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Comparison of inotropic and chronotropic effects of vasoactive intestinal peptide in isolated dog atria
Journal of the Autonomic Nervous System 61 Ž1996. 257–263
Comparison of inotropic and chronotropic effects of vasoactive intestinal peptide in isolated dog atria Don W. Wallick a , Sherry L. Stuesse b
b,)
a DiÕision of InÕestigatiÕe Medicine, Mt. Sinai Medical Center, CleÕeland, OH 44106, USA Neurobiology Department, Northeastern Ohio UniÕersities College of Medicine, P.O. Box 95, Rootstown, OH 44272-0095, USA
Received 25 September 1995; revised 20 June 1996; accepted 17 July 1996
Abstract The positive chronotropic and inotropic effects of vasoactive intestinal peptide, VIP, were studied in an isolated canine right atrial preparation. Atria were removed, maintained in a bath, and perfused with Tyrode’s solution. Contractile force and atrial depolarization were measured. VIP Ž18.8–600 pmol. was injected into a cannulated sinoatrial nodal artery and dose response curves were obtained. The mean EC 50 was similar for the inotropic and the chronotropic responses Ž136 and 144 pmol, respectively.. Time courses of the onset and of recovery from the responses were measured. Times for onset of VIP effects were similar but, once the effect was initiated, rate of development of the response and recovery time from the responses were dose dependent. The increases in atrial rate lasted two to four times longer than did the increases in contractile force. Recovery from the chronotropic and inotropic responses to VIP differ, suggesting that the intracellular responses are coupled differently to the receptors. The responses to VIP were compared to those of 100 pmol isoproterenol, another positive chronotropic and inotropic agent. Isoproterenol was a slightly more potent chronotropic and inotropic agent than VIP. Desensitization of the responses was determined. Repeated exposures to VIP decreased the chronotropic response but not the inotropic response to VIP. There was no significant decrease in responsiveness to isoproterenol. Keywords: Autonomic nervous system; Parasympathetic; Cardiac; Contractile force; Neuropeptide; Heart rate; Atrium; Isoproterenol; Receptors
1. Introduction Vasoactive intestinal peptide ŽVIP. is a 28 amino acid polypeptide that may coexist with acetylcholine in parasympathetic postganglionic nerve terminals in the heart w8,17,18,29–31x. Two major types of VIP receptors have been characterized in the heart; one that has a high affinity for VIP and the second that has a high affinity for secretin, a VIP-like molecule w6x. The former is found in Cynomolgus monkeys, humans, and dogs, while the latter is found in rats. Thus dogs may be an appropriate model for research into the clinical effects of this peptide. In betablocked, atropinized dogs, vagal efferent stimulation increased heart rate and contractile force w25x. This effect may be due to the release of VIP w21x. VIP is released from dog atria when parasympathetic nerves are stimulated at a high frequency w13,14x. However, the physiological actions
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of VIP on atria have not been well characterized. VIP that is administered intravenously or directly into an appropriate coronary artery increases heart rate and cardiac contractile force w2,21,15,28x. VIP also has a positive inotropic action on isolated dog atrial and ventricular muscles w22x and a positive inotropic effect on paced, isolated pieces of human atria ww9xx. In the studies reported herein, we further characterize the inotropic and chronotropic responses of isolated dog atria to VIP. We compare dose response curves, the time course of responses, and the response to repeated exposures to low levels of VIP or to isoproterenol, a positive chronotropic and inotropic agent that activates beta adrenergic receptors.
2. Materials and methods Mongrel dogs of either sex Ž18–27 kg. were premedicated with morphine Ž1–2 mgrkg, i.m.. and anesthetized with alpha-chloralose Ž100 mgrkg, i.v... Thirty-five dogs were used. The right side of the chest was opened at the
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fifth intercostal space. The pericardium was opened, and a cradle was made by suturing the edges of the pericardium to the chest wall. We isolated the sinoatrial nodal artery and cannulated the artery with a 22-gauge catheter filled with Heplock Ž100 units of heparinrml.. The azygos vein, inferior vena cava, and superior vena cava were tied. The entire free wall of the right atrium, including the SA node, was removed from the dog and immersed in cold, modified Tyrode’s solution Ž120 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl 2 , 1.1 mM MgCl 2 , 0.5 mM NaH 2 PO4 , 25 mM NaHCO 2 , 5.4 mM dextrose, 0.06 mM L-ascorbic acid, bubbled with 95% O 2 –5%CO 2 .. Removal of the atrial appendage and the blockage of blood flow to the heart resulted in death while the animal was completely anesthetized, and the body was incinerated. Care and use of the dogs was in accordance with guidelines set down by the National Institutes of Health and the American Association for Accreditation of Laboratory Animal Care. We cauterized any severed branches of the SA nodal artery. The atrium was transferred to a tissue bath, superfused with Tyrode’s solution, and the SA nodal artery was perfused with warm, oxygenated Tyrode’s solution Ž358C.. The perfusion pressure was maintained at about 80 mmHg by altering the pump speed Žfrom 1 to 4 mlrmin.. In some experiments, atropine Ž30 nM. and propranolol Ž330 nM. were added to the Tyrode’s solution to block the muscarinic and beta-adrenergic receptors, respectively. Betaadrenergic blockade does not diminish the VIP effect on atrial and ventricular pacemakers w23,24x. VIP is not known to be released independently of acetylcholine, however, in some systems, atropine, a muscarinic receptor blocker, may actually enhance VIP release via presynaptic modulation w18,31x. Vasoactive intestinal peptide, VIP, ŽaPGAS350, Bachchem. was dissolved in distilled water Ž1 mgrml. and stored at y708C in 20 m l aliquots. VIP was diluted with Tyrode’s just before use and 0.1 ml was injected into a port immediately upstream from the sinoatrial node perfusate ŽFig. 1.. Concentrations Ž18.8–600 pmol. were administered randomly except for the highest which was always given last. Isoproterenol HCl was diluted to a concentration of 10y6 M with Tyrode’s and kept on ice during the course of the experiment until 100 pmol in 0.1 ml was given. VIP or isoproterenol was administered no more frequently than every 20 min and longer intervals Žup to 30 min. were allowed after the two highest concentrations of VIP. The time intervals were sufficient for atrial rate and contractile strength to return to control levels before a chemical was given again. Approximately 5 min after each injection, the atrial chamber was drained and rinsed with fresh Tyrode’s. In those experiments in which we investigated desensitization by VIP or isoproterenol, respectively, 100 pmol of isoproterenol or 150 pmol of VIP were injected into the SA artery at the beginning and at the end of the experiment to serve as controls. To record atrial depolarization, a bipolar electrode was
Fig. 1. Apparatus used to record from isolated right atrial preparations.
