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Effects of adrenergic and cholinergic drugs on splenic arteries and veins from hooded seals (Cystophora cristata)
Comparative Biochemistry and Physiology Part A 120 (1998) 277 – 281
Effects of adrenergic and cholinergic drugs on splenic arteries and veins from hooded seals (Cystophora cristata) A. Cabanac *, L.P. Folkow, A.S. Blix Department of Arctic Biology and Institute of Medical Biology, Uni6ersity of Tromsø, Tromsø, Norway Received 15 October 1997; received in revised form 13 January 1998; accepted 28 January 1998
Abstract Isolated ring preparations of arteries and veins from hooded seal spleens were subjected in vitro to adrenaline (A), noradrenaline (NA), isoprenaline (Iso), and acetylcholine (ACh), alone or in combination with the blockers phentolamine (Phe), propranolol (Pro), and atropine (Atr). Both arteries and veins constricted in response to A (the estimated effective dose required for half-maximal response (ED50) was 3.3 and 0.2 mM, for arteries and veins, respectively) and NA (estimated ED50 was 1.5 and 0.6 mM, for arteries and veins, respectively), but these effects were abolished when the drugs were given in combination with the a-adrenoceptor blocker Phe. The responses of arteries and veins to ACh and the b-adrenoceptor agonist Iso were minor and inconsistent, and were completely abolished when combined with their respective blockers (Atr and Pro, respectively). The ED50 for both A and NA are quite high in relation to normal plasma levels of A and NA in seals. This implies that these vessels (and, hence, the supply of blood to the spleen) primarily are subjected to neurogenic, rather than humoral physiological control. © 1998 Elsevier Science Inc. All rights reserved. Keywords: Pinnipeds; Diving; Spleen; Splenic vascular resistance; Vasomotor control; In vitro; Adrenaline; Noradrenaline; Isoprenaline; Acetylcholine; Vasoconstriction; Vasodilation; Vessels
1. Introduction A series of dramatic cardiovascular changes take place in seals when they dive for extended periods of time. These changes include a profound peripheral vasoconstriction which is accompanied by intense bradycardia [4]. The changes serve to reduce the rate of utilization of endogenous oxygen stores, and thereby extend the submersion period. Another change which may be observed is a substantial increase in the number of circulating red blood cells [9,27]. Several authors [6,8,19,26,27,33] have suggested that the spleen is the origin of the increased hematocrit during diving, and that this mechanism probably is important for the ability of these mammals to dive repeatedly and for * Corresponding author. Present address: 263 route du fleuve, Pointe-au-pe`re, Que´bec G5M 1K7, Canada. 0742-8413/98/$19.00 © 1998 Elsevier Science Inc. All rights reserved. PII S1095-6433(98)00029-4
extended periods of time. The proposed function of splenic contraction and release of (oxygenated) red blood cells has been that this increases the available blood oxygen store [32], or that it improves the oxygencarrying capacity and, hence, reduces surface time during repeated diving [10]. Recently, Cabanac and coworkers [7] demonstrated in vitro that the spleens of the hooded seal (Cystophora cristata) and the harp seal (Phoca groenlandica) are capable of active and forceful contraction under aadrenergic stimulation. However, that study did not include any examination of the vasomotor responses of the splenic vasculature. Such responses are obviously important since they are the means by which blood supply to the spleen—which determines both splenic filling and subsequent drainage—is controled. The purpose of the present study, therefore, was to investigate the physiological control of the splenic vas-
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A. Cabanac et al. / Comparati6e Biochemistry and Physiology, Part A 120 (1998) 277–281
culature by studying in vitro the effects of adrenergic and cholinergic drugs on splenic arteries and veins from the hooded seal. Arteries and veins of about 2-mm diameter were chosen in preference to smaller pre-capillary vessels, for two reasons. First, these vessels were observed to display vigorous contractile responses upon perfusion of isolated intact spleens with modified Krebs’ solutions containing adrenaline (A) (Cabanac, Folkow and Blix, unpublished observations). Second, it has been demonstrated repeatedly that vasomotor control of the blood supply to several tissues/organs of diving animals is exercised at the level of arteries, rather than in the pre-capillary arterioles [5,16,31]. In this way, vasoconstriction can be maintained for extended periods of time during diving, in spite of the accumulation of vasodilator metabolites in the ischemic tissues [16].
