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Microcirculatory effects of prostacyclin (PGI2) in the hamster cheek pouch
MICROVASCULAR
RESEARCH
18,
245-254 (1979)
Microcirculatory Effects of Prostacyclin in the Hamster Cheek Pouch GERALD Department
A. HIGGS, DAVID
of Prostaglandin
C. CARDINAL, SALVADOR AND JOHN R. VANE
Research, Beckenham. Received
Wellcome Research Laboratories, Kent BR3 3BS. England August
(PGI,) MONCADA, Langley
Court,
16, 1978
The effects of prostacyclin (PGI,) on small blood vessels in the hamster cheek pouch microcirculation were studied microscopically. Prostacyclin applied systemically or locally caused an increase in the diameter of precapillary arterioles (lo-50 pm diameter) with inherent or induced tone. It was more potent than prostaglandin PGE,, PGEz, or bradykinin. In animals treated with indomethacin (4 mg/kg po), the relative vasodilator potency of prostacyclin to PGE, and PGE, was increased. The cyclic endoperoxide precursor of prostaglandins, PGH,, and the stable chemical breakdown product of prostacyclin, 6-oxo-PGF,,, were also dilators but were less potent than prostacyclin itself. In some experiments, responses of arterioles to PGH, were biphasic, a long-lasting dilatation being preceded by a short-lasting vasoconstriction. The postcapillary venules (20-50 pm diameter) in this preparation were inactive and did not respond by dilatation or constriction to PGI,, PGE,, PGE,, PGH,, 6-0x0-PGF,,, bradykinin, or noradrenaline.
INTRODUCTION Prostaglandins (PGs) of the E series are potent vasodepressor agents in a number of species including man, while F-type prostaglandins are vasoconstrictors in some vascular beds (for review see Messina et al. (1976)). The potent systemic cardiovascular actions of prostaglandins, coupled with their ability to modulate the effects of other vasoactive substances, suggests that prostaglandins could play an important part in the control of blood pressure and flow. Evidence is now emerging which indicates that the stable prostaglandins are not the major oxygenation products of their fatty acid precursors. The cyclic endoperoxides, PGG2 and PGHz, which are intermediates in the generation of prostaglandins from arachidonic acid (Hamberg et al., 1974) can also be converted to thromboxane A, (TXA2; Hamberg et al., 1975) or prostacyclin (PG12; Moncada et al., 1976a). These two unstable substances have opposing biological actions; TXAz contracts isolated vascular smooth *muscle, induces platelet aggregation in vitro (Hamberg et al., 1975; Bunting ef al., 1976b), and is a vasoconstrictor in the dog (Dustinget al., 1978), while PGI, relaxes isolated vascular smooth muscle, is a potent inhibitor of platelet aggregation in vitro (Moncada et al., 1976a; Bunting et al., 1976a), and reduces peripheral vascular resistance in the dog and other species (Armstrong et al., 1977). Prostacyclin has now been demonstrated to inhibit 245 002b2862!7~050245-10$0230!0 Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in U.S.A.
246
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intravascular thrombus formation in vivo and prolong bleeding time (Higgs et nl., 1977; Ubatuba et al., 1979). The cyclooxygenase which generates prostaglandin endoperoxides i’s widely distributed in mammalian tissues, but the enzymes which isomerise the endoperoxides differ from tissue to tissue. Thromboxanes are the predominant products of cyclooxygenase in platelets (Needleman et al., 1976) and polymorphonuclear leukocytes (Higgs et al., 1976; Goldstein ef al., 1977; Davison et al., 1978) while in blood vessel walls prostacyclin is the major metabolite of prostaglandin endoperoxides (Moncada ef al., 1976a; Salmon et al., 1978). Prostaglandins from the E series dilate precapillary vessels in the microcirculation of the rat (Kaley and Weiner, 1968), hamster (Siggins, 1972), and cat (Welch et al., 1974). In this paper we have investigated the direct effects of prostacyclin, its precursor PGH,, and its chemical degradation product 6-oxo-PGF,, (Johnson et al., 1976) on blood vessels in the microcirculation of the hamster cheek pouch. Some of these results have been communicated to the Physiological Society (Higgs et al., 1978b). METHODS Hamster cheek pouch preparation. The microcirculation of the hamster cheek pouch was studied using the method of Duling et al. (1968). Male golden hamsters (90- 150 g) were anaesthetized with sodium pentobarbitone (75 mg/kg ip), and their everted right cheek pouches were immersed in a well (9 ml) in a Perspex microscope stage. Anaesthesia was maintained by intravenous injection of sodium pentobarbitone via a cannula in the left jugular vein and mean systemic arterial blood pressure was monitored via a cannula in the left carotid artery. The cheek pouch preparation was superfused at 5 mYmin with a Krebs’ bicarbonate solution (1 litre of glass-distilled water containing 7.01 g NaCl, 1.68 g NaHCO,, 0.3503 g KCl, 0.2437 g MgC12, 0.2271 g CaCl, * 6H20, and 1.8 g D-glucose) at 