sutured near the SA node. In some experiments, a second bipolar electrode was sutured to the atrium for pacing the tissue. A special purpose analog board was used to measure the cardiac cycle length ŽA–A intervals in ms.. A Walton Brodie strain gauge was sutured to the free wall between the SA nodal artery and the atrial appendage to record atrial contractile force. The strain gauge arch was oriented parallel to the vena cavae. The atrial muscle was stretched approximately 50% and the strain gauge was then sutured to the muscle, the attachment procedure advocated by the manufacturer ŽWarren Research Products, Charleston, SC.. Although variations in attachment and size of the atrial muscle will affect the absolute magnitude of the response, our changes are normalized by expressing the results as percent of control. Atrial contractile force, the atrial electrogram, A–A interval, and perfusion pressure were displayed on a Gould TA2000 recorder and recorded on a MacLab 8 system. The A–A interval was then converted to an atrial rate. The Marquardt–Levenberg algorithm for nonlinear curve fitting ŽNFIT version 1.1, University of Texas. was used to fit a line to the dose response curves and regression coefficients and EC 50 were calculated from this curve. For determining time course of inotropic or chronotropic effects, we measured times from the injection. As in previous studies from other laboratories w16,20x, we used 50% recovery time as an index of recovery from a cardiac response. Data for time course of the responses were compared by analysis of variance with paired comparisons done by use of Tukey’s post hoc analysis. Data are expressed as means "S.E.M. A probability of F 0.05 was considered significant. The preparation that we used contained the atrial tissue normally perfused by the sinoatrial nodal artery. Under our perfusion conditions, the tissue was stable Žas measured by a change in control atrial rate and contractile force of less than 10%. and viable over a period of at least 4 h. We used this preparation because the effects of substances could be evaluated without the confounding effects of degradation in the blood w27x and because known amounts
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of chemicals could be administered directly into the sinoatrial nodal artery.
3. Results In the absence of stimulation or drug administration, for all our experiments, the mean control atrial rate was 102 " 2 beats per minute Ž n s 35. with a range of 62–125 beats per minute. In 6 dogs, we measured the atrial chronotropic and inotropic responses to administration of VIP through the sinoatrial nodal artery. The percent increases in contractile force were greater than percent changes in atrial rate for any given concentration of VIP ŽFig. 2.. The positive chronotropic effect was almost maximal at 300 pmol of VIP. In contrast, the inotropic effect did not plateau at 300 pmol of VIP but was maximal at 600 pmol. The mean effective concentrations at which 50% of the maximal responses were obtained ŽEC 50 . was similar for atrial rate and for contractile force, 144 pmol and 136 pmol, respectively.
Fig. 2. Effect of VIP on atrial rate Ždotted line. and contractile force Žsolid line. in isolated atria from 6 dogs. Mean percent change from control is plotted at each VIP concentration. Closed arrow denotes the mean EC 50 for the inotropic effect; open arrow denotes the mean EC 50 for the chronotropic effect. The control atrial rate was 102"5 beats per minute. Positive S.E.M. are given for the inotropic effect, negative S.E.M. for the chronotropic effect. Lines fitted by use of the Marquardt– Levenberg algorithm for nonlinear curves.
3.1. Effects of changes in atrial rate on contractile force The development of contractile force in cardiac tissue depends on the frequency of contraction w10x. On the average, the highest dose of VIP resulted in an increase in atrial rate of 90 beats per minute and an increase in contractile force of 173% ŽFig. 2.. To determine what portion of the increase in contractile force with VIP administration was due to the increase in atrial rate alone, we paced the atrium at various rates and examined the relationship between atrial rate and contractile force Ž n s 7.. Increasing the atrial rate from 100 to 190 beats per minute had a moderate effect on contractile force in 4 dogs Žan average increase of 22%. and a larger effect in 3 dogs Žan average increase of 71%.. The linear regression equations fitted to the data were y s 0.23 x y 22 Ž r 2 s 0.64. for the moderately responding group and y s 0.76 x y 73 Ž r 2 s 0.80. for the atria that showed a large dependency of change in contractile force Ž y . on atrial rate Ž x .. Thus on the average about 13–40% of the inotropic effect of VIP can be attributed to the chronotropic alterations caused by VIP. 3.2. Repeated application of a dose of VIP or isoproterenol In these experiments, we used isoproterenol Ž100 pmol. and VIP Ž75 or 150 pmol.. In one series of experiments, the atria were challenged with isoproterenol at the beginning and at the end of 5 injections of VIP. In another series of experiments, the order of presentation of chemicals was reversed Ž n s 6 for each paradigm.. Increases in atrial rate were similar for the 150 pmole dose of VIP and the 100 pmole dose of isoproterenol ŽFig. 3.. Isoproterenol was a
more potent positive inotropic agent than VIP because it caused larger contractile force changes for a smaller amount of chemical ŽFig. 3, panels B and D.. The initial chronotropic response to application of 150 pmol of VIP was larger than subsequent responses to VIP ŽFig. 3, panel A.. In comparing the first to the second injection of 150 pmol of VIP, the difference is not quite statistically significant Ž P s 0.062., but the decrease in response is significant when comparing the first to the third injection and to subsequent injections Ž P - 0.030.. In our paired comparisons, no other significant differences were found Žpanel A, Fig. 3.. Chronotropic responses to repeated injections of isoproterenol appeared to decrease in magnitude over the course of the experiment; the trend was not significant ŽFig. 3, panel C.. Inotropic responses to repeated application of either chemical remained statistically unchanged over the course of five applications ŽFig. 3, panels B and D.. However, the inotropic and chronotropic responses at the end of the experiment were significantly less than that at the beginning of the experiment Žcompare first and last bars in each panel of Fig. 3. in all cases except when comparing the inotropic responses caused by VIP ŽFig. 3, panel D.. Application of the lower amount of VIP Ž75 pmol. resulted in no significant differences between responses Žnot shown.. The prior application of isoproterenol did not affect the responsiveness to VIP or vice versa. 3.3. Time course The onset of the chronotropic and inotropic changes due to VIP application were not significantly different: 13.6 " 1.1 s versus 15.5 " 0.6 s at 75 pmol of VIP, and 11.5 " 1.1
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Fig. 3. Effect of repeated exposures to 150 pmoles VIP Žpanels A and B, filled bars. or to 100 pmoles of isoproterenol Žpanels C and D, open bars. on atrial rate Žleft panels. and contractile force Žright panels. in isolated tissue. In the top panels, isoproterenol was given at the beginning and the end of the experiment Žopen bars.. In the bottom panels, VIP was given at the beginning and the end of the experiment Žfilled bars.. Values are expressed as percent of the control response. Bars represent means with positive S.E.M., n s 6 per group. In upper left panel, asterisks denote changes in responses that are significantly different from the first VIP response Ž P F 0.05..