2. Materials and methods A total of four hooded seals (C. cristata), two of each sex, were caught in drift-ice east of Greenland (the West Ice, 71°N, 18°W) during expeditions with R.V. ‘Jan Mayen’ in March/April 1994 and 1995. Two pups which were about 1-week old, weighing 26 and 24 kg, respectively, were killed and used immediately on board the research vessel. Two others were brought alive to the Department of Arctic Biology at Tromsø, Norway, and kept indoors in a large freshwater tank, under simulated natural light conditions (70°N) until used, at which time they weighed 82 and 67 kg, respectively. The animals were fed capelin (Mallotus 6illosus) twice a day. All seals were killed instantaneously by use of a ‘hakapik’, immediately followed by bleeding, without any use of anesthetics, as authorized by Norwegian Veterinary Authorities. All animals were taken under permits issued by the Norwegian Government, and the experiments were approved by the Norwegian Committee on Ethics in Animal Experimentation. The spleens were quickly removed and transferred into a bath containing 0.9% NaCl solution for in vitro preparation. The description of the approach for splenectomy and subsequent handling of the spleen is given in [7]. Small rings (: 5 mm long and 2 – 3 mm diameter) of splenic arteries and veins were excised from the surface region of the spleen and carefully cleaned from extraneous tissue. The ring samples were mounted in separate 30-ml organ baths, as described by Johnsen and Folkow [20], and connected to force displacement transducers (Type FT03; Grass Medical Instruments, MA) for isometric recordings of tension. Contact with the endothelial surface was carefully avoided in this process in order to preserve endothelial integrity. The transducer signals fed via an amplifier (Universal Am-
plifier installed into a Type 5900 Signal Conditioner; Gould, OH) into a thermal array recorder (TA 4000; Gould, OH) and the responses were recorded as a continuous analog curve. The baths were filled with modified Krebs solution (see below) and kept at constant temperature (37.49 0.4°C) by circulating a jacket which surrounded the bath with thermostatically controled water. The saline inside the baths was oxygenated by bubbling with a gas mixture containing 95% O2 and 5% CO2. After each step in any experiment, the saline was changed three times to rinse the vessels and the baths. Before any drug injections were made, the vessels were pre-stretched for 1 h at resting tension-levels of :1 g for the veins and : 4 g for the arteries. These pre-stretch levels are similar to those employed in studies of similar-sized and -typed vessels in previous studies [1,23]. Drugs were carefully added directly into the baths by use of a 20–200 ml micro-pipette (Finnpipette, Labsystems, Helsinki, Finland). The following experiments were carried out in succession on both arteries and veins during the sessions.
2.1. Experiment 1 Vessels were subjected to graded concentrations of A (0.4, 2.5, 7.0, 29.3 and 141× 10 − 7 M) and noradrenaline (NA) (24.3 and 153 × 10 − 7 M) separately.
2.2. Experiment 2 NA (153× 10 − 7 M) was added 4–6 min after the a-adrenergic blocker phentolamine (Phe) had been added to yield concentrations \ 1450 × 10 − 7 M).
2.3. Experiment 3 Vessels were subjected to graded concentrations of acetylcholine (ACh) (5.6, 140, 174, 196, 331 and 421 × 10 − 7 M) alone, and 2–3 min after atropine (Atr) had been added to yield concentrations of 1400–2190 × 10 − 7 M).
2.4. Experiment 4 Vessels were subjected to graded concentrations of the b-adrenergic agonist isoprenaline (Iso) (2.6, 26 and 260 × 10 − 7 M).
2.5. Experiment 5 NA (153 × 10 − 7 M) was added 5 min before ACh or Iso were added to yield concentrations of 5.6 or 140 × 10 − 7 M, and 2.6 or 260 × 10 − 7 M, respectively. The following drugs were used in the experiments: A, Atr, NA, (1 mg.ml − 1, Nycomed Pharma, Oslo, Norway), Iso (0.2 mg.ml − 1, Nycomed Pharma, Oslo, Nor-
A. Cabanac et al. / Comparati6e Biochemistry and Physiology, Part A 120 (1998) 277–281
way), ACh (10 mg.ml − 1, Nycomed Pharma, Oslo, Norway), and Phe (Regitine 10 mg.ml − 1, Ciba-Geigy, Basel, Switzerland). The composition of the physiological saline (in mM) was: 119.0 NaCl, 25.0 NaHCO3, 4.7 KCl, 2.5 CaCl2, 1.18 KH2PO4, 1.17 MgSO4, 5.5 glucose, and 0.027 EDTA.
3. Results When subjected to graded concentrations of A and NA, both arteries and veins displayed graded constriction responses (Fig. 1). The responses were monotonic functions of the drug doses, and reached a plateau for each dose. The maximal contraction tension for arteries was : 40 g and for veins :10 g. The dose – response curves for A and NA were similar for the arteries (Student t-test on the slopes of the transformed log–log regression lines: t= 1.12, P \ 0.05). The slope of the transformed log–log regression line for the veins was smaller for A than for NA (t=3.50 – 3.18, P B 0.05). However, the difference was very small (Fig. 1), and the effect of the two catecholamines is obviously similar. The arteries constricted about four times more strongly than the veins when subjected to identical doses of A or NA. The effective dose required for a half-maximal response (ED50) was estimated to be 3.3 and 1.5 mM for arteries, and 0.2 and 0.6 mM for veins, in response to A and NA, respectively.