36” which was prebubbled with 5% carbon dioxide in oxygen. In some experiments, animals were treated with indomethacin (4 mg/kg orally), 1-2 hr before cheek pouch eversion. Blood vessels in the microcirculation of the cheek pouch were examined microscopically using long-working-distance objectives (10, 20, or 32x) attached to a fixed-stage microscope (Leitz, Laborlux). By using 10 or 25x eyepieces the microcirculation could be studied at magnifications of 100-800. The preparation was viewed on a IO-in. television monitor (Hitachi, VM126AK) by coupling a lightweight, closed-circuit television camera (Hitachi, HV62K) to the microscope phototube. Vasoactive substances dissolved in Tris buffer (50 n-&f, pH 9.0) or saline (0.9% w/v) were applied to the preparation by intravenous injection (0.01-0.05 mYmin), infusion into the superfusing Krebs’ (0.05 mYmin), or by single injection (lo-50 ~1) into the Krebs’ immediately above the blood vessels being observed. Prostacyclin sodium salt (Whittaker, 1977) and PGH, were kept at 0” before use. Measurement of changes in blood vessel diameter. Changes in the diameter of blood vessels induced by vasoactive substances were recorded by including a videotape recorder (National, NV-3030E) in the television circuit. The television screen was calibrated for each magnification using a stage micrometer (2 mm, 200
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divisions; Leitz). By showing individual frames from the videotape recording, blood vessel diameters were measured directly from the television screen at OS- to lo-set intervals. A continuous, automatic record of changes in the width of the red cell column in blood vessels was obtained by attaching a photometric device to the television screen (Cardinal and Higgs, 1978). The device, which responds to increases and decreases in the amount of light transmitted from the screen during constriction and dilatation of blood vessels, was connected to a potentiometric pen recorder via a signal conditioning unit. At the beginning of each experiment the output of the photometric device was calibrated by taking direct measurements from the screen. Measurement of mean blood velocity. Mean blood velocity in venules and arterioles was measured using the method of Begent and Born (1970). Intravascular thrombus formation was observed in venules following the local application of adenosine diphosphate by iontophoresis, and in arterioles following localised electrical damage (Higgs et al., 1978a). The movement of embolised thrombi, downstream from their site of formation, was recorded on videotape. By reviewing the tape in slow motion the velocity of the thrombus (which was assumed to be a function of mean blood velocity) along a measured length of blood vessel was calculated. RESULTS After preparation of the hamster cheek pouch, a 30- to 45min equilibration period was allowed before suitable blood vessels were selected for observation. In healthy preparations, the mean blood velocity in precapillary arterioles (lo-50 pm diameter) was 2-7 mmlsec and in postcapillary venules (30-50 pm diameter), l-2 mm/set. About half the arterioles observed had inherent tone which could often be recognised by a rhythmic vasomotion or by their response to dilators. In these vessels, local injections of PGI, (0.1-20 ng), PGE, (lo-100 ng), PGE, (0.1-1.0 kg), 6-oxo-PGF,, (0.1-0.5 kg), or bradykinin (lo-100 ng) into the superfusing Krebs’ solution caused dose-dependent dilatation (Figs. 1, 2, and 3). Prostacyclin was approximately 8 times more potent than PGE, and 80 times more potent than PGE,. In three experiments, where animals were pretreated with indomethacin (2 hr before everting the cheek pouch), the relative potency of prostacyclin to PGE, or PGE, increased by about lo-fold. In these experiments, the dose-response curve of prostacyclin was shifted to the left whereas the dose-response curves for PGE, and PGEz remained unchanged. The cyclic endoperoxide PGH, (SO-500 ng) caused a dose-dependent dilatation of arterioles with inherent tone and in some experiments this response was preceded by a short-lasting constriction. PGD, or PGF,, (0.1-1.0 pg) did not produce significant changes in arteriolar diameter. Dilatation of precapillary vessels induced by single injections of prostaglandins into the superfusing Krebs’ solution lasted for 3-13 min, and tachyphylaxis was not observed with any of the vasoactive substances used. Precapillary vessels which did not respond to PGEl (500 ng), PGIz (100 ng), or bradykinin (100 ng) were assumed to be maximally dilated. In all the arterioles observed, locally applied noradrenaline (0. l- 10 ng) caused a dose-dependent
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FIG. 1. Response of a precapillary arteriole to prostacyclin. (A) An arteriole (22 pm diameter) with inherent tone, 3 min before the injection of 5 ng PGI, into the Krebs’ solution immediately above the vessel. (B) The same vessel % set after the injection of prostacyclin.
