Acta physiol. scand. 1973. 93. 139-141 From the Department of Pharmacology, Karolinska Institutet, and the Department of Clinical Physiology at Karolinska Sjukhuset, Stockholm, Sweden
Effect of Substance P on Blood Flow in Canine Adipose Tissue and Skeletal Muscle BY and SUNEROSELL BENGTPERNOW
i n 1931 Euler and Gaddum described smooth muscle stimulating and hypotensive effects of extracts from horse brain and intestine. The active factor was called Substance P (SP). Intravenous administration of purified preparations of SP caused a rapid fall in blood pressure in the rabbit and cat, which was not blocked by atropine, antihistamines or ganglionic blocking agents (Pernow 1953 a). The hypotensive effect of SP was presumed to be due to peripheral vasodilation; this was later partially confirmed by studies in man, where a n increase in skin and muscle blood flow was demonstrated during infusion of SP into the brachial artery (Lofstrom, Pernow and Wahren 1965). Recently, Chang and Leeman (1970) isolated an undecapeptide from bovine hypothalamus with biological properties indistinguishable from those described earlier for SP, which is known to be present in large amounts in the hypothalamus (Pernow 1953 b). This undecapeptide is now available in synthetic form (Tregear et al. 1971, Fisher et al. 1974), which opens up greater possibilities for pharmacological studies. The present paper reports some actions of synthetic SP on the circulation in skeletal muscle and adipose tissue in the dog.
Methods 7 dogs, anesthetized with sodium pentobarbital (30 mg/kg b.wt., with supplement when necessary) were used. Arterial blood gases were analyzed in most animals and, if necessary, artificial positive pressure breathing was instituted. The subcutaneous adipose tissue in the inguinal region of female dogs was prepared as described earlier (Rosell 1966). Blood flow was measured by means of a drop counter in the main artery. The vein draining the adipose tissue was cannulated with polyethylene tubing and the venous outflow was collected for the analysis of glycerol (Laurel1 and Tibbling 1966) and free fatty acids (FFA) (Trout et al. 1960). FFA and glycerol were also measured in arterial samples in order to be able to calculate net outflow. In some experiments the adipose tissue was placed in a plethysmograph (&erg and Rosell 1967) in order to be able to determine the capillary filtration coefficient (CFC). To measure skeletal muscle blood flow the gracilis muscle was isolated from surrounding tissue and blood flow was measured by means of a drop counter in the main artery (Renkin 139
140
BENGT PERNOW AND SUNE ROSELL
Fig. 1. Canine subcutaneous adipose tissue (28 g). Intra-arterial injection of Substance P.
and Rosell 1962). Arterial blood pressure was monitored from a common carotid artery. Heparin was administered i.a. Synthetic SP was kindly placed at our disposal by Professor K. Folkers, Institute for Biomedical Research, University of Texas, Austin, Texas. It was dissolved in physiological saline and given in close arterial injections.
Results SP injected i.a. in doses from 10 ng caused a dose-related vasodilatation in subcutaneous adipose tissue (Fig. 1) and in skeletal muscle. The increase in blood flow was of short duration ( 1-2 min) and did not show any tachyphylaxis. Attempts to block the vasodilatation were not successful. Histamine receptor blocking agents (mepyramine 1 mg La., burimamide 200 pg, methiamide 240 pg La.) and adrenergic blocking agents (propranolol 700 pg, La.; dihydroergotamine 300 pg, i.a.1 as well as cystein di-/)-naphthylamide (0.25 mg La.) were tested. The latter substance has been reported to block the effect of SP on intestinal motility (Stern and HukoviC 1961). As a measurement of the functional capillary surface area or capillary permeability, CFC was determined in the adipose tissue. Doses which induced vasodilatation increased C F C by 10-20 per cent (2 expts.). I n 3 expts. the net outflow of glycerol and/or FFA from the adipose tissue was measured. SP injected i.a. in doses of 0.2-40 ng or infused (13-26 ng/min La.) did not change the net outflow of glycerol or FFA, indicating that there was no change in the lipolytic rate. The lipolysis induced by sympathetic nerve stimulation (4 Hz) was not affected by infusion of SP during the stimulation period. Discussion The present results demonstrate that synthetic SP is a very active vasodilator in adipose tissue and skeletal muscle, with a potency quite comparable to that of prostaglandins of the E-type, so far the most potent vasodilator tested in adipose tissue (Fredholm, t)berg and Rosell 1970). Thus the threshold dose of PGE, and SP, respectively, were found t o
EFFECT OF SUBSTANCE P ON BLOOD FLOW IN CANINE ADIPOSE TISSUE AND SKELETAL MUSCLE
141
be quite comparable on the weight basis, but since SP is a much larger molecule (molecular weights 354 and 1 340 respectively) it is even more active than PG on molecular basis. The circulatory effect of SP was not blocked by adrenergic blocking agents or antihistamines, which is consistent with the results obtained in man (Lofstrom et al. 1965). These results, together with the absence of tachyphylaxis, indicate that the vasodilatation is a direct effect of SP on the vascular smooth muscles and not due to release of any biogenic substance, such as histamine or catecholamines. This is further supported by the finding that SP did not induce any lipolysis, since both catecholamines and histamine are lipolytic agents in canine subcutaneous adipose tissue (Rosell 1966, Fredholm, Meng, Rosell 1968). Johnson and Erdos (1973) found that SP releases histamine from isolated rat mast cells. Our results indicate that such an action does not seem to be of any importance in canine adipose tissue. The 10-20 per cent increase in CFC found in the present experiments is presumably due to an increase in the functional capillary surface area due to dilatation of the precapillary sphincter region. This study was supported by grants from the Swedish Medical Research Council (04X-4495 and 4OX-3518) and Karolinska Institutet.
References CHANG,M. M. and S. E. LEEMAN, Isolation of a sialogogic peptide from bovine hypothalamic tissue and its characterization as substance P. J . b i d . Chem. 1970. 245. 4784-4790. An unidentified depressor substance in certain tissue extracts. J . EULER,v., U. S. and J. H. GADDUM, Physiol. (Lond.) 1931. 72. 74-87. FREDHOLM, B. B., B. C)BERGand S. ROSELL,Effects of vasoactive drugs on circulation in canine subcutaneous adipose tissue. Acra physiol. scand. 1970. 79. 564-574. FREDHOLM, B. B., H. C. MENGand S. ROSELL,Release of free fatty acids from canine subcutaneous adipose tissue by histamine and compound 48/80. Life Sci. 1968. 7. 1209-1215. FISHER,G. H., J. HUMPHRIES, K. FOLKERS, B. PERNOW and C. Y. BOWERS, Synthesis and some biological activities of substance P. J . Med. Chem. 1974. 17. 843-852. JOHNSON, A. R. and E. ERDOS,Release of histamine from mast c:lls by vasoactive ceptides. Proc. SOC. exp. B i d . (N.Y . ) 1973. 142. 1252-1256. LAURELL, S. and 0.TIBBLING, An enzymatic fluorimetric icrornethod for the determination of glyccrol. Clin. chim. Aera. 1966. 13. 317-322. LOFSTROM,B., B. PERNOW and J. WAHREN, Vasodilating action of substance P in the human forearm. Acta physiol. scand. 1965. 63. 31 1-324. &ERG, B. and S. ROSELL,Sympathetic control of consecutive vascular sections in canine subcutaneous adipose tissue. Acra physiol. scand. 1967. 71. 47-56. B. Studies on substans P. Purification, occurrence and biological actions. Acra phvsiol. stasd. PERNOW., 1953a. 29. Suppl. 105. PERNOW,B., Distribution of substans P in the central and peripheral nervous system. Nature (Lond.) 1953b. 171. 746-747. The influence of sympathetic adrenergic vasoconstrictor nerves on transport RENKIN,E. M. and S. ROSELL, of diffusible solutes from blood to tissues in skeletal muscle. Acra physiol. scand. 1962. 54. 223-240. ROSELL,S., Release of free fatty acids from subcutaneous adipose tissue in dogs following sympathetic nerve stimulation. Acta physiol. srand. 1966. 67. 343-351. STERN,P. and S. H U K O V ISpecific ~, antagonists of Substance P. In Symposium on Substance P. Proc. Sci. SOC.Bosnia Herzegouina. 1961. 1. 83-88.
TREGEAR, G. W.. H. D. NIALL,J. T. POTTS,S. E. LEEMAN and M. M. CHANG,Synthesis of Substance P. Nature New B i d . 1971. 232. 87-89. TROUT,P. L., H. ESTESand S. J. FRIEDBERG, Titration of free fatty acids of plasma. A study of current methods and a now modification. J. Lipid. Res. 1960. 1. 199-202.
Effect of Substance P on Blood Flow in Canine Adipose Tissue and Skeletal Muscle BY and SUNEROSELL BENGTPERNOW
i n 1931 Euler and Gaddum described smooth muscle stimulating and hypotensive effects of extracts from horse brain and intestine. The active factor was called Substance P (SP). Intravenous administration of purified preparations of SP caused a rapid fall in blood pressure in the rabbit and cat, which was not blocked by atropine, antihistamines or ganglionic blocking agents (Pernow 1953 a). The hypotensive effect of SP was presumed to be due to peripheral vasodilation; this was later partially confirmed by studies in man, where a n increase in skin and muscle blood flow was demonstrated during infusion of SP into the brachial artery (Lofstrom, Pernow and Wahren 1965). Recently, Chang and Leeman (1970) isolated an undecapeptide from bovine hypothalamus with biological properties indistinguishable from those described earlier for SP, which is known to be present in large amounts in the hypothalamus (Pernow 1953 b). This undecapeptide is now available in synthetic form (Tregear et al. 1971, Fisher et al. 1974), which opens up greater possibilities for pharmacological studies. The present paper reports some actions of synthetic SP on the circulation in skeletal muscle and adipose tissue in the dog.
Methods 7 dogs, anesthetized with sodium pentobarbital (30 mg/kg b.wt., with supplement when necessary) were used. Arterial blood gases were analyzed in most animals and, if necessary, artificial positive pressure breathing was instituted. The subcutaneous adipose tissue in the inguinal region of female dogs was prepared as described earlier (Rosell 1966). Blood flow was measured by means of a drop counter in the main artery. The vein draining the adipose tissue was cannulated with polyethylene tubing and the venous outflow was collected for the analysis of glycerol (Laurel1 and Tibbling 1966) and free fatty acids (FFA) (Trout et al. 1960). FFA and glycerol were also measured in arterial samples in order to be able to calculate net outflow. In some experiments the adipose tissue was placed in a plethysmograph (&erg and Rosell 1967) in order to be able to determine the capillary filtration coefficient (CFC). To measure skeletal muscle blood flow the gracilis muscle was isolated from surrounding tissue and blood flow was measured by means of a drop counter in the main artery (Renkin 139
140
BENGT PERNOW AND SUNE ROSELL
Fig. 1. Canine subcutaneous adipose tissue (28 g). Intra-arterial injection of Substance P.
and Rosell 1962). Arterial blood pressure was monitored from a common carotid artery. Heparin was administered i.a. Synthetic SP was kindly placed at our disposal by Professor K. Folkers, Institute for Biomedical Research, University of Texas, Austin, Texas. It was dissolved in physiological saline and given in close arterial injections.
Results SP injected i.a. in doses from 10 ng caused a dose-related vasodilatation in subcutaneous adipose tissue (Fig. 1) and in skeletal muscle. The increase in blood flow was of short duration ( 1-2 min) and did not show any tachyphylaxis. Attempts to block the vasodilatation were not successful. Histamine receptor blocking agents (mepyramine 1 mg La., burimamide 200 pg, methiamide 240 pg La.) and adrenergic blocking agents (propranolol 700 pg, La.; dihydroergotamine 300 pg, i.a.1 as well as cystein di-/)-naphthylamide (0.25 mg La.) were tested. The latter substance has been reported to block the effect of SP on intestinal motility (Stern and HukoviC 1961). As a measurement of the functional capillary surface area or capillary permeability, CFC was determined in the adipose tissue. Doses which induced vasodilatation increased C F C by 10-20 per cent (2 expts.). I n 3 expts. the net outflow of glycerol and/or FFA from the adipose tissue was measured. SP injected i.a. in doses of 0.2-40 ng or infused (13-26 ng/min La.) did not change the net outflow of glycerol or FFA, indicating that there was no change in the lipolytic rate. The lipolysis induced by sympathetic nerve stimulation (4 Hz) was not affected by infusion of SP during the stimulation period. Discussion The present results demonstrate that synthetic SP is a very active vasodilator in adipose tissue and skeletal muscle, with a potency quite comparable to that of prostaglandins of the E-type, so far the most potent vasodilator tested in adipose tissue (Fredholm, t)berg and Rosell 1970). Thus the threshold dose of PGE, and SP, respectively, were found t o
EFFECT OF SUBSTANCE P ON BLOOD FLOW IN CANINE ADIPOSE TISSUE AND SKELETAL MUSCLE
141
be quite comparable on the weight basis, but since SP is a much larger molecule (molecular weights 354 and 1 340 respectively) it is even more active than PG on molecular basis. The circulatory effect of SP was not blocked by adrenergic blocking agents or antihistamines, which is consistent with the results obtained in man (Lofstrom et al. 1965). These results, together with the absence of tachyphylaxis, indicate that the vasodilatation is a direct effect of SP on the vascular smooth muscles and not due to release of any biogenic substance, such as histamine or catecholamines. This is further supported by the finding that SP did not induce any lipolysis, since both catecholamines and histamine are lipolytic agents in canine subcutaneous adipose tissue (Rosell 1966, Fredholm, Meng, Rosell 1968). Johnson and Erdos (1973) found that SP releases histamine from isolated rat mast cells. Our results indicate that such an action does not seem to be of any importance in canine adipose tissue. The 10-20 per cent increase in CFC found in the present experiments is presumably due to an increase in the functional capillary surface area due to dilatation of the precapillary sphincter region. This study was supported by grants from the Swedish Medical Research Council (04X-4495 and 4OX-3518) and Karolinska Institutet.
References CHANG,M. M. and S. E. LEEMAN, Isolation of a sialogogic peptide from bovine hypothalamic tissue and its characterization as substance P. J . b i d . Chem. 1970. 245. 4784-4790. An unidentified depressor substance in certain tissue extracts. J . EULER,v., U. S. and J. H. GADDUM, Physiol. (Lond.) 1931. 72. 74-87. FREDHOLM, B. B., B. C)BERGand S. ROSELL,Effects of vasoactive drugs on circulation in canine subcutaneous adipose tissue. Acra physiol. scand. 1970. 79. 564-574. FREDHOLM, B. B., H. C. MENGand S. ROSELL,Release of free fatty acids from canine subcutaneous adipose tissue by histamine and compound 48/80. Life Sci. 1968. 7. 1209-1215. FISHER,G. H., J. HUMPHRIES, K. FOLKERS, B. PERNOW and C. Y. BOWERS, Synthesis and some biological activities of substance P. J . Med. Chem. 1974. 17. 843-852. JOHNSON, A. R. and E. ERDOS,Release of histamine from mast c:lls by vasoactive ceptides. Proc. SOC. exp. B i d . (N.Y . ) 1973. 142. 1252-1256. LAURELL, S. and 0.TIBBLING, An enzymatic fluorimetric icrornethod for the determination of glyccrol. Clin. chim. Aera. 1966. 13. 317-322. LOFSTROM,B., B. PERNOW and J. WAHREN, Vasodilating action of substance P in the human forearm. Acta physiol. scand. 1965. 63. 31 1-324. &ERG, B. and S. ROSELL,Sympathetic control of consecutive vascular sections in canine subcutaneous adipose tissue. Acra physiol. scand. 1967. 71. 47-56. B. Studies on substans P. Purification, occurrence and biological actions. Acra phvsiol. stasd. PERNOW., 1953a. 29. Suppl. 105. PERNOW,B., Distribution of substans P in the central and peripheral nervous system. Nature (Lond.) 1953b. 171. 746-747. The influence of sympathetic adrenergic vasoconstrictor nerves on transport RENKIN,E. M. and S. ROSELL, of diffusible solutes from blood to tissues in skeletal muscle. Acra physiol. scand. 1962. 54. 223-240. ROSELL,S., Release of free fatty acids from subcutaneous adipose tissue in dogs following sympathetic nerve stimulation. Acta physiol. srand. 1966. 67. 343-351. STERN,P. and S. H U K O V ISpecific ~, antagonists of Substance P. In Symposium on Substance P. Proc. Sci. SOC.Bosnia Herzegouina. 1961. 1. 83-88.
TREGEAR, G. W.. H. D. NIALL,J. T. POTTS,S. E. LEEMAN and M. M. CHANG,Synthesis of Substance P. Nature New B i d . 1971. 232. 87-89. TROUT,P. L., H. ESTESand S. J. FRIEDBERG, Titration of free fatty acids of plasma. A study of current methods and a now modification. J. Lipid. Res. 1960. 1. 199-202.