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The general a-adrenoceptor antagonist Phe, when given to both arteries and veins before a near-maximal dose of NA, blocked at the least 99% of the control responses to the latter. ACh was found to cause very small changes in vascular tension in both arteries and veins. Moreover, the tension changes varied in direction both with the dose given, and from experiment to experiment. (The largest observed relaxation was −12% of the prestretch tension ( : 1 g) in a vein, while the largest tension developed by a constricting artery or vein was only about 3.0% of the tension developed by the vessels when the highest dose of NA was given). Atr blocked the effects of ACh in both arteries and veins. The b-adrenergic agonist Iso, likewise, caused very minor responses which differed quantitatively and qualitatively both in arteries and veins, and between animals. (The largest observed relaxation was − 5.7 and − 10% of the pre-stretch tension in the artery (: 4g) and in the vein ( : 1 g), respectively, while the largest tension developed by a constricting artery or vein was only about 1.2 and 1.6%, respectively, of the tension developed by the vessels when the highest dose of NA was given). Finally, arteries and veins were stimulated to constrict by administration of a sub-maximal dose of NA and then subjected to graded doses of both ACh and Iso. Again, the responses were very minor and differed quantitatively and qualitatively both in arteries and veins, and between animals, for both of the drugs.
4. Discussion
Fig. 1. Tension changes (g) in arteries and veins, sampled from the outer layers of the spleen of four hooded seals, in response to increasing doses of adrenaline (A) and noradrenaline (NA) (mol l − 1). Data represent means9 S.E. ( ) A to veins, () A to arteries, ( ) NA to veins, and () NA to arteries.
The present study has shown that A and NA both cause concentration-dependent constriction of arterial and venous splenic vessels of hooded seals. These responses were abolished by the general a-adrenoceptor blocker Phe. The constrictor response of pinniped splenic arteries to a-adrenoceptor stimulation is therefore similar to the responses previously reported for several terrestrial mammals such as rodents, dogs, cats, and humans [13,28]. However, constriction of splenic veins, as presently demonstrated in the hooded seal, has previously been reported to be non-significant or even absent in terrestrial species [15,21,29]. The ratio of the arterial to venous resistance is an important determinant of splenic hydrostatic pressure, and therefore control of splenic venous tone is central in the control of splenic filling and emptying. The insignificant response in splenic veins of some terrestrial mammals probably represents a weak response, rather than an insignificant one, and reflects less dependence on the spleen as a blood-storing organ. Data on plasma levels of catecholamines in hooded seals have not been published, but data for some other
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pinniped species are available. Thus, basal plasma A and NA levels of :0.65 and :0.75 nM, respectively, have been observed in venous blood from resting gray seals, Halichoerus grypus, (Lohman and Folkow, unpublished), of : 1.0 and :2.0 nM, respectively, in venous blood from resting harbor seals, Phoca 6itulina [17], while the corresponding values for resting Weddell seals, Leptonychotes weddellii, were : 0.5 and : 1.1 nM, respectively [18]. In connection with diving, however, plasma catecholamine levels of seals may increase dramatically. For example, diving gray seals displayed peak plasma concentrations of A and NA of 4.6–33 and 8.3–13 nM, respectively (Lohman and Folkow, unpublished), while concentrations in diving harbor seals reached levels of 5 – 25 and 5 – 17 nM, respectively, depending on dive duration [17]. Still, the high levels found during diving are much lower than the estimated ED50 values of both arteries and veins in the present in vitro study. Similar disproportionate differences between the sensitivity of vessel responses to catecholamines in vitro, and normal/realistic plasma catecholamine levels, have been demonstrated previously. For example, Nilsson [22] reported the effective dose required for ED50 for NA in the in vitro preparation of the superior mesenteric artery of the rat, to be 0.82 mM, on average, while rat plasma NA levels average : 40 nM [11]. The low sensitivity of the in vitro preparation in relation to the plasma catecholamine levels normally encountered simply reflects that the vessels are less likely to be influenced by circulating catecholamines [2]. In such cases, exogenous catecholamines primarily act upon adrenoceptors which are associated with the varicosities of sympathetic neurons, and (particularly in ring vessel preparations) must penetrate towards the muscle cells from the adventitia side and pass through the nerve plexus before being able to exert their action, in which case they are susceptible to both neuronal and extraneuronal uptake which reduces the effective concentration at the effector cell ([14]). Similar high ED50 values have been observed in a variety of vessels and species (e.g. in the angular oculi veins of reindeer (ED50 for NA of 1.7 mM [20]); in the facial vein of rabbits (ED50 for NA of 0.114 mM [25]). Given the similarity in ED50 values between the cited studies and those of the present study, we conclude that vasomotor activity in splenic arteries and veins of hooded seals is subjected to neurogenic rather than humoral control. Administration of the b-adrenoceptor agonist Iso caused the seal splenic vessels to respond only weakly, or not at all. The weak responses, when present, were either on the dilatory or constrictor side in the unstimulated pre-stretched vessels, while the vessels which were pre-constricted by NA usually displayed weak constriction when Iso was added. The pre-stretched, unstimulated vessels may have had a myogenic tone that was
too low for any further dilatation to show, but this could not have been the case in those experiments in which the vessels were already constricted following NA injection. Contradictory responses to Iso in spleen vessels from cats [3], and dogs [24], have also been reported. The same argument applies with regard to ACh, which also produced weak and ambiguous responses, depending on dose and preparation [12]. This suggests that neither cholinergic nor b-adrenoceptormediated mechanisms are important for either constrictor or dilator mechanisms in either arteries or veins in the hooded seal spleen. This is in contrast to cats, where b-adrenoceptors were reported to be prominent in the responses to catecholamines [30]. In conclusion, the present study suggests that in the hooded seal the arterial blood supply to the spleen, as well as its venous drainage, is under a-adrenergic sympathetic nervous control, while b-adrenergic and cholinergic mechanisms appear to be of minor importance for the control of splenic vascular resistance.