s versus 12.4 " 0.6 s at 300 pmol VIP, n s 8 per dose. However, once the response was initiated, the time course of changes in atrial rate and contractile force were dose dependent. At 75 pmol of VIP, peak heart rates were
reached at 50 " 4 s and peak inotropic responses occurred at 47 " 2 s. When the dose of VIP was increased to 300 pmol, peak heart rates were reached at 35 " 3 s while peak inotropic changes took 27 " 1 s Ž n s 8; Fig. 4.. Peak heart
Fig. 4. Time course of changes in atrial rate Žpanels A and C. and contractile force Žpanels B and D. after injections of VIP. Injections were at time 0. Times to initiation of a response, to 50% of the peak response, and to 50% recovery were plotted. The solid lines connect the mean responses at each of the times. The upper dotted line connects the mean values plus the standard errors for the time courses of the chronotropic and the inotropic responses; the bottom dotted line is the mean values minus the standard errors. Note that the dotted lines show the variability in both the x and the y axes. n s 8 per group.
D.W. Wallick, S.L. Stuesser Journal of the Autonomic NerÕous System 61 (1996) 257–263
rate and contractile force were reached faster at higher doses of VIP. Recovery from the positive inotropic and chronotropic effects of VIP administration also differed in time course. We measured the times for 50% recovery. At 75 pmol of VIP, atrial rate recovered by 50% from the peak positive chronotropic response at 143 " 16 s, whereas contractile force took about half as long, 78 " 5 s Žpanels A and B, Fig. 4.. At 300 pmol recovery from the chronotropic effect was increased to 242 " 43 s but the inotropic effect was not significantly changed: 60 " 16 s Žpanels C and D, Fig. 4.. Thus as the concentration increases, it takes longer for the atrium to recover from chronotropic effects but the inotropic recovery times are unchanged. At the highest VIP dose, changes in atrial rate lasted about four times longer than changes in contractile force ŽFig. 4..
4. Discussion In our isolated right atrial preparation, exogenous infusion of VIP directly into the sinoatrial nodal artery increased atrial rate. A positive chronotropic effect for VIP was previously demonstrated in intact dogs w21,23,32x, in an isolated rat atrial preparation w6x, and in blood perfused dog atria w15x. Force of contraction was also augmented by VIP, as previously demonstrated in intact dogs w22,32x, dog atria w15x, and in human right atria in vitro w9x. Karasawa determined the effect of VIP on atrial contractility and heart rate in a blood perfused isolated atrial preparation w15x. Maximal changes in atrial rate and in contractile force are similar in our study and that of Karasawa w15x. However, the highest doses of VIP that we and Karasawa used produced responses that are considerably larger than those obtained in intact atropinized and beta-blocked dogs with high vagal stimulation levels Ž20–25% increases in heart rate with 20 Hz stimulation. w13x. Our dose response curves for the effect of VIP on atrial rate are similar to those described by Karasawa who did not report EC 50 values. In dogs, VIP immunoreactive fibers and terminals are denser in the sinoatrial nodal area than in the rest of the atrium w29,30x. This innervation probably originates in parasympathetic ganglia and is thus intrinsic to the heart. VIP immunoreactivity is lower for ventricles than for other parts of the heart w8,30x. If the threshold of the VIP response were proportional to density of innervation, one would expect a chronotropic response at a lower dose than an inotropic one, but the thresholds did not differ. The percent changes in contractile force are greater than the percent changes in atrial rate at the higher concentrations of VIP, greater than 150 pmol ŽFig. 2.. This confirms previous observations w15x. Although there is a positive correlation between increases in atrial rate and increases in contractile force when the atrial preparation was paced, the increase in contractile force due to chronotropic changes
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alone is not large enough to account for all the inotropic response to VIP. The time courses of the chronotropic effects are dose dependent, i.e. as the VIP concentration increases, it takes longer for the heart rate to return to control level. The time course for recovery from the positive inotropic effect was not dose dependent. This lack of correlation may be due to the massaging effect caused by the movement of the heart on the washout of neurotransmitters released into the extracellular space w20x. This massaging effect has been demonstrated in dogs: When sympathetic nerves are stimulated at one level in dogs whose hearts are paced at various levels, the decay time from the positive inotropic response is negatively correlated with heart rate Žas heart rate increases, recovery is faster and thus time needed for 50% recovery decreases.. With repeated short-term exposures to VIP, we found that the atrial chronotropic response was decreased, suggesting a desensitization. No chronotropic desensitization was seen