PROSTACYCLIN
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MICROCIRCULATION
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PG12
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6-0~0
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FIG. 2. Continuous records of changes in arteriole diameter induced by the local application of prostaglandin E, (PGE,), prostacyclin (PGI,), or 6-oxo-prostaglandin F,, (6-oxo-PGF,,). The traces were obtained using a photometric device (Cardinal and Higgs, 1978) attached to a television screen.
constriction (Figs. 4 and 5); 10 ng caused a mean decrease in diameter of 53 k 5% (mean + SEM; n = 10; range = 28-74). Tone could be induced in maximally dilated arterioles by including noradrenaline (0. l-l .O rig/ml) in the superfusing Krebs’ solution. Precapillary vessels with induced tone responded to PGIP, PGE,, 6-oxo-PGF,,, and PGH, by dilatation. The doses of locally applied vasoactive substances which were required to produce maximal constriction or dilatation of arterioles did not significantly affect mean systemic arterial blood pressure, but supramaximal doses of noradrenaline (100 ng) or PGI, (1 pg) were associated with small pressor or depressor responses, respectively (k 10 mm Hg).
FIG. 3. Vasodilator responses of arterioles to locally applied prostaglandins. Each point is the mean of 3-12 responses of arterioles in different animals and the bars represent -c I SEM.
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FIG. 4. Response of a postcapillary venule (top) and a precapillary arteriole to noradrenaline. (A) The vessels (approximately 35 pm diameter) 20 set before the injection of 10 ng noradrenaline into the Krebs’ solution immediately above the vessels. (B) The same vessels 48 set after the injection of noradrenaline.
PROSTACYCLIN
AND
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MICROCIRCULATION
l-Ong NA
251
2 Min
long NA
FIG. 5. A continuous record of changes in arteriole diameter induced by the local application of noradrenaline (NA).
Intravenous infusions of PGI, (5-50 ng 100 g-l min-‘) or PGE, (100-500 ng 100 g-’ min-‘) dilated the precapillary vessels in the cheek pouch microcirculation and caused a dose-dependent fall in mean systemic arterial blood pressure. PGI, (50 ng 100 g-l min-‘) or PGE, (500 ng 100 g-l mu-‘) reduced mean systemic arterial blood pressure by about 30 mm Hg. Intravenous infusions of 6-oxo-PGF,, (500 ng 100 g-l min-‘) did not significantly alter the tone of blood vessels in the microcirculation or lower systemic blood pressure. The postcapillary venules were insensitive to all the vasoactive substances used; Fig. 4 illustrates the complete occlusion of a precapillary arteriole in response to the local application of 10 ng noradrenaline, while the diameter of an adjacent venule remained unchanged. DISCUSSION These results demonstrate that prostacyclin dilates precapillary arterioles with inherent tone and reverses noradrenaline-induced constriction in these vessels. Applied locally, prostacyclin is a more potent vasodilator than the E-type prostaglandins and this activity is not due to its chemical degradation product 6-0x0PGF,,, which is also a vasodilator but is less potent than prostacyclin itself. Because indomethacin was given before cheek pouch eversion, it was not possible to measure its effects on vascular tone, but the increased sensitivity of arterioles to prostacyclin following indomethacin treatment may be explained by an inhibition of endogenous prostacyclin formation. Indomethacin causes a similar increase in the sensitivity of isolated smooth muscle preparations to prostaglandins (Eckenfels and Vane, 1972). The reversal of noradrenaline-induced arteriole constriction by PGE, and PGE, confirms the observations of Westwick and Lewis (1976) using this preparation. Prostaglandins dilate postcapillary vessels in the rat microcirculation (Messina et nl., 1974) and the complete insensitivity to vasoactive substances of venules in this preparation confirms the observations of Duling et al. (1968). The biphasic response obtained with PGH, is similar to that reported for PGG, (Lewis et al., 1977) in hamster cheek pouch arterioles, but in the rat cremaster microcirculation PGH, is only a dilator (Messina et al., 1977). A biphasic response to PGG, and PGH, has been observed in isolated vascular smooth muscle preparations (Bunting et al., 1976b; Dusting et al., 1977) and the relaxation component
252