Acknowledgements We wish to thank the crew of R/V ‘Jan Mayen’ and students and scientists at the Department of Arctic Biology, for their cooperation and valuable help in connection with field and laboratory work. This study was supported in part by the Norwegian Research Council, grant no. 106925.110
References [1] Bevan JA. Some characteristics of the isolated sympathetic nerve-pulmonary artery preparation of the rabbit. J Pharmacol Exp Ther 1962;137:213 – 8. [2] Bevan JA, Bevan RD, Duckles, SP. Adrenergic regulation of vascular smooth muscle. In: Handbook of Physiological Society. Bethesda: American Physiological Society, 1980:515–566. [3] Bickerton RK. The response of isolated strips of cat spleen to sympathomimetic drugs and their antagonists. J Pharmacol Exp Ther 1963;142:99 – 110. [4] Blix AS, Folkow B. Cardiovascular adjustments to diving in mammals and Birds. In: Shepherd JT, Abboud FM, editors. Handbook of Physiology. Bethesda: American Physiological Society, 1983:917 – 46. [5] Bron KM, Murdaugh HV Jr., Millen JE, Lenthall R, Raskin P, Robin ED. Arterial constrictor response in a diving mammal. Science 1966;152:540 – 3. [6] Bryden MM, Lim GHK. Blood parameters of the southern elephant seal (Mirounga leonina, Linn.) in relation to diving. Comp Biochem Physiol 1969;28:139 – 48. [7] Cabanac A, Folkow LP, Blix AS. Volume capacity and contraction control of the seal spleen. J Appl Physiol 1997;82:1989 – 94. [8] Castellini JM, Castellini MA. Estimation of splenic volume and its relationship to long-duration apnea in seals. Physiol Zool 1993;66:619 – 27.
A. Cabanac et al. / Comparati6e Biochemistry and Physiology, Part A 120 (1998) 277–281 [9] Castellini MA, Costa DP, Huntley A. Hematocrit variation during sleep apnea in elephant seal pups. Am J Physiol 1986;251:R429– 31. [10] Castellini MA, Davis RW, Kooyman GL. Blood chemistry regulation during repetitive diving in Weddell seals. Physiol Zool 1988;61:379 – 86. [11] Chin AK, Evonuk E. Changes in plasma catecholamine and corticosterone levels after muscular exercise. J Appl Physiol 1971;30:205 – 7. [12] Daly MDB, Scott MJ. The effects of acetylcholine on the volume and vascular resistance of the dog’s spleen. J Physiol 1961;156:246 – 59. [13] Davies BN, Withrington PG. The actions of drugs on the smooth muscle of capsule and blood vessels of the spleen. Pharmacol Rev 1973;25:373–413. [14] DelaLande IS, Frewin D, Waterson JG. The influence of sympathetic innervation on vascular sensitivity to noradrenaline. Br J Pharmacol 1967;31:82–93. [15] Fleming WW, Parpart AK. Effects of topically applied epinephrine, norepinephrine and histamine on the intermediate circulation of the mouse spleen. Angiology 1958;9:294– 302. [16] Folkow B, Fuxe K, Sonnenschein RR. Responses of skeletal musculature in its vasculature during ‘diving’ in the duck: peculiarities of the adrenergic vasoconstrictor innervation. Acta Physiol Scand 1966;67:327–42. [17] Hance AJ, Robin ED, Halter JB, Lewiston N, Robin DA, Cornell L, Caligiuri M, Theodore J. Hormonal changes and enforced diving in the harbor seal Phoca 6itulina. II. Plasma catecholamines. Am J Physiol 1982;242:R528–32. [18] Hochachka PW, Liggins GC, Guyton GP, Schneider RC, Stanek KS, Hurford WE, Creasy RK, Zapol DG, Zapol WM. Hormonal regulatory adjustments during voluntary diving in Weddell seals. Comp Biochem Physiol B Biochem Mol Biol 1995;112B:361– 75. [19] Hurford WE, Hochachka PW, Schneider RC, Guyton GP, Stanek KS, Zapol DG, Liggins GC, Zapol WM. Splenic contraction, catecholamine release and blood volume redistribution during diving in the Weddell seal. J Appl Physiol 1996;80:298 – 306.