with repeated exposures to isoproterenol. In contrast, we found no inotropic desensitization with either exposure to VIP or to isoproterenol. Anderson and his colleagues found that in open-chest, paced and anesthetized dogs, continuous 90 min infusions of VIP or of isoproterenol reduced subsequent responsiveness of left ventricular muscle to the homologous chemical w3x. Although the chemical concentrations that we used in our experiments are similar to those used in Anderson’s study w30x, our experiments differ from theirs in that we used much shorter exposure times, which may mimic physiological conditions more closely than do the conditions in their experiments. Any desensitization of VIP responses probably occurs by binding of VIP to receptors and subsequent internalization w26x. Repeated applications of VIP in dogs, which leads to desensitization of the inotropic response, does not change the inotropic effects of forskolin, which stimulates adenylate cyclase w3x. Thus application of VIP does not act by desensitizing the adenylate cyclase subunit that is stimulated by forskolin. Moreover, in human colonic carcinoma cells, short-term exposure to VIP causes rapid Žhalf-time of minutes. internalization of VIP receptors w11,26x. If desensitization occurs at the receptor level, one might have expected both the atrial chronotropic and inotropic responses to VIP to be affected. This is not the case. Our differences in VIP desensitization of the inotropic and the chronotropic responses of dog atrium implies that either there are differences in the receptors for these two responses, or that the biochemical cascade that is initiated differs for the two. This linkage from receptor activation to response occurs via activation of a VIP receptor that is linked to a G protein and results in increased intracellular levels of cyclic AMP w1,5,6,11,26x. VIP ultimately affects pacemaker rate in dog Purkinje fibers by shifting the pacemaker If current w4x. In membranous preparations from right atria of dogs, VIP is about as efficient as
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in stimulating cyclic AMP production w6x. We find that in the dog right atrium, isoproterenol, which also acts via G-protein activation and cyclic amp increases w7x, is slightly more effective than VIP in increasing heart rate and contractile force ŽFig. 3.. This corroborates Anderson’s observations on ventricular contractility w2x. In rat brain tissue, three types of VIP receptor have been described. It is doubtful that all these types of VIP receptors exist in canine cardiac tissue because one of the central nervous system receptors binds secretin w19x and infusion of secretin does not affect canine cardiac responses w6x. Two types of VIP binding sites have been described in dog ventricles w12x, but the VIP receptors in dog atrial muscle and in pacemaker tissue have not yet been characterized. The role of VIP in modulating chronotropic and inotropic responses in the heart is still not known. However, if the chronotropic effect is rapidly desensitized by exposure to small amounts of VIP, modulation must begin at the receptor level and be dependent on VIP concentration. DL-isoproterenol
Acknowledgements We thank Jason Frankhouser for his excellent laboratory assistance and David Igel for assistance in statistics. Supported by a grant from the National American Heart Association and by a Research Challenge grant from the Ohio Board of Regents.
References w1x Amiranoff, B. and Rosselin, G., VIP receptors and control of cyclic AMP production. In: S.I. Said ŽEd.., Vasoactive Intestinal Peptide, Raven Press, New York, 1982, pp. 307–322. w2x Anderson, F.L., Kralios, A.C., Hershberger, R. and Bristow, M.R., Effect of vasoactive intestinal peptide on myocardial contractility and coronary blood flow in the dog: comparison with isoproterenol and forskolin, J. Cardiovasc. Pharmacol., 12 Ž1988. 365–371. w3x Anderson, F.L., Kralios, A.C., Hershberger, R. and Bristow, M.R., Desensitization of myocardial but not coronary VIP receptor-mediated responses in dog, Am. J. Physiol., 255 Ž1988. H601–H607. w4x Chang, F., Yu, H. and Cohen, I.S., Actions of vasoactive intestinal peptide and neuropeptide Y on the pacemaker current in canine Purkinje fibers, Circ. Res., 74 Ž1994. 157–162. w5x Chatelain, P., Robberecht, P., Waelbroeck, M., DeNeef, P., Cumus, J.C., Huu, A.N., Roba, J. and Christophe, J., Topographical distribution of the secretin and VIP-stimulated adenylate cyclase system in the heart of five animal species, Pflugers Arch., 397 Ž1983. 100–105. ¨ w6x Christophe, J., Waelbroeck, M., Chatelain, P. and Robberecht, P., Heart receptors for VIP, PHI and secretin are able to activate adenylate cyclase and to mediate inotropic and chronotropic effects. Species variations and physiopathology, Peptides, 5 Ž1984. 341–353. w7x Drummond, G.I. and Severson, D.L., Cyclic nucleotides and cardiac function, Circ. Res., 44 Ž1979. 145–151. w8x Forssmann, W.G., Triepel, J., Doffner, C., Heym, C.H., Cuevas, P., Noble, M.I.M. and Yanaihara, W., Vasoactive intestinal peptide in the heart, Ann. N.Y. Acad. Sci., 527 Ž1988. 405–420.