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of this response is abolished by 15-hydroperoxyarachidonic acid, a selective inhibitor of prostacyclin production in vitro (Moncada et al., 1976b). These authors have suggested that cyclic endoperoxides have a direct contractor effect on vascular smooth muscle and that any relaxation is due to conversion to prostacyclin in the tissue. Blood vessels from a number of species, including the hamster (Higgs et al., 1978a) and man (Moncada et al., 1977), convert cyclic endoperoxides to prostacyclin, and under optimal conditions, over 80% of PGH2 is converted to prostacyclin by aortic microsomal preparations (Moncada et al., 1976a; Salmon et al., 1978). Furthermore, the isomerase which converts prostaglandin endoperoxides to PGE or PGD is not present in pig aorta (Salmon et al., 1978). This indicates that PG14, rather than PGEB, is the natural product of blood vessel arachidonate cyclooxygenase. The observations that prostaglandins augment vasodilatation and antagonise vasoconstriction have led to the theory that cyclooxygenase activation plays an important part in vascular homeostasis. The demonstration in this paper that prostacyclin is a potent vasodilator is consistent with this proposal and adds force to the concept that the prostaglandin involved in the regulation of blood vessel responses is prostacyclin. Prostaglandins are thought to be inflammatory mediators and some of the most convincing support for this theory is drawn from the evidence that PGEs have potent vasodilator properties. It is argued that the observed release of PGEs in acute inflammation accounts for the characteristic erythema and enhanced vascular permeability. Williams and Peck (1977) have demonstrated that permeabilityincreasing mediators act independently of the cyclooxygenase pathway but that the vasodilator component of an inflammatory response is abolished by cyclooxygenase inhibitors. It seems likely, therefore, that prostacyclin released from inflamed blood vessels accounts for the vasodilatation seen in acute inflammation. REFERENCES ARMSTRONG, J. M., CHAPPLE, D., DUSTING, G. J., HUGHES, R., MONCADA, S., AND VANE, J. R. (1977). Cardiovascular actions of prostacyclin (PGI,) in chloralose anaesthetized dogs. Brif. J. Pharmacol. 61, 136P. BEGENT, N. A., AND BORN, G. V. R. (1970). Growth rate in vivo of platelet thrombi, produced by iontophoresis of ADP as a function of mean blood flow velocity. Nature (London) 227, 926. BUNTING, S., GRYGLEWSKI, R., MONCADA, S., AND VANE, J. R. (1976a). Arterial walls generate from prostaglandin endoperoxides a substance (prostaglandin X) which relaxes strips of mesenteric and coeliac arteries and inhibits platelet aggregation. Prostaglandins 12, 897. BUNTING, S., MONCADA, S., AND VANE, J. R. (1976b). The effects of prostaglandin endoperoxides and thromboxane A2 on strips of rabbit coeliac artery and certain other smooth muscle preparations. Brit. J. Pharmacol. 57, 462P. CARDINAL, D. C., AND HIGGS, G. A. (1978). A photometric device for measuring blood vessel diameter in the microcirculation. J. Physiol. 275, 5P. DAVISON, E. M., FORD-HUTCHINSON, A. W., SMITH, M. J. H., AND WALKER, J. R. (1978). The release of thromboxane B, by rabbit peritoneal polymorphonuclear leucocytes. &it. J. Pharmacol. 63, 407P. DULING, B. R., BERNE, R. M., AND BORN, G. V. R. (1968). Microiontophoretic application of vasoactive agents to the microcirculation of the hamster cheek pouch. Microvasc. Res. 1, 158. DUSTING, G. J., LATTIMER, N., MONCADA, S., AND VANE, J. R. (1977). Prostaglandin X, the vascular metabolite of arachidonic acid responsible for relaxation of bovine coronary artery strips. Brif. J. Pharmncol. 59, 4431.
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G. .I., MONCADA, S., AND VANE, J. R. (1978). Vascular actions of arachidonic acid and its metabolites in perfused mesenteric and femoral beds of the dog. Eur. J. Pharmacol. 49, 65. ECKENFELS, A., AND VANE, J. R. (1972). Prostaglandins, oxygen tension and smooth muscle tone. Brit. J. Pharmacol. 45, 451. GOLDSTEIN, I. M., MALMSTEN, C. L., KAPLAN, H. B., JINDAHL, H., SAMUELSSON, B., AND WEISSMANN, G. (1977). Thromboxane generation by stimulated human granulocytes: Inhibition by glucocorticoids and superoxide dismutase. C/in. Res. 25. 518A. HAMBERG, M., SVENSSON, J., AND SAMUELSSON, B. (1975). Thromboxanes: A new group of biologically active compounds derived from prostaglandin endoperoxides. Proc. Nat. Acad. Sci. USA 72, DUSTING,
2994.
M., SVENSSON, J., WAKABAYASHI, T., AND SAMUELSSON, B. (1974). Isolation and structure of two prostaglandin endoperoxides that cause platelet aggregation. Proc. Nat. Acad. Sci. USA 71, 345. HIGGS, G. A., BUNTING, S., MONCADA, S., AND VANE, J. R. (1976). Polymorphonuclear leukocytes produce thromboxane A,-like activity during phagocytosis. Prostaglandins 12, 749. HIGGS, E. A., HIGGS, G. A., MONCADA, S., AND VANE, J. R. (1978a). Prostacyclin (PGI,) inhibits the formation of platelet thrombi in arterioles and venules of the hamster cheek pouch. Brit. J. HAMBERG,
Pharmacol.