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[20] Johnsen HK, Folkow LP. Vascular control of brain cooling in reindeer. Am J Physiol 1988;254:R730– 9. [21] MacKenzie DWJ, Wipple AO, Wintersteiner MP. Studies on the microscopic anatomy and physiology of living transilluminated mammalian spleens. Am J Anat 1941;68:397 – 456. [22] Nilsson H. Differential responses in consecutive sections of the arterial system. Acta Physiol Scand 1984;121:353 – 61. [23] Ohgushi M, Yasue H, Kugiyama K, Murohara T, Sakaino N. Contraction and endothelium dependent relaxation via adrenoceptors are variable in various pig arteries. Cardiovasc Res 1993;27:779 – 84. [24] Ojiri Y, Noguchi K, Chibana T, Sakanashi M. Effects of adrenergic stimulants on the splenic diameter, haemoglobin content and haematocrit in anaesthetized dogs: determination of the adrenoceptor subtype responsible for changes in the splenic diameter. Acta Physiol Scand 1993;149:31 – 9. [25] Pegram BL, Bevan RD, Bevan JA. Facial vein of the rabbit: neurogenic vasodilation mediated by adrenergic receptors. Circ Res 1976;39:854 – 60. [26] Ponganis PJ, Kooyman GL, Sartoris D, Jobsis P. Pinniped splenic volumes. Am J Physiol 1992;262:R322– 5. [27] Qvist J, Hill RD, Schneider RC, Falke KJ, Liggins GC, Guppy M, Elliot RL, Hochachka PW, Zapol WM. Hemoglobin concentrations and blood gas tensions of free-diving Weddell seals. J Appl Physiol 1986;61:1560 – 9. [28] Reilly FD. Innervation and vascular pharmacodynamics of the mammalian spleen. Experientia 1985;41:187 – 92. [29] Reilly FD, McCuskey RS. Studies of the hemopoietic microenvironment. VI. Regulatory mechanisms in the splenic microvascular system of mice. Microvasc Res 1977;13:79 – 90. [30] Ross G. Effects of catecholamines on splenic blood flow in the cat. Am J Physiol 1967;213:1079– 83. [31] White FN, Ikeda M, Elsner RW. Adrenergic innervation of large arteries in the seal. Comp Gen Pharmacol 1973;4:271–6. [32] Zapol WM. Diving adaptations of the Weddell seal. Sci Am 1987;256:80 – 5. [33] Zapol WM, Hill RD, Qvist J, Falke K, Schneider RC, Liggins GC, Hochachka PW. Arterial gas tensions and hemoglobin concentrations of the freely diving Weddell seal. Undersea Biomed Res 1989;16:363 – 73.
Effects of adrenergic and cholinergic drugs on splenic arteries and veins from hooded seals (Cystophora cristata) A. Cabanac *, L.P. Folkow, A.S. Blix Department of Arctic Biology and Institute of Medical Biology, Uni6ersity of Tromsø, Tromsø, Norway Received 15 October 1997; received in revised form 13 January 1998; accepted 28 January 1998
Abstract Isolated ring preparations of arteries and veins from hooded seal spleens were subjected in vitro to adrenaline (A), noradrenaline (NA), isoprenaline (Iso), and acetylcholine (ACh), alone or in combination with the blockers phentolamine (Phe), propranolol (Pro), and atropine (Atr). Both arteries and veins constricted in response to A (the estimated effective dose required for half-maximal response (ED50) was 3.3 and 0.2 mM, for arteries and veins, respectively) and NA (estimated ED50 was 1.5 and 0.6 mM, for arteries and veins, respectively), but these effects were abolished when the drugs were given in combination with the a-adrenoceptor blocker Phe. The responses of arteries and veins to ACh and the b-adrenoceptor agonist Iso were minor and inconsistent, and were completely abolished when combined with their respective blockers (Atr and Pro, respectively). The ED50 for both A and NA are quite high in relation to normal plasma levels of A and NA in seals. This implies that these vessels (and, hence, the supply of blood to the spleen) primarily are subjected to neurogenic, rather than humoral physiological control. © 1998 Elsevier Science Inc. All rights reserved. Keywords: Pinnipeds; Diving; Spleen; Splenic vascular resistance; Vasomotor control; In vitro; Adrenaline; Noradrenaline; Isoprenaline; Acetylcholine; Vasoconstriction; Vasodilation; Vessels
1. Introduction A series of dramatic cardiovascular changes take place in seals when they dive for extended periods of time. These changes include a profound peripheral vasoconstriction which is accompanied by intense bradycardia [4]. The changes serve to reduce the rate of utilization of endogenous oxygen stores, and thereby extend the submersion period. Another change which may be observed is a substantial increase in the number of circulating red blood cells [9,27]. Several authors [6,8,19,26,27,33] have suggested that the spleen is the origin of the increased hematocrit during diving, and that this mechanism probably is important for the ability of these mammals to dive repeatedly and for * Corresponding author. Present address: 263 route du fleuve, Pointe-au-pe`re, Que´bec G5M 1K7, Canada. 0742-8413/98/$19.00 © 1998 Elsevier Science Inc. All rights reserved. PII S1095-6433(98)00029-4
extended periods of time. The proposed function of splenic contraction and release of (oxygenated) red blood cells has been that this increases the available blood oxygen store [32], or that it improves the oxygencarrying capacity and, hence, reduces surface time during repeated diving [10]. Recently, Cabanac and coworkers [7] demonstrated in vitro that the spleens of the hooded seal (Cystophora cristata) and the harp seal (Phoca groenlandica) are capable of active and forceful contraction under aadrenergic stimulation. However, that study did not include any examination of the vasomotor responses of the splenic vasculature. Such responses are obviously important since they are the means by which blood supply to the spleen—which determines both splenic filling and subsequent drainage—is controled. The purpose of the present study, therefore, was to investigate the physiological control of the splenic vas-
278
A. Cabanac et al. / Comparati6e Biochemistry and Physiology, Part A 120 (1998) 277–281
culature by studying in vitro the effects of adrenergic and cholinergic drugs on splenic arteries and veins from the hooded seal. Arteries and veins of about 2-mm diameter were chosen in preference to smaller pre-capillary vessels, for two reasons. First, these vessels were observed to display vigorous contractile responses upon perfusion of isolated intact spleens with modified Krebs’ solutions containing adrenaline (A) (Cabanac, Folkow and Blix, unpublished observations). Second, it has been demonstrated repeatedly that vasomotor control of the blood supply to several tissues/organs of diving animals is exercised at the level of arteries, rather than in the pre-capillary arterioles [5,16,31]. In this way, vasoconstriction can be maintained for extended periods of time during diving, in spite of the accumulation of vasodilator metabolites in the ischemic tissues [16].
2. Materials and methods A total of four hooded seals (C. cristata), two of each sex, were caught in drift-ice east of Greenland (the West Ice, 71°N, 18°W) during expeditions with R.V. ‘Jan Mayen’ in March/April 1994 and 1995. Two pups which were about 1-week old, weighing 26 and 24 kg, respectively, were killed and used immediately on board the research vessel. Two others were brought alive to the Department of Arctic Biology at Tromsø, Norway, and kept indoors in a large freshwater tank, under simulated natural light conditions (70°N) until used, at which time they weighed 82 and 67 kg, respectively. The animals were fed capelin (Mallotus 6illosus) twice a day. All seals were killed instantaneously by use of a ‘hakapik’, immediately followed by bleeding, without any use of anesthetics, as authorized by Norwegian Veterinary Authorities. All animals were taken under permits issued by the Norwegian Government, and the experiments were approved by the Norwegian Committee on Ethics in Animal Experimentation. The spleens were quickly removed and transferred into a bath containing 0.9% NaCl solution for in vitro preparation. The description of the approach for splenectomy and subsequent handling of the spleen is given in [7]. Small rings (: 5 mm long and 2 – 3 mm diameter) of splenic arteries and veins were excised from the surface region of the spleen and carefully cleaned from extraneous tissue. The ring samples were mounted in separate 30-ml organ baths, as described by Johnsen and Folkow [20], and connected to force displacement transducers (Type FT03; Grass Medical Instruments, MA) for isometric recordings of tension. Contact with the endothelial surface was carefully avoided in this process in order to preserve endothelial integrity. The transducer signals fed via an amplifier (Universal Am-
plifier installed into a Type 5900 Signal Conditioner; Gould, OH) into a thermal array recorder (TA 4000; Gould, OH) and the responses were recorded as a continuous analog curve. The baths were filled with modified Krebs solution (see below) and kept at constant temperature (37.49 0.4°C) by circulating a jacket which surrounded the bath with thermostatically controled water. The saline inside the baths was oxygenated by bubbling with a gas mixture containing 95% O2 and 5% CO2. After each step in any experiment, the saline was changed three times to rinse the vessels and the baths. Before any drug injections were made, the vessels were pre-stretched for 1 h at resting tension-levels of :1 g for the veins and : 4 g for the arteries. These pre-stretch levels are similar to those employed in studies of similar-sized and -typed vessels in previous studies [1,23]. Drugs were carefully added directly into the baths by use of a 20–200 ml micro-pipette (Finnpipette, Labsystems, Helsinki, Finland). The following experiments were carried out in succession on both arteries and veins during the sessions.
2.1. Experiment 1 Vessels were subjected to graded concentrations of A (0.4, 2.5, 7.0, 29.3 and 141× 10 − 7 M) and noradrenaline (NA) (24.3 and 153 × 10 − 7 M) separately.
2.2. Experiment 2 NA (153× 10 − 7 M) was added 4–6 min after the a-adrenergic blocker phentolamine (Phe) had been added to yield concentrations \ 1450 × 10 − 7 M).
2.3. Experiment 3 Vessels were subjected to graded concentrations of acetylcholine (ACh) (5.6, 140, 174, 196, 331 and 421 × 10 − 7 M) alone, and 2–3 min after atropine (Atr) had been added to yield concentrations of 1400–2190 × 10 − 7 M).
2.4. Experiment 4 Vessels were subjected to graded concentrations of the b-adrenergic agonist isoprenaline (Iso) (2.6, 26 and 260 × 10 − 7 M).
2.5. Experiment 5 NA (153 × 10 − 7 M) was added 5 min before ACh or Iso were added to yield concentrations of 5.6 or 140 × 10 − 7 M, and 2.6 or 260 × 10 − 7 M, respectively. The following drugs were used in the experiments: A, Atr, NA, (1 mg.ml − 1, Nycomed Pharma, Oslo, Norway), Iso (0.2 mg.ml − 1, Nycomed Pharma, Oslo, Nor-
A. Cabanac et al. / Comparati6e Biochemistry and Physiology, Part A 120 (1998) 277–281
way), ACh (10 mg.ml − 1, Nycomed Pharma, Oslo, Norway), and Phe (Regitine 10 mg.ml − 1, Ciba-Geigy, Basel, Switzerland). The composition of the physiological saline (in mM) was: 119.0 NaCl, 25.0 NaHCO3, 4.7 KCl, 2.5 CaCl2, 1.18 KH2PO4, 1.17 MgSO4, 5.5 glucose, and 0.027 EDTA.
3. Results When subjected to graded concentrations of A and NA, both arteries and veins displayed graded constriction responses (Fig. 1). The responses were monotonic functions of the drug doses, and reached a plateau for each dose. The maximal contraction tension for arteries was : 40 g and for veins :10 g. The dose – response curves for A and NA were similar for the arteries (Student t-test on the slopes of the transformed log–log regression lines: t= 1.12, P \ 0.05). The slope of the transformed log–log regression line for the veins was smaller for A than for NA (t=3.50 – 3.18, P B 0.05). However, the difference was very small (Fig. 1), and the effect of the two catecholamines is obviously similar. The arteries constricted about four times more strongly than the veins when subjected to identical doses of A or NA. The effective dose required for a half-maximal response (ED50) was estimated to be 3.3 and 1.5 mM for arteries, and 0.2 and 0.6 mM for veins, in response to A and NA, respectively.
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The general a-adrenoceptor antagonist Phe, when given to both arteries and veins before a near-maximal dose of NA, blocked at the least 99% of the control responses to the latter. ACh was found to cause very small changes in vascular tension in both arteries and veins. Moreover, the tension changes varied in direction both with the dose given, and from experiment to experiment. (The largest observed relaxation was −12% of the prestretch tension ( : 1 g) in a vein, while the largest tension developed by a constricting artery or vein was only about 3.0% of the tension developed by the vessels when the highest dose of NA was given). Atr blocked the effects of ACh in both arteries and veins. The b-adrenergic agonist Iso, likewise, caused very minor responses which differed quantitatively and qualitatively both in arteries and veins, and between animals. (The largest observed relaxation was − 5.7 and − 10% of the pre-stretch tension in the artery (: 4g) and in the vein ( : 1 g), respectively, while the largest tension developed by a constricting artery or vein was only about 1.2 and 1.6%, respectively, of the tension developed by the vessels when the highest dose of NA was given). Finally, arteries and veins were stimulated to constrict by administration of a sub-maximal dose of NA and then subjected to graded doses of both ACh and Iso. Again, the responses were very minor and differed quantitatively and qualitatively both in arteries and veins, and between animals, for both of the drugs.
4. Discussion
Fig. 1. Tension changes (g) in arteries and veins, sampled from the outer layers of the spleen of four hooded seals, in response to increasing doses of adrenaline (A) and noradrenaline (NA) (mol l − 1). Data represent means9 S.E. ( ) A to veins, () A to arteries, ( ) NA to veins, and () NA to arteries.
The present study has shown that A and NA both cause concentration-dependent constriction of arterial and venous splenic vessels of hooded seals. These responses were abolished by the general a-adrenoceptor blocker Phe. The constrictor response of pinniped splenic arteries to a-adrenoceptor stimulation is therefore similar to the responses previously reported for several terrestrial mammals such as rodents, dogs, cats, and humans [13,28]. However, constriction of splenic veins, as presently demonstrated in the hooded seal, has previously been reported to be non-significant or even absent in terrestrial species [15,21,29]. The ratio of the arterial to venous resistance is an important determinant of splenic hydrostatic pressure, and therefore control of splenic venous tone is central in the control of splenic filling and emptying. The insignificant response in splenic veins of some terrestrial mammals probably represents a weak response, rather than an insignificant one, and reflects less dependence on the spleen as a blood-storing organ. Data on plasma levels of catecholamines in hooded seals have not been published, but data for some other
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pinniped species are available. Thus, basal plasma A and NA levels of :0.65 and :0.75 nM, respectively, have been observed in venous blood from resting gray seals, Halichoerus grypus, (Lohman and Folkow, unpublished), of : 1.0 and :2.0 nM, respectively, in venous blood from resting harbor seals, Phoca 6itulina [17], while the corresponding values for resting Weddell seals, Leptonychotes weddellii, were : 0.5 and : 1.1 nM, respectively [18]. In connection with diving, however, plasma catecholamine levels of seals may increase dramatically. For example, diving gray seals displayed peak plasma concentrations of A and NA of 4.6–33 and 8.3–13 nM, respectively (Lohman and Folkow, unpublished), while concentrations in diving harbor seals reached levels of 5 – 25 and 5 – 17 nM, respectively, depending on dive duration [17]. Still, the high levels found during diving are much lower than the estimated ED50 values of both arteries and veins in the present in vitro study. Similar disproportionate differences between the sensitivity of vessel responses to catecholamines in vitro, and normal/realistic plasma catecholamine levels, have been demonstrated previously. For example, Nilsson [22] reported the effective dose required for ED50 for NA in the in vitro preparation of the superior mesenteric artery of the rat, to be 0.82 mM, on average, while rat plasma NA levels average : 40 nM [11]. The low sensitivity of the in vitro preparation in relation to the plasma catecholamine levels normally encountered simply reflects that the vessels are less likely to be influenced by circulating catecholamines [2]. In such cases, exogenous catecholamines primarily act upon adrenoceptors which are associated with the varicosities of sympathetic neurons, and (particularly in ring vessel preparations) must penetrate towards the muscle cells from the adventitia side and pass through the nerve plexus before being able to exert their action, in which case they are susceptible to both neuronal and extraneuronal uptake which reduces the effective concentration at the effector cell ([14]). Similar high ED50 values have been observed in a variety of vessels and species (e.g. in the angular oculi veins of reindeer (ED50 for NA of 1.7 mM [20]); in the facial vein of rabbits (ED50 for NA of 0.114 mM [25]). Given the similarity in ED50 values between the cited studies and those of the present study, we conclude that vasomotor activity in splenic arteries and veins of hooded seals is subjected to neurogenic rather than humoral control. Administration of the b-adrenoceptor agonist Iso caused the seal splenic vessels to respond only weakly, or not at all. The weak responses, when present, were either on the dilatory or constrictor side in the unstimulated pre-stretched vessels, while the vessels which were pre-constricted by NA usually displayed weak constriction when Iso was added. The pre-stretched, unstimulated vessels may have had a myogenic tone that was
too low for any further dilatation to show, but this could not have been the case in those experiments in which the vessels were already constricted following NA injection. Contradictory responses to Iso in spleen vessels from cats [3], and dogs [24], have also been reported. The same argument applies with regard to ACh, which also produced weak and ambiguous responses, depending on dose and preparation [12]. This suggests that neither cholinergic nor b-adrenoceptormediated mechanisms are important for either constrictor or dilator mechanisms in either arteries or veins in the hooded seal spleen. This is in contrast to cats, where b-adrenoceptors were reported to be prominent in the responses to catecholamines [30]. In conclusion, the present study suggests that in the hooded seal the arterial blood supply to the spleen, as well as its venous drainage, is under a-adrenergic sympathetic nervous control, while b-adrenergic and cholinergic mechanisms appear to be of minor importance for the control of splenic vascular resistance.
Acknowledgements We wish to thank the crew of R/V ‘Jan Mayen’ and students and scientists at the Department of Arctic Biology, for their cooperation and valuable help in connection with field and laboratory work. This study was supported in part by the Norwegian Research Council, grant no. 106925.110
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