w9x Franco-Cereceda, A., Bengtsson, L. and Lundberg, J.M., Inotropic effects of calcitonin gene-related, vasoactive intestinal polypeptide and somatostatin on the human right atrium in vitro, European J. Pharmacol., 134 Ž1987. 69–76. w10x Freeman, G.L., Little, W.C. and O’Rourke, R.A., Influence of heart rate on left ventricular performance in conscious dogs, Circ. Res., 61 Ž1987. 455–464. w11x Gozes, I. and Brenneman, D.E., VIP: Molecular Biology and Neurobiological Function. In: Molecular Neurobiology, 3 Ž1989. 201–236. w12x Hershberger, R.E., Anderson, F.L. and Bristow, M.R., Vasoactive intestinal peptide receptor in failing human ventricular myocardium exhibits increased affinity and decreased density, Circ. Res., 65 Ž1989. 283–294. w13x Hill, M.R.S., Wallick, D.W., Martin, P.J. and Levy, M.N., Frequency dependence of vasoactive intestinal polypeptide release and vagally induced tachycardia in the canine heart, J. Auton. Nerv. Syst., 43 Ž1993. 117–122. w14x Hill, M.R.S., Wallick, D.W., Martin, P.J. and Levy, M.N., Effects of repetitive vagal stimulation on heart rate and on cardiac vasoactive intestinal polypeptide efflux, Am. J. Physiol., 168 Ž1995. H1939– H1946. w15x Karasawa, Y., Furukawa, Y., Ren, L.M., Takei, M., Murakami, M., Narita, M. and Chiba, S., Cardiac responses to VIP and VIP-ergiccholinergic interaction in isolated dog heart preparations, European J. Pharmacol., 187 Ž1990. 9–17. w16x Johnson, G.L. and Kahn, Jr., J.B., Cocaine and antihistaminic compounds: comparisons of effects of some cardiovascular actions of norepinephrine, tyramine and bretylium, J. Pharmacol. Exp. Ther., 152 Ž1966. 458–468. w17x Loffelholz, K. and Pappana, A.J., The parasympathetic neuroeffector ¨ junction of the heart, Pharmacol. Rev., 27 Ž1985. 1–24. w18x Lundberg, J.M., Evidence for coexistence of vasoactive polypeptide ŽVIP. and acetylcholine in neurons of cat exocrine glands, Acta Physiol. Scand., 112 Ž1981. 1–57. w19x Martin, J.-L., Feinstein, D.L., Yu, N., Sorg, O., Rossier, C. and Magistretti, P.J., VIP receptor subtypes in mouse cerebral cortex: evidence for a differential localization in astrocytes, microvessels and synaptosomal membranes, Brain Res., 587 Ž1992. 1–12. w20x Masuda, Y. and Levy, M.N., Heart rate as a determinant of the decay rate of the cardiac inotropic response to sympathetic nervous activity, Canadian J. Physiol. Pharmacol., 61 Ž1983. 1374–1381. w21x Rigel, D.F., Effects of neuropeptides on heart rate in dogs: comparison of VIP, PHI, NPY, CGRP, and NT, Am. J. Physiol., 255 Ž1988. H311–H317. w22x Rigel, D.F., Grupp, I.L., Balasubramaniam, A. and Grupp, G., Contractile effects of cardiac neuropeptides in isolated canine atrial and ventricular muscles, Am. J. Physiol., 257 Ž1989. H1082–H1087. w23x Rigel, D.F. and Lathrop, D.A., Vasoactive intestinal polypeptide facilitates atrioventricular nodal conduction and shortens atrial ventricular refractory periods in conscious and anesthetized dogs, Circ. Res., 67 Ž1990. 1323–1333. w24x Rigel, D.F. and Lathrop, D.A., Vasoactive intestinal polypeptide enhances automaticity of supraventricular pacemakers in anesthetized dogs, Am. J. Physiol., 261 Ž1991. H463–H468. w25x Rigel, D.F., Lipson, D. and Katona, P.G., Excess tachycardia; heart rate after antimuscarinic agents in conscious dogs, Am. J. Physiol., 246 Ž1984. H168–H173. w26x Rosselin, G., Anteunis, A., Astesano, A., Boissard, C., Gali, P., Hejblum, G. and Marie, J.C., Regulation of the vasoactive intestinal peptide receptor, Ann. N.Y. Acad. Sci., 527 Ž1988. 220–237. w27x Strauss, E., Keltz, T.N. and Yalow, R.S., Enzymatic degradation of VIP. In: S.I. Said ŽEd.., Vasoactive Intestinal Peptide, Raven Press, New York, 1982, pp. 333–339. w28x Unverferth, D.V., O’Dorisio, T.M., Wuir, W.W., White, W.W., Miller, M.M., Hamlin, R.L. and Magorien, R.D., Effect of vasoactive intestinal polypeptide on the canine cardiovascular system, J. Lab. Clin. Med., 106 Ž1985. 542–550.
D.W. Wallick, S.L. Stuesser Journal of the Autonomic NerÕous System 61 (1996) 257–263 w29x Weihe, E. and Reinecke, M., Peptidergic innervation of the mammalian sinus nodes: vasoactive intestinal polypeptide, neurotensin, substance P, Neurosci. Lett., 26 Ž1981. 283–288. w30x Weihe, E., Reinecke, M. and Forssmann, W.G., Distribution of vasoactive intestinal polypeptide-like immunoreactivity in the mammalian heart: Interrelation with neurotensin- and substance P-like immunoreactive nerves, Cell Tiss. Res., 236 Ž1984. 527–540.
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Comparison of inotropic and chronotropic effects of vasoactive intestinal peptide in isolated dog atria Don W. Wallick a , Sherry L. Stuesse b
b,)
a DiÕision of InÕestigatiÕe Medicine, Mt. Sinai Medical Center, CleÕeland, OH 44106, USA Neurobiology Department, Northeastern Ohio UniÕersities College of Medicine, P.O. Box 95, Rootstown, OH 44272-0095, USA
Received 25 September 1995; revised 20 June 1996; accepted 17 July 1996
Abstract The positive chronotropic and inotropic effects of vasoactive intestinal peptide, VIP, were studied in an isolated canine right atrial preparation. Atria were removed, maintained in a bath, and perfused with Tyrode’s solution. Contractile force and atrial depolarization were measured. VIP Ž18.8–600 pmol. was injected into a cannulated sinoatrial nodal artery and dose response curves were obtained. The mean EC 50 was similar for the inotropic and the chronotropic responses Ž136 and 144 pmol, respectively.. Time courses of the onset and of recovery from the responses were measured. Times for onset of VIP effects were similar but, once the effect was initiated, rate of development of the response and recovery time from the responses were dose dependent. The increases in atrial rate lasted two to four times longer than did the increases in contractile force. Recovery from the chronotropic and inotropic responses to VIP differ, suggesting that the intracellular responses are coupled differently to the receptors. The responses to VIP were compared to those of 100 pmol isoproterenol, another positive chronotropic and inotropic agent. Isoproterenol was a slightly more potent chronotropic and inotropic agent than VIP. Desensitization of the responses was determined. Repeated exposures to VIP decreased the chronotropic response but not the inotropic response to VIP. There was no significant decrease in responsiveness to isoproterenol. Keywords: Autonomic nervous system; Parasympathetic; Cardiac; Contractile force; Neuropeptide; Heart rate; Atrium; Isoproterenol; Receptors
1. Introduction Vasoactive intestinal peptide ŽVIP. is a 28 amino acid polypeptide that may coexist with acetylcholine in parasympathetic postganglionic nerve terminals in the heart w8,17,18,29–31x. Two major types of VIP receptors have been characterized in the heart; one that has a high affinity for VIP and the second that has a high affinity for secretin, a VIP-like molecule w6x. The former is found in Cynomolgus monkeys, humans, and dogs, while the latter is found in rats. Thus dogs may be an appropriate model for research into the clinical effects of this peptide. In betablocked, atropinized dogs, vagal efferent stimulation increased heart rate and contractile force w25x. This effect may be due to the release of VIP w21x. VIP is released from dog atria when parasympathetic nerves are stimulated at a high frequency w13,14x. However, the physiological actions
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of VIP on atria have not been well characterized. VIP that is administered intravenously or directly into an appropriate coronary artery increases heart rate and cardiac contractile force w2,21,15,28x. VIP also has a positive inotropic action on isolated dog atrial and ventricular muscles w22x and a positive inotropic effect on paced, isolated pieces of human atria ww9xx. In the studies reported herein, we further characterize the inotropic and chronotropic responses of isolated dog atria to VIP. We compare dose response curves, the time course of responses, and the response to repeated exposures to low levels of VIP or to isoproterenol, a positive chronotropic and inotropic agent that activates beta adrenergic receptors.
2. Materials and methods Mongrel dogs of either sex Ž18–27 kg. were premedicated with morphine Ž1–2 mgrkg, i.m.. and anesthetized with alpha-chloralose Ž100 mgrkg, i.v... Thirty-five dogs were used. The right side of the chest was opened at the
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fifth intercostal space. The pericardium was opened, and a cradle was made by suturing the edges of the pericardium to the chest wall. We isolated the sinoatrial nodal artery and cannulated the artery with a 22-gauge catheter filled with Heplock Ž100 units of heparinrml.. The azygos vein, inferior vena cava, and superior vena cava were tied. The entire free wall of the right atrium, including the SA node, was removed from the dog and immersed in cold, modified Tyrode’s solution Ž120 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl 2 , 1.1 mM MgCl 2 , 0.5 mM NaH 2 PO4 , 25 mM NaHCO 2 , 5.4 mM dextrose, 0.06 mM L-ascorbic acid, bubbled with 95% O 2 –5%CO 2 .. Removal of the atrial appendage and the blockage of blood flow to the heart resulted in death while the animal was completely anesthetized, and the body was incinerated. Care and use of the dogs was in accordance with guidelines set down by the National Institutes of Health and the American Association for Accreditation of Laboratory Animal Care. We cauterized any severed branches of the SA nodal artery. The atrium was transferred to a tissue bath, superfused with Tyrode’s solution, and the SA nodal artery was perfused with warm, oxygenated Tyrode’s solution Ž358C.. The perfusion pressure was maintained at about 80 mmHg by altering the pump speed Žfrom 1 to 4 mlrmin.. In some experiments, atropine Ž30 nM. and propranolol Ž330 nM. were added to the Tyrode’s solution to block the muscarinic and beta-adrenergic receptors, respectively. Betaadrenergic blockade does not diminish the VIP effect on atrial and ventricular pacemakers w23,24x. VIP is not known to be released independently of acetylcholine, however, in some systems, atropine, a muscarinic receptor blocker, may actually enhance VIP release via presynaptic modulation w18,31x. Vasoactive intestinal peptide, VIP, ŽaPGAS350, Bachchem. was dissolved in distilled water Ž1 mgrml. and stored at y708C in 20 m l aliquots. VIP was diluted with Tyrode’s just before use and 0.1 ml was injected into a port immediately upstream from the sinoatrial node perfusate ŽFig. 1.. Concentrations Ž18.8–600 pmol. were administered randomly except for the highest which was always given last. Isoproterenol HCl was diluted to a concentration of 10y6 M with Tyrode’s and kept on ice during the course of the experiment until 100 pmol in 0.1 ml was given. VIP or isoproterenol was administered no more frequently than every 20 min and longer intervals Žup to 30 min. were allowed after the two highest concentrations of VIP. The time intervals were sufficient for atrial rate and contractile strength to return to control levels before a chemical was given again. Approximately 5 min after each injection, the atrial chamber was drained and rinsed with fresh Tyrode’s. In those experiments in which we investigated desensitization by VIP or isoproterenol, respectively, 100 pmol of isoproterenol or 150 pmol of VIP were injected into the SA artery at the beginning and at the end of the experiment to serve as controls. To record atrial depolarization, a bipolar electrode was
Fig. 1. Apparatus used to record from isolated right atrial preparations.
sutured near the SA node. In some experiments, a second bipolar electrode was sutured to the atrium for pacing the tissue. A special purpose analog board was used to measure the cardiac cycle length ŽA–A intervals in ms.. A Walton Brodie strain gauge was sutured to the free wall between the SA nodal artery and the atrial appendage to record atrial contractile force. The strain gauge arch was oriented parallel to the vena cavae. The atrial muscle was stretched approximately 50% and the strain gauge was then sutured to the muscle, the attachment procedure advocated by the manufacturer ŽWarren Research Products, Charleston, SC.. Although variations in attachment and size of the atrial muscle will affect the absolute magnitude of the response, our changes are normalized by expressing the results as percent of control. Atrial contractile force, the atrial electrogram, A–A interval, and perfusion pressure were displayed on a Gould TA2000 recorder and recorded on a MacLab 8 system. The A–A interval was then converted to an atrial rate. The Marquardt–Levenberg algorithm for nonlinear curve fitting ŽNFIT version 1.1, University of Texas. was used to fit a line to the dose response curves and regression coefficients and EC 50 were calculated from this curve. For determining time course of inotropic or chronotropic effects, we measured times from the injection. As in previous studies from other laboratories w16,20x, we used 50% recovery time as an index of recovery from a cardiac response. Data for time course of the responses were compared by analysis of variance with paired comparisons done by use of Tukey’s post hoc analysis. Data are expressed as means "S.E.M. A probability of F 0.05 was considered significant. The preparation that we used contained the atrial tissue normally perfused by the sinoatrial nodal artery. Under our perfusion conditions, the tissue was stable Žas measured by a change in control atrial rate and contractile force of less than 10%. and viable over a period of at least 4 h. We used this preparation because the effects of substances could be evaluated without the confounding effects of degradation in the blood w27x and because known amounts
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of chemicals could be administered directly into the sinoatrial nodal artery.
3. Results In the absence of stimulation or drug administration, for all our experiments, the mean control atrial rate was 102 " 2 beats per minute Ž n s 35. with a range of 62–125 beats per minute. In 6 dogs, we measured the atrial chronotropic and inotropic responses to administration of VIP through the sinoatrial nodal artery. The percent increases in contractile force were greater than percent changes in atrial rate for any given concentration of VIP ŽFig. 2.. The positive chronotropic effect was almost maximal at 300 pmol of VIP. In contrast, the inotropic effect did not plateau at 300 pmol of VIP but was maximal at 600 pmol. The mean effective concentrations at which 50% of the maximal responses were obtained ŽEC 50 . was similar for atrial rate and for contractile force, 144 pmol and 136 pmol, respectively.
Fig. 2. Effect of VIP on atrial rate Ždotted line. and contractile force Žsolid line. in isolated atria from 6 dogs. Mean percent change from control is plotted at each VIP concentration. Closed arrow denotes the mean EC 50 for the inotropic effect; open arrow denotes the mean EC 50 for the chronotropic effect. The control atrial rate was 102"5 beats per minute. Positive S.E.M. are given for the inotropic effect, negative S.E.M. for the chronotropic effect. Lines fitted by use of the Marquardt– Levenberg algorithm for nonlinear curves.
3.1. Effects of changes in atrial rate on contractile force The development of contractile force in cardiac tissue depends on the frequency of contraction w10x. On the average, the highest dose of VIP resulted in an increase in atrial rate of 90 beats per minute and an increase in contractile force of 173% ŽFig. 2.. To determine what portion of the increase in contractile force with VIP administration was due to the increase in atrial rate alone, we paced the atrium at various rates and examined the relationship between atrial rate and contractile force Ž n s 7.. Increasing the atrial rate from 100 to 190 beats per minute had a moderate effect on contractile force in 4 dogs Žan average increase of 22%. and a larger effect in 3 dogs Žan average increase of 71%.. The linear regression equations fitted to the data were y s 0.23 x y 22 Ž r 2 s 0.64. for the moderately responding group and y s 0.76 x y 73 Ž r 2 s 0.80. for the atria that showed a large dependency of change in contractile force Ž y . on atrial rate Ž x .. Thus on the average about 13–40% of the inotropic effect of VIP can be attributed to the chronotropic alterations caused by VIP. 3.2. Repeated application of a dose of VIP or isoproterenol In these experiments, we used isoproterenol Ž100 pmol. and VIP Ž75 or 150 pmol.. In one series of experiments, the atria were challenged with isoproterenol at the beginning and at the end of 5 injections of VIP. In another series of experiments, the order of presentation of chemicals was reversed Ž n s 6 for each paradigm.. Increases in atrial rate were similar for the 150 pmole dose of VIP and the 100 pmole dose of isoproterenol ŽFig. 3.. Isoproterenol was a
more potent positive inotropic agent than VIP because it caused larger contractile force changes for a smaller amount of chemical ŽFig. 3, panels B and D.. The initial chronotropic response to application of 150 pmol of VIP was larger than subsequent responses to VIP ŽFig. 3, panel A.. In comparing the first to the second injection of 150 pmol of VIP, the difference is not quite statistically significant Ž P s 0.062., but the decrease in response is significant when comparing the first to the third injection and to subsequent injections Ž P - 0.030.. In our paired comparisons, no other significant differences were found Žpanel A, Fig. 3.. Chronotropic responses to repeated injections of isoproterenol appeared to decrease in magnitude over the course of the experiment; the trend was not significant ŽFig. 3, panel C.. Inotropic responses to repeated application of either chemical remained statistically unchanged over the course of five applications ŽFig. 3, panels B and D.. However, the inotropic and chronotropic responses at the end of the experiment were significantly less than that at the beginning of the experiment Žcompare first and last bars in each panel of Fig. 3. in all cases except when comparing the inotropic responses caused by VIP ŽFig. 3, panel D.. Application of the lower amount of VIP Ž75 pmol. resulted in no significant differences between responses Žnot shown.. The prior application of isoproterenol did not affect the responsiveness to VIP or vice versa. 3.3. Time course The onset of the chronotropic and inotropic changes due to VIP application were not significantly different: 13.6 " 1.1 s versus 15.5 " 0.6 s at 75 pmol of VIP, and 11.5 " 1.1
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Fig. 3. Effect of repeated exposures to 150 pmoles VIP Žpanels A and B, filled bars. or to 100 pmoles of isoproterenol Žpanels C and D, open bars. on atrial rate Žleft panels. and contractile force Žright panels. in isolated tissue. In the top panels, isoproterenol was given at the beginning and the end of the experiment Žopen bars.. In the bottom panels, VIP was given at the beginning and the end of the experiment Žfilled bars.. Values are expressed as percent of the control response. Bars represent means with positive S.E.M., n s 6 per group. In upper left panel, asterisks denote changes in responses that are significantly different from the first VIP response Ž P F 0.05..
s versus 12.4 " 0.6 s at 300 pmol VIP, n s 8 per dose. However, once the response was initiated, the time course of changes in atrial rate and contractile force were dose dependent. At 75 pmol of VIP, peak heart rates were
reached at 50 " 4 s and peak inotropic responses occurred at 47 " 2 s. When the dose of VIP was increased to 300 pmol, peak heart rates were reached at 35 " 3 s while peak inotropic changes took 27 " 1 s Ž n s 8; Fig. 4.. Peak heart
Fig. 4. Time course of changes in atrial rate Žpanels A and C. and contractile force Žpanels B and D. after injections of VIP. Injections were at time 0. Times to initiation of a response, to 50% of the peak response, and to 50% recovery were plotted. The solid lines connect the mean responses at each of the times. The upper dotted line connects the mean values plus the standard errors for the time courses of the chronotropic and the inotropic responses; the bottom dotted line is the mean values minus the standard errors. Note that the dotted lines show the variability in both the x and the y axes. n s 8 per group.
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rate and contractile force were reached faster at higher doses of VIP. Recovery from the positive inotropic and chronotropic effects of VIP administration also differed in time course. We measured the times for 50% recovery. At 75 pmol of VIP, atrial rate recovered by 50% from the peak positive chronotropic response at 143 " 16 s, whereas contractile force took about half as long, 78 " 5 s Žpanels A and B, Fig. 4.. At 300 pmol recovery from the chronotropic effect was increased to 242 " 43 s but the inotropic effect was not significantly changed: 60 " 16 s Žpanels C and D, Fig. 4.. Thus as the concentration increases, it takes longer for the atrium to recover from chronotropic effects but the inotropic recovery times are unchanged. At the highest VIP dose, changes in atrial rate lasted about four times longer than changes in contractile force ŽFig. 4..
4. Discussion In our isolated right atrial preparation, exogenous infusion of VIP directly into the sinoatrial nodal artery increased atrial rate. A positive chronotropic effect for VIP was previously demonstrated in intact dogs w21,23,32x, in an isolated rat atrial preparation w6x, and in blood perfused dog atria w15x. Force of contraction was also augmented by VIP, as previously demonstrated in intact dogs w22,32x, dog atria w15x, and in human right atria in vitro w9x. Karasawa determined the effect of VIP on atrial contractility and heart rate in a blood perfused isolated atrial preparation w15x. Maximal changes in atrial rate and in contractile force are similar in our study and that of Karasawa w15x. However, the highest doses of VIP that we and Karasawa used produced responses that are considerably larger than those obtained in intact atropinized and beta-blocked dogs with high vagal stimulation levels Ž20–25% increases in heart rate with 20 Hz stimulation. w13x. Our dose response curves for the effect of VIP on atrial rate are similar to those described by Karasawa who did not report EC 50 values. In dogs, VIP immunoreactive fibers and terminals are denser in the sinoatrial nodal area than in the rest of the atrium w29,30x. This innervation probably originates in parasympathetic ganglia and is thus intrinsic to the heart. VIP immunoreactivity is lower for ventricles than for other parts of the heart w8,30x. If the threshold of the VIP response were proportional to density of innervation, one would expect a chronotropic response at a lower dose than an inotropic one, but the thresholds did not differ. The percent changes in contractile force are greater than the percent changes in atrial rate at the higher concentrations of VIP, greater than 150 pmol ŽFig. 2.. This confirms previous observations w15x. Although there is a positive correlation between increases in atrial rate and increases in contractile force when the atrial preparation was paced, the increase in contractile force due to chronotropic changes
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alone is not large enough to account for all the inotropic response to VIP. The time courses of the chronotropic effects are dose dependent, i.e. as the VIP concentration increases, it takes longer for the heart rate to return to control level. The time course for recovery from the positive inotropic effect was not dose dependent. This lack of correlation may be due to the massaging effect caused by the movement of the heart on the washout of neurotransmitters released into the extracellular space w20x. This massaging effect has been demonstrated in dogs: When sympathetic nerves are stimulated at one level in dogs whose hearts are paced at various levels, the decay time from the positive inotropic response is negatively correlated with heart rate Žas heart rate increases, recovery is faster and thus time needed for 50% recovery decreases.. With repeated short-term exposures to VIP, we found that the atrial chronotropic response was decreased, suggesting a desensitization. No chronotropic desensitization was seen with repeated exposures to isoproterenol. In contrast, we found no inotropic desensitization with either exposure to VIP or to isoproterenol. Anderson and his colleagues found that in open-chest, paced and anesthetized dogs, continuous 90 min infusions of VIP or of isoproterenol reduced subsequent responsiveness of left ventricular muscle to the homologous chemical w3x. Although the chemical concentrations that we used in our experiments are similar to those used in Anderson’s study w30x, our experiments differ from theirs in that we used much shorter exposure times, which may mimic physiological conditions more closely than do the conditions in their experiments. Any desensitization of VIP responses probably occurs by binding of VIP to receptors and subsequent internalization w26x. Repeated applications of VIP in dogs, which leads to desensitization of the inotropic response, does not change the inotropic effects of forskolin, which stimulates adenylate cyclase w3x. Thus application of VIP does not act by desensitizing the adenylate cyclase subunit that is stimulated by forskolin. Moreover, in human colonic carcinoma cells, short-term exposure to VIP causes rapid Žhalf-time of minutes. internalization of VIP receptors w11,26x. If desensitization occurs at the receptor level, one might have expected both the atrial chronotropic and inotropic responses to VIP to be affected. This is not the case. Our differences in VIP desensitization of the inotropic and the chronotropic responses of dog atrium implies that either there are differences in the receptors for these two responses, or that the biochemical cascade that is initiated differs for the two. This linkage from receptor activation to response occurs via activation of a VIP receptor that is linked to a G protein and results in increased intracellular levels of cyclic AMP w1,5,6,11,26x. VIP ultimately affects pacemaker rate in dog Purkinje fibers by shifting the pacemaker If current w4x. In membranous preparations from right atria of dogs, VIP is about as efficient as
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in stimulating cyclic AMP production w6x. We find that in the dog right atrium, isoproterenol, which also acts via G-protein activation and cyclic amp increases w7x, is slightly more effective than VIP in increasing heart rate and contractile force ŽFig. 3.. This corroborates Anderson’s observations on ventricular contractility w2x. In rat brain tissue, three types of VIP receptor have been described. It is doubtful that all these types of VIP receptors exist in canine cardiac tissue because one of the central nervous system receptors binds secretin w19x and infusion of secretin does not affect canine cardiac responses w6x. Two types of VIP binding sites have been described in dog ventricles w12x, but the VIP receptors in dog atrial muscle and in pacemaker tissue have not yet been characterized. The role of VIP in modulating chronotropic and inotropic responses in the heart is still not known. However, if the chronotropic effect is rapidly desensitized by exposure to small amounts of VIP, modulation must begin at the receptor level and be dependent on VIP concentration. DL-isoproterenol
Acknowledgements We thank Jason Frankhouser for his excellent laboratory assistance and David Igel for assistance in statistics. Supported by a grant from the National American Heart Association and by a Research Challenge grant from the Ohio Board of Regents.
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w31x Whittaker, W.P., Vasoactive intestinal polypeptide ŽVIP. as a cholinergic co-transmitter: some recent results, Cell Biology International Reports, 13 Ž1989. 1039–1052. w32x Xenopoulos, N.P. and Applegate, R.J., Effects of vasoactive intestinal peptide on myocardial performance, Am. J. Physiol., 266 Ž1994. H399–H405.