63, 535.
MONCADA, S., AND VANE, J. R. (1977). Prostacyclin (PGI,) inhibits the formation of platelet thrombi induced by adenosine diphosphate (ADP) in vivo. Brit. J. Pharmacol. 61, 137P. HIGGS, G. A., MONCADA, S., AND VANE, J. R. (1978b). Prostacyclin as a potent dilator of arterioles in the hamster cheek pouch. J. Physiol. 275, 30P. JOHNSON, R. A., MORTON, D. R., KINNER, J. H., GORMAN, R. R., MCGUIRE, J. C., SUN, F. F., WHITTAKER, N., BUNTING, S., SALMON, J. A., MONCADA, S., AND VANE, J. R. (1976). The chemical structure of prostaglandin X (prostacyclin). Prostaglandins 12, 915. KALEY, G., AND WEINER, R. (1968). Microcirculatory studies with prostaglandin E,. In “Prostaglandin Symposium of the Worcester Foundation for Experimental Biology” (P. W. Ramwell and J. E. Shaw, eds.), p. 321. Wiley-Interscience, New York. LEWIS, G. P., WESTWICK, J., AND WILLIAMS, T. J. (1977). Microvascular responses produced by the prostaglandin endoperoxide PGG, in vivo. Brit. J. Pharmacol. 59, 442P. MESSINA, E. J., RODENBURG, J., SLOMIANY, B. L., ROBERTS, A. M., HINTZE, T. H., AND KALEY, G. (1977). Microcirculatory effects of arachidonic acid and an authentic prostaglandin endoperoxide (PGH,). The Physiologist, 20, 63. MESSINA, E. J., WEINER, R., AND KALEY, G. (1974). Microcirculatory effects of prostaglandins E,, El and A, in the rat mesentery and cremaster muscle. Microvasc. Res. 8, 77. MESSINA, E. J., WEINER, R., AND KALEY, G. (1976). Prostaglandins and local circulatory control. HIGGS,
Fed.
G. A.,
Proc.
35, 2367.
S., GRYGLEWSKI, R., BUNTING, S., AND VANE, J. R. (1976a). An enzyme isolated from arteries transforms prostaglandin endoperoxides to an unstable substance that inhibits platelet aggregation. Nature (London) 263, 663. MONCADA, S., GRYGLEWSKI, R., BUNTING, S., AND VANE, J. R. (1976b). A lipid peroxide inhibits the enzyme in blood vessel microsomes that generates from prostaglandin endoperoxides the substance (prostaglandin X) which prevents platelet aggregation. Prostaglandins 12, 715. MONCADA, S., HIGGS, E. A., AND VANE, J. R. (1977). Human arterial and venous tissues generate prostacyclin (prostaglandin X), a potent inhibitor of platelet aggregation. Lancer 1, 18. NEEDLEMAN, P., MONCADA, S., BUNTING, S., VANE, J. R., HAMBERG, M., AND SAMUELSSON, B. (1976). Identification of an enzyme in platelet microsomes which generates thromboxane A, from prostaglandin endoperoxides. Nature (London) 261, 558. SALMON, J. A., SMITH, D. R., FLOWER, R. J., MONCADA, S., AND VANE, J. R. (1978). Further studies on the enzymatic conversion of prostaglandin endoperoxide into prostacyclin by porcine aorta microsomes. Biochim. Biophys. Acta 523, 250. SIGGINS, G. R. (1972). Prostaglandins and the microvascular system: Physiological and histochemical correlations. In “Prostaglandins in Cellular Biology” (P. W. Ramwell and B. B. Phaniss, eds.), p. 451. Plenum, New York/London. UBATUBA, F., MONCADA, S., AND VANE, J. R. (1979). The effect of prostacyclin (PGI,) on platelet behaviour, thrombus formation in vivo and bleeding time. Thromb. Haemorrh. 41, 425. MONCADA,
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WELCH, K. M. A., KNOWLES, L., AND SPIRA, P. (1974). Local effects of prostaglandins on cat pial arteries. Eur. J. Pharmacol. 25, 155. WESTWICK, J., AND LEWIS, G. P. (1976). Reversal of noradrenaline induced constriction of hamster cheek pouch arterioles by prostaglandins and their metabolites. In “IXth European Conference of Microcirculation, Antwerp”; Bibl. Anat. 16, 466. WILLIAMS, T. J., AND PECK, M. J. (1977). Role of prostaglandin-mediated vasodilatation in inflammation. Nature (London) 270, 530. WHITTAKER, N. (1977). A synthesis of prostacyclin sodium salt. Tetrahedron Left. 32, 2805.
RESEARCH
18,
245-254 (1979)
Microcirculatory Effects of Prostacyclin in the Hamster Cheek Pouch GERALD Department
A. HIGGS, DAVID
of Prostaglandin
C. CARDINAL, SALVADOR AND JOHN R. VANE
Research, Beckenham. Received
Wellcome Research Laboratories, Kent BR3 3BS. England August
(PGI,) MONCADA, Langley
Court,
16, 1978
The effects of prostacyclin (PGI,) on small blood vessels in the hamster cheek pouch microcirculation were studied microscopically. Prostacyclin applied systemically or locally caused an increase in the diameter of precapillary arterioles (lo-50 pm diameter) with inherent or induced tone. It was more potent than prostaglandin PGE,, PGEz, or bradykinin. In animals treated with indomethacin (4 mg/kg po), the relative vasodilator potency of prostacyclin to PGE, and PGE, was increased. The cyclic endoperoxide precursor of prostaglandins, PGH,, and the stable chemical breakdown product of prostacyclin, 6-oxo-PGF,,, were also dilators but were less potent than prostacyclin itself. In some experiments, responses of arterioles to PGH, were biphasic, a long-lasting dilatation being preceded by a short-lasting vasoconstriction. The postcapillary venules (20-50 pm diameter) in this preparation were inactive and did not respond by dilatation or constriction to PGI,, PGE,, PGE,, PGH,, 6-0x0-PGF,,, bradykinin, or noradrenaline.
INTRODUCTION Prostaglandins (PGs) of the E series are potent vasodepressor agents in a number of species including man, while F-type prostaglandins are vasoconstrictors in some vascular beds (for review see Messina et al. (1976)). The potent systemic cardiovascular actions of prostaglandins, coupled with their ability to modulate the effects of other vasoactive substances, suggests that prostaglandins could play an important part in the control of blood pressure and flow. Evidence is now emerging which indicates that the stable prostaglandins are not the major oxygenation products of their fatty acid precursors. The cyclic endoperoxides, PGG2 and PGHz, which are intermediates in the generation of prostaglandins from arachidonic acid (Hamberg et al., 1974) can also be converted to thromboxane A, (TXA2; Hamberg et al., 1975) or prostacyclin (PG12; Moncada et al., 1976a). These two unstable substances have opposing biological actions; TXAz contracts isolated vascular smooth *muscle, induces platelet aggregation in vitro (Hamberg et al., 1975; Bunting ef al., 1976b), and is a vasoconstrictor in the dog (Dustinget al., 1978), while PGI, relaxes isolated vascular smooth muscle, is a potent inhibitor of platelet aggregation in vitro (Moncada et al., 1976a; Bunting et al., 1976a), and reduces peripheral vascular resistance in the dog and other species (Armstrong et al., 1977). Prostacyclin has now been demonstrated to inhibit 245 002b2862!7~050245-10$0230!0 Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in U.S.A.
246
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ET
AL.
intravascular thrombus formation in vivo and prolong bleeding time (Higgs et nl., 1977; Ubatuba et al., 1979). The cyclooxygenase which generates prostaglandin endoperoxides i’s widely distributed in mammalian tissues, but the enzymes which isomerise the endoperoxides differ from tissue to tissue. Thromboxanes are the predominant products of cyclooxygenase in platelets (Needleman et al., 1976) and polymorphonuclear leukocytes (Higgs et al., 1976; Goldstein ef al., 1977; Davison et al., 1978) while in blood vessel walls prostacyclin is the major metabolite of prostaglandin endoperoxides (Moncada ef al., 1976a; Salmon et al., 1978). Prostaglandins from the E series dilate precapillary vessels in the microcirculation of the rat (Kaley and Weiner, 1968), hamster (Siggins, 1972), and cat (Welch et al., 1974). In this paper we have investigated the direct effects of prostacyclin, its precursor PGH,, and its chemical degradation product 6-oxo-PGF,, (Johnson et al., 1976) on blood vessels in the microcirculation of the hamster cheek pouch. Some of these results have been communicated to the Physiological Society (Higgs et al., 1978b). METHODS Hamster cheek pouch preparation. The microcirculation of the hamster cheek pouch was studied using the method of Duling et al. (1968). Male golden hamsters (90- 150 g) were anaesthetized with sodium pentobarbitone (75 mg/kg ip), and their everted right cheek pouches were immersed in a well (9 ml) in a Perspex microscope stage. Anaesthesia was maintained by intravenous injection of sodium pentobarbitone via a cannula in the left jugular vein and mean systemic arterial blood pressure was monitored via a cannula in the left carotid artery. The cheek pouch preparation was superfused at 5 mYmin with a Krebs’ bicarbonate solution (1 litre of glass-distilled water containing 7.01 g NaCl, 1.68 g NaHCO,, 0.3503 g KCl, 0.2437 g MgC12, 0.2271 g CaCl, * 6H20, and 1.8 g D-glucose) at 36” which was prebubbled with 5% carbon dioxide in oxygen. In some experiments, animals were treated with indomethacin (4 mg/kg orally), 1-2 hr before cheek pouch eversion. Blood vessels in the microcirculation of the cheek pouch were examined microscopically using long-working-distance objectives (10, 20, or 32x) attached to a fixed-stage microscope (Leitz, Laborlux). By using 10 or 25x eyepieces the microcirculation could be studied at magnifications of 100-800. The preparation was viewed on a IO-in. television monitor (Hitachi, VM126AK) by coupling a lightweight, closed-circuit television camera (Hitachi, HV62K) to the microscope phototube. Vasoactive substances dissolved in Tris buffer (50 n-&f, pH 9.0) or saline (0.9% w/v) were applied to the preparation by intravenous injection (0.01-0.05 mYmin), infusion into the superfusing Krebs’ (0.05 mYmin), or by single injection (lo-50 ~1) into the Krebs’ immediately above the blood vessels being observed. Prostacyclin sodium salt (Whittaker, 1977) and PGH, were kept at 0” before use. Measurement of changes in blood vessel diameter. Changes in the diameter of blood vessels induced by vasoactive substances were recorded by including a videotape recorder (National, NV-3030E) in the television circuit. The television screen was calibrated for each magnification using a stage micrometer (2 mm, 200
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divisions; Leitz). By showing individual frames from the videotape recording, blood vessel diameters were measured directly from the television screen at OS- to lo-set intervals. A continuous, automatic record of changes in the width of the red cell column in blood vessels was obtained by attaching a photometric device to the television screen (Cardinal and Higgs, 1978). The device, which responds to increases and decreases in the amount of light transmitted from the screen during constriction and dilatation of blood vessels, was connected to a potentiometric pen recorder via a signal conditioning unit. At the beginning of each experiment the output of the photometric device was calibrated by taking direct measurements from the screen. Measurement of mean blood velocity. Mean blood velocity in venules and arterioles was measured using the method of Begent and Born (1970). Intravascular thrombus formation was observed in venules following the local application of adenosine diphosphate by iontophoresis, and in arterioles following localised electrical damage (Higgs et al., 1978a). The movement of embolised thrombi, downstream from their site of formation, was recorded on videotape. By reviewing the tape in slow motion the velocity of the thrombus (which was assumed to be a function of mean blood velocity) along a measured length of blood vessel was calculated. RESULTS After preparation of the hamster cheek pouch, a 30- to 45min equilibration period was allowed before suitable blood vessels were selected for observation. In healthy preparations, the mean blood velocity in precapillary arterioles (lo-50 pm diameter) was 2-7 mmlsec and in postcapillary venules (30-50 pm diameter), l-2 mm/set. About half the arterioles observed had inherent tone which could often be recognised by a rhythmic vasomotion or by their response to dilators. In these vessels, local injections of PGI, (0.1-20 ng), PGE, (lo-100 ng), PGE, (0.1-1.0 kg), 6-oxo-PGF,, (0.1-0.5 kg), or bradykinin (lo-100 ng) into the superfusing Krebs’ solution caused dose-dependent dilatation (Figs. 1, 2, and 3). Prostacyclin was approximately 8 times more potent than PGE, and 80 times more potent than PGE,. In three experiments, where animals were pretreated with indomethacin (2 hr before everting the cheek pouch), the relative potency of prostacyclin to PGE, or PGE, increased by about lo-fold. In these experiments, the dose-response curve of prostacyclin was shifted to the left whereas the dose-response curves for PGE, and PGEz remained unchanged. The cyclic endoperoxide PGH, (SO-500 ng) caused a dose-dependent dilatation of arterioles with inherent tone and in some experiments this response was preceded by a short-lasting constriction. PGD, or PGF,, (0.1-1.0 pg) did not produce significant changes in arteriolar diameter. Dilatation of precapillary vessels induced by single injections of prostaglandins into the superfusing Krebs’ solution lasted for 3-13 min, and tachyphylaxis was not observed with any of the vasoactive substances used. Precapillary vessels which did not respond to PGEl (500 ng), PGIz (100 ng), or bradykinin (100 ng) were assumed to be maximally dilated. In all the arterioles observed, locally applied noradrenaline (0. l- 10 ng) caused a dose-dependent
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FIG. 1. Response of a precapillary arteriole to prostacyclin. (A) An arteriole (22 pm diameter) with inherent tone, 3 min before the injection of 5 ng PGI, into the Krebs’ solution immediately above the vessel. (B) The same vessel % set after the injection of prostacyclin.
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249
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FIG. 2. Continuous records of changes in arteriole diameter induced by the local application of prostaglandin E, (PGE,), prostacyclin (PGI,), or 6-oxo-prostaglandin F,, (6-oxo-PGF,,). The traces were obtained using a photometric device (Cardinal and Higgs, 1978) attached to a television screen.
constriction (Figs. 4 and 5); 10 ng caused a mean decrease in diameter of 53 k 5% (mean + SEM; n = 10; range = 28-74). Tone could be induced in maximally dilated arterioles by including noradrenaline (0. l-l .O rig/ml) in the superfusing Krebs’ solution. Precapillary vessels with induced tone responded to PGIP, PGE,, 6-oxo-PGF,,, and PGH, by dilatation. The doses of locally applied vasoactive substances which were required to produce maximal constriction or dilatation of arterioles did not significantly affect mean systemic arterial blood pressure, but supramaximal doses of noradrenaline (100 ng) or PGI, (1 pg) were associated with small pressor or depressor responses, respectively (k 10 mm Hg).
FIG. 3. Vasodilator responses of arterioles to locally applied prostaglandins. Each point is the mean of 3-12 responses of arterioles in different animals and the bars represent -c I SEM.
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HIGGS ET AL.
FIG. 4. Response of a postcapillary venule (top) and a precapillary arteriole to noradrenaline. (A) The vessels (approximately 35 pm diameter) 20 set before the injection of 10 ng noradrenaline into the Krebs’ solution immediately above the vessels. (B) The same vessels 48 set after the injection of noradrenaline.
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MICROCIRCULATION
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251
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FIG. 5. A continuous record of changes in arteriole diameter induced by the local application of noradrenaline (NA).
Intravenous infusions of PGI, (5-50 ng 100 g-l min-‘) or PGE, (100-500 ng 100 g-’ min-‘) dilated the precapillary vessels in the cheek pouch microcirculation and caused a dose-dependent fall in mean systemic arterial blood pressure. PGI, (50 ng 100 g-l min-‘) or PGE, (500 ng 100 g-l mu-‘) reduced mean systemic arterial blood pressure by about 30 mm Hg. Intravenous infusions of 6-oxo-PGF,, (500 ng 100 g-l min-‘) did not significantly alter the tone of blood vessels in the microcirculation or lower systemic blood pressure. The postcapillary venules were insensitive to all the vasoactive substances used; Fig. 4 illustrates the complete occlusion of a precapillary arteriole in response to the local application of 10 ng noradrenaline, while the diameter of an adjacent venule remained unchanged. DISCUSSION These results demonstrate that prostacyclin dilates precapillary arterioles with inherent tone and reverses noradrenaline-induced constriction in these vessels. Applied locally, prostacyclin is a more potent vasodilator than the E-type prostaglandins and this activity is not due to its chemical degradation product 6-0x0PGF,,, which is also a vasodilator but is less potent than prostacyclin itself. Because indomethacin was given before cheek pouch eversion, it was not possible to measure its effects on vascular tone, but the increased sensitivity of arterioles to prostacyclin following indomethacin treatment may be explained by an inhibition of endogenous prostacyclin formation. Indomethacin causes a similar increase in the sensitivity of isolated smooth muscle preparations to prostaglandins (Eckenfels and Vane, 1972). The reversal of noradrenaline-induced arteriole constriction by PGE, and PGE, confirms the observations of Westwick and Lewis (1976) using this preparation. Prostaglandins dilate postcapillary vessels in the rat microcirculation (Messina et nl., 1974) and the complete insensitivity to vasoactive substances of venules in this preparation confirms the observations of Duling et al. (1968). The biphasic response obtained with PGH, is similar to that reported for PGG, (Lewis et al., 1977) in hamster cheek pouch arterioles, but in the rat cremaster microcirculation PGH, is only a dilator (Messina et al., 1977). A biphasic response to PGG, and PGH, has been observed in isolated vascular smooth muscle preparations (Bunting et al., 1976b; Dusting et al., 1977) and the relaxation component
252
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ET
AL.
of this response is abolished by 15-hydroperoxyarachidonic acid, a selective inhibitor of prostacyclin production in vitro (Moncada et al., 1976b). These authors have suggested that cyclic endoperoxides have a direct contractor effect on vascular smooth muscle and that any relaxation is due to conversion to prostacyclin in the tissue. Blood vessels from a number of species, including the hamster (Higgs et al., 1978a) and man (Moncada et al., 1977), convert cyclic endoperoxides to prostