, KC1 4.8 mM, KH,PO, 1.2 mM, Hepes 25 mM, glucose 5 mM, bovine serum albumin 20 g/l, pH 7.4, equilibrated with 0,) and incubated in silicone-treated Erlenmeyer flasks in a rotating waterbath shaker at 37’C. After 20 min CaCl, and MgSO, (final concentration 1 mM) were added and incubation was continued until further use.Cell numbers were determined in a Fuchs-Rosenthal chamber; cell viability was checked by determination of the percentage of rod-shaped cells and averaged SO-95:/, under all incubation conditions. Routinely, 4-5 x lo6 cells were obtained from one heart. Insulin-deficient diabetes was induced by injecting (i.p.) citrate-buffered streptozotocin (pH 4.5) at a dosageof 60 mgjkg body weight. Control animals received a similar injection of the vehicle alone. All animals were kept for 3 weeks on a normal rat chow and water ad libitum. After that time cardiac myocytes were prepared. Blood sampleswere collected from the vena renalis immediately before starting perfusion of the heart and taken for analysisof plasma glucose (Eckel et al., 1987) and glycosylated haemoglobin (HbA1), which were used for verification of the diabetic state. HbA, was monitored using a column separation system obtained from IS0 Lab., Akron, Ohio, USA,

Receptor binding assays

Alpha- or beta-adrenergic binding were monitored by incubating isolated cardiac myocytes (2.4 x lo5 or 9 x lo3 cells/ml, respectively) from either diabetic or non-diabetic rats with

Alpha-adrenoceptors

and

[3H]WB-4101 (0.1-1.5 no) or [“‘I]iodocyanopindolol (0.01-0.8 nM) in Hepes buffer at 37°C for 30 or 60 min, respectively (equilibrium conditions). Incubations were terminated by the oil centrifugation technique as described in previous publications from this laboratory (Eckel et al., 1983; Eckel and Reinauer, 1990). Non-specific binding was determined by performing parallel incubations in the presence of prazosin ( 10 PM) or proprano101 (1 PM), respectively, and all data were corrected for non-specific binding.

Free

Calcium

in Myocytes

619

which was determined for each cell preparation by addition of buffer, recording of fluorescencesignalsfor 5 min, and calibration asoutlined above. Where indicated, the calculated increase in Cai2+ was corrected for this value, All data analysis was run on an IBM Personal Computer by using Graphpad (ISI, Philadelphia, USA) statistical software. Receptor binding data were analyzed by nonlinear regressionusing Marquardt algorithm and equations for rectangular (one receptor population) or double rectangular (two receptor populations) hyperbola. Significance of reported differences was evaluated by using Determination of Cai2 + the null hypothesis and t statistics for paired Myocytes were loaded with quin2 or indo-l data. Corresponding significance levels are by incubating 4 x 10’ cells/ml in Hepes indicated in the legendsto Figures. buffer for 30 min at 37°C in the presence of quin2 acetoxymethylester (quinP/AM) (final concentration 20 PM) or inde1 acetoxymethResults ylester (indel /AM) (final concentration 5 Determination of adrenergic receptors PM). Cells were then washed 3 times at room temperature with Hepesbuffer and kept in the Freshly isolated ventricular cardiomyocytes dark for 10 min. Immediately before starting contain functional adrenergic receptors which the measurementsthe cells were washedagain belong to the alpha,- and beta,-subtype and checked for viability. Cells exhibiting less (Brown el al., 1985; Buxton and Brunton, than 900/, rod-shape morphology were not 1985). Cardiac myocytes from streptozotocinused for Ca.2+ determinations. Fluorescence diabetic animals have been found to exhibit a was determined in a Perkin Elmer 650-40 reduced number of beta-receptors (Nishio ef spectrofluorometer by keeping the cells in a al., 1988); alterations of alpha-receptors, howthermostatted cuvette at 37°C stirred by a ever, have not been studied in this cellular studies using the magnetic bar. Measurements were performed system. Binding at 339 and 33 1 nM excitation and 490 and 410 alpha 1-selective antagonist WB-4 101 are summarized in Table 1. Non-linear regression nm emissionwavelengths for quin2 and inde 1, respectively. The heavy metal chelator analysis of binding data revealed the presence TPEN (0.5 I(M) was included in order to of one population of alpha,-adrenoceptors with a number of 1.3 x IO5 receptors per cell. stabilize the fluorescence signals. Calibration was performed after each experimental run Neither receptor number nor binding affinity and Cai2+ was calculated according to Tsien of alpha-adrenoceptors were found to be significantly affected by insulin-deficient diaet al. (1982) using the equation [Cai’+] = & betes. For comparison, determinations of F) where K,, equals 115 CF Cnin)liFmax and 240 nM for quin2 and indel , respectively. beta-adrenoceptors were performed using the ~nlaxis the maximal fluorescence, which was samecellular preparation. In agreement with obtained after cell lysis with 50 PM digitonin an earlier report (Nishio et al., 1988)) the and addition of CaCl, (10 mM). Fmin was number of beta-receptors decreased by 50’,,, calculated after quenching with MnCl, (0.3 in cardiocytes from diabetic rats (Table I 1. mM). Intracellular dye concentrations were calculated to be 1.2 mM for quin2 and 350 pM Effects 0Jmethoxamine on Cai2+ for indo-1, in agreement with recent reports in the literature (for review see Cobbold and Initial determinations of Cai2+ using the Rink, 1987). The leakage of dye from loaded fluorescent dye quin2 showed a basal value of cellsproduces an artifactual increasein Cai2+, 134 & 16 nM (n=5), whichincreased by 176’,,,

J. Eckel et al.

620

to 371 + 66 nM upon depolarization of the cells by addition of 50 mM KCl, in good agreement with numerous reports in the literature. However, although proven to be responsive to experimental changes in Cai’ + , quin2 loaded cells exhibited no alterations of Cai2 + after alpha-adrenergic stimulation by methoxamine (data not shown), confirming an earlier report by Movsesian et al. ( 1985). In order to exclude potential problems related to calcium buffering by quin2 (Cobbold and Rink, 1987), the cells were loaded with indocan be used at much lower 1, which concentrations. Figure 1 shows a representative experiment in which Ca,‘+ was determined after adjusting the extracellular calcium concentration to 2.5 mM and addition of methoxamine at 100 PM, a dosage which is known to be maximally effective in producing a positive inotropic response (Heyliger et al., 1982). In this particular experiment Cai2 ’ was found to increase by 3 1 o/o from 118 mu to 154 nM after exposure to methoxamine with a maximal response reached by 4 - 5 min. Responsiveness of cells at that time point was verified by addition of KC1 (Fig. 1). Addition of buffer instead of methoxamine or combined addition of methoxamine and prazosin resulted in a significantly lower artifactual increase in Cai’+, which is most probably due to the

leakage of indo-l from the cells (not shown in Fig. 1, but see Fig. 2) In 34 separate cell preparations basal Cal’+ was calculated to be 154 + 4 nM (Fig. 2). CaiZi raised to 192 + 10 nM (n=15) after treatment of myocytes with methoxamine for 5 min. The leakage of dye resulted in a smaller but significant increase in the basal value to 169 f 5 nM (n = 17) (Fig. 2). In all experiments, however, the increase in Ca,‘+ induced by alpha-adrenergic stimulation was significantly higher than the leakage value. Differentiation between enhanced i&x and mobilization of intracellular calcium The increase in Cai ” elicited by exposure to methoxamine might be mediated by a release from intracellular stores (mobilization), by an enhanced influx from the extracellular environment or both. As shown in Figure 3, treatment of cells with the calcium antagonist nifedipine did not modify the alphaadrenergic response. Furthermore, even after reduction of extracellular calcium to a very low level by chelation with EGTA, an unaltered increase in Cai ‘+ has been observed (Fig. 3). These data suggest that the alpha-

5.0

Dqtonln

t CCICI,

t TABLE

1. Characteristics receptors in normal and rats.

of alphaand cardiac myocytes streptozotocin-diabetic

Control

betafrom

7.5 x 10-10 1.3 x 10s

b

Diabetes

Alpha,-receptor KD (mol/l) receptors/cell

MnCk2

r--

4

10.4

x 10-10

1.4 x

10s

COCI, I mln ii

Beta,-receptor KD (mol/l) receptors/cell

7.4 x 10-l' 4.7 x 105

5.6 x 10-l’ 2.3 x IO'*

Alpha- or beta-adrenergic binding was quantified using [3H]WE4101 or [‘2SI]iodocyanopindolol at equilibrium conditions as outlined in Methods. All parameters have been calculated by non-linear curve fitting to mean binding data obtained from at least three separate cell preparations. ‘Significantly different from control with

P < 0.05.

FIGURE 1. Typical fluorescence recording from indoloaded cardiomyocytes. After addition of CaCI, (2.5 mM), methoxamine (100 PM) was added and the signal was recorded for 5 min. At that time point responsiveness was verified by addition of KC1 (50 mM). Also shown is the calibration procedure which is described in detail in the Methods section. Calculations of Cai2+ were performed using the maximal fluorescence, F,,,,,,and Fmin which is calculated aAer quenching with MnCI,. An appropriate scale for Cai 2+ is shown on left ordinate.

Alpha-adrenoceptors

and Free Calciam

n=34

n= 15

in Myocytes

621

n=17

: l

T

z

200

.5 + 3 150

IOC Basal

Methoxamine

Leakage

were calculated from fluorescence recordings FIGURE 2. Effect of methoxamine on Ca, 2f . Calcium concentrations of indo-l loaded cardiomyocytes as detailed in Figure 1. Cells were incubated for 5 min either with methoxamine or after addition of an equivalent amount of buffer (leakage). Horizontal bars indicate mean values of the indicated number of experiments.

q Basal
0.05. “Not significantly different from control

(P > 0.05).

0

Methoxamme

lsoproterenol

FIGURE 4. Alphaand beta-adrenergir mediated increase in Ca,a+ in cardiac myocytes from control and streptozotocin-diabetic rats. Indc-l loaded cells were incubated for 5 min with methoxamine (100 PM), isoproterenol (IO0 PM) or KC1 (50 mu) plus isoproterenol. Car’+ was then determined from fluorescence recordings as outlined in Figure 1. Data are mean values k S.E.M. (n = 3-l 7) corrected for leakage. *Not significantly different from leakage (P > 0.05).

in ventricular cardiomyocytes after incubation of cells with the alpha-selective agonist methoxamine. This increase was clearly significant over the background value, which had to be carefully controlled in each experimental run due to the inevitable leakage of dye from the loaded cells (for discussion, see Cobbold and Rink, 1987). The average for leakage increase in Cai * ’ after correction can be calculated to be lO-2Oo/o. Thus, the magnitude of this alpha-adrenergic effect is considerably smaller when compared to an increase in Cai2+ by 30--40% observed after beta-stimulation [this paper and Sheu et al., 1987). This, however, is in excellent agreement with the pure contribution of alphaadrenoceptors to the maximal inotropic effect in cardiac tissue, which was found to be about one half of the effect of beta-stimulation (Osnes et al., 1989). It has to be kept in mind that the Cai2+ as determined in the present study using a suspension of quiescent isolated cardiomyocytes, mainly reflects the diastolic level of Ca,‘+ with the contribution of very few spontaneously beating cells. Much caution is needed when comparing these results to determinations of Ca2+ transients performed in electrically stimulated, contracting multicellular preparations from cardiac tissue

Alpha-adrenoceptors

and

(Endoh and Blinks, 1988) or in the whole heart (Auffermann et al., 1989), since regulalion of Ca,‘+ in these experimental designs may involve additional systems which are not operative in an isolated quiescent cardiomyocyte. Nevertheless, our findings agree with the recent report by Auffermann et al. ( 1989)) who observed an elevation in diastolic Ca,‘+ and an increase in the amplitude of Ca2+ transients by alpha-adrenoceptor agents, when applying indosurface fluorometry to the isolated perfused rabbit heart. An increase in the amplitude of Ca2+ transients by alphaadrenergic stimulation has first been reported by Endoh and Blinks (1988) in their studies on isolated papillary muscles. This effect, however, has been found to be only 6.8% of that produced by isoproterenol, whereas the maximum increase in force ofcontraction produced by alpha-stimulation was about 50% of that elicited via beta-adrenoceptors. This led to the conclusion that the positive inotropic effect of alpha-adrenoceptor stimulation is in large part due to an increase in myofibrillar sensitivity to Ca2+ (Endoh and Blinks, 1988). From our findings we would conclude that the increase in Cai2 + produced by alpha-agonists should not be underestimated and that it may he an equal important component of the positive inotropic effect. Certainly, additional components like protein kinase C, which has been implicated in the regulation of cardiac contractility (Movsesian et al., 1985) and has been shown to be activated by alpha,-adrenoceptors in heart myocytes (Henrich and Simpson, 1988), may also be involved. The second major finding of the present investigation consists in the observation that the increase in Cai2+ was independent of extracellular calcium and not related to the activity of the slow calcium channel. Thus, at least quiescent alphain myocytes adrenoceptor stimulation leads to a release of calcium from intracellular stores. This agrees with a recent study by Hartmann et al. (1988), who showed that the positive inotropic effect of alpha-receptor stimulation in single feline myocytes is not produced by inward calcium current. Similar conclusions were also reached by Hescheler et al. (1988) using ventricular myocytes from rabbit heart. In contrast to the effect of methoxamine, the increase in Cat’+

Free

Calcium

in Myocytes

623

due to beta-stimulation was found to be completely mediated by activation of the calcium channel (Table 2), in agreement with the well established effect of beta-adrenoceptors on Ca2 + influx. The present study provides no information on the source of calcium mobilized intracellularly in response to alpha-adrenoceptor stimulation. Recent evidence indicates an increase of IPs (Poggioli et al., 1986: Buxton and Doggwiler, 1988) and inositol ( 1,3,4,5) tetrakisphosphate (Schmitz et al., 1989) in cardiac tissue resulting from alpha-adrenergic stimulation. Despite the well established role of IP, as a second messenger molecule which mediates release of calcium from intracellular stores in a variety of tissues (Berridge and Irvine, 1984), the function of IP, in cardiac tissue remains a matter of controversy ~Movsesian et al., 1985; Nosek et al., 1986). However, exclusive mobilization of intracellular calcium by alpha-adrenoceptor stimulation, as shown in the present study, in combination with a documented rise in IP, preceding the increase in force of contraction (Scholz et al., 1988), lends further support to the assumption that IP, is involved in excitation-contraction couphng in cardiac muscle. Interaction between adrenergic receptors may be of specific impact for certain conditions in which the proportion of functional receptors is changed. The present study shows that insulin-deficient diabetes may be such a condition. Thus the number of alpha,-receptors was found to be unaltered in diabetic animals with a decrease in the number of beta-receptors by 5046 resulting in a proportionate decrease of beta- and alphareceptors from 3.6 to 1.6. This differs from the data of Heyliger et al. (1982) who observed a parallel decrease of beta- and alpha-receptors by 3o-4Oo/o in papillary muscle from diabetic rat. The contractile response of this tissue preparation towards methoxamine was completely lost. Similar data were recently reported by Horackova and Murphy ( 1988) in their studies on single isolated rat ventricular myocytes. Thus, these authors described a full suppression of the positive inotropic effects of alpha- and beta-agonists in cells from diabetic rats even at high concentrations of the hormones. Interestingly, an enhanced sensitivity of the diabetic lamb heart to alpha-

624

J. Eckel et al.

adrenoceptor stimulation has been observed by Downing et al. ( 1983)) although the mechanistic basisfor this finding has not been examined. Our data suggest that the increase in Cat’+ mediated by a maximal stimulation of alpha- or beta-adrenoceptors is not affected by streptozotocin diabetes despite a decrease in the number of beta-receptors. In the present study a 3 week period of diabetes has been chosen. This has been shown to produce fundamental changesin hormonal responsiveness and receptor characteristics of these cells (Eckel and Reinauer, 1990). In contrast, Horackova and Murphy (1988) have used a loweek diabetic model which may, at least partly, explain the lossof inotropic response. In summary, our investigations have shown

that alpha,-adrenoceptor stimulation in cardiac myocytes produces a significant increase in Cai2+ involving mobilization of intracellular calcium stores. Further studies will be needed to clarify the complete mechanism of this hormonal effect and to elucidate its potential significance for alpha-adrenergic regulation of cardiac function and metabolism. Acknowledgement

This work was supported by the Ministerium fur Wissenschaft und Forschung des Landes Nordrhein-Westfalen, the Bundesministerium fur Jugend, Familie und Gesundheit, and the Deutsche Forschungsgemeinschaft (EC 64/1-l).

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Alpha-adrenoceptorsandFreeCalciuminMyocytes

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HA, MAZZOCCA NJ, KLEIMAN RB, HOUSER SR (1988) Effects of phenylephrine on calcium current and of feline ventricular myocytes. Am J Physiol 255: H1173-H1180. HENRICH CJ, SIMPSON PC (1988) Differential acute and chronic response of protein kinase C in cultured neonatal rat heart myocytes to alpha,-adrenergic and phorbol ester stimulation. J Mol Cell Cardiol20: 1081-1085. HESCHELER J, NAWRATH H, TANG M, TRAUTWEIN W (1988) Adrenoceptor-mediated changes of excitation and contraction in ventricular heart muscle from guinea-pigs and rabbits. J Physiol 397: 657670. HEYLIGER CE, PIERCE GN, SINGAL PK, BEAMISH RE, DHALLA NS (1982) Cardiac alpha- and beta-adrenergic receptor alterations in diabetic cardiomyopathy. Basic Res Cardiol 77: 61&618. HORACKOVA M, MURPHY MG (1988) Effects of chronic diabetes mellitus on the electrical and contractile activities, 4sCa2f transport, fatty acid profiles and ultrastructure of isolated rat ventricular myocytes. Pfliigrrs .%rch 411: 564572. MOVSESIAN MA, THOMAS AP, SELAK M, WILLIAMSON JR (1985) Inositol trisphosphate does not release Ca2+ from permeabilized cardiac myocytes and sarcoplasmic reticulum. FEBS Lett 185: 328-332. NISHIO Y, KASHIWAGI A, KIDA Y, KODAMA M, AEIE N, SAEKI Y, SHICETA Y (1988) Deficiency ofcardiac beta-adrenergic receptor in streptozotocin-induced diabetic rats. Diabetes 37: 1181lll87. NOSEK TM, WILLIAMS MF, ZEICLER ST, GODT R (1986) Inositol trisphosphate enhances calcium release in skinned cardiac and skeletal muscle. Am J Physiol 250: C807-Call. OSNES JB, AASS H, SKOMEDAL T (1989) Adrenoceptors in myocardial regulation: concomitant contribution from both alpha- and beta-adrenoceptor stimulation to the inotropic response. Basic Res Cardiol 84 (Suppl. I): 9-l 7. POGGIOLI J, SULPICE JC, VASSART G (1986) I nositol phosphate production following alpha,-adrenergic, muccarinir or electrical stimulation in isolated rat heart. FEBS Lett 206: 292-298. SCHMITZ W, KOHL C, NEUMANN J, SCHOLZ H, SCHOLZ J (1989) On the mechanism of positive inotropic effects of alphaadrenoceptor agonists. Basic Res Cardiol 84 (Suppl. 1): 23-33. SCHOLZ H, BRUCKNER R, M~CCE A, REUPCKE C (1986) Myocardial alpha-adrmoceptors and positive inotropy. J Mol Cell Cardiol 18 (Suppl. 5): 7987. SCHOLZ J, SCHAEFER B, SCHMITZ W, SCHOLZ H, STEINFATH M, LOHSE M, SCHWABE U, PURUNEN J 1988) Alpha,-adrenoceptor-mediated positive inotropic effect and inositol trisphosphate increase in mammalian heart. J Pharmacol Exp Ther 245: 327-335. SHEU S-S, SHARMA VK, KORTH M (1987) Voltage dependent effects of isoproterenol on cytosolic Ca + + concentration in rat heart. Am J Physiol 252: H697-H703. ‘ISIEN RW (1977) Cyclic AMP and contractile activity in heart. Adv Cycl Nucl Res 8: 368-420 TSIEN RY, POZZAN T, RINK TJ (1982) Calcium homeostasis in intact lymphocytes: cytoplasmic free calcium monitored with a new, intracellularly trapped fluorescent indicator. J Cell Biol m: 325-334. VOGEL SM, TERZIC A (1989) Alpha-adrenergic regulation of action potentials in isolated rat cardiomyocytes. Eur J Pharmacol 164: 23 I-239.

HARTMANN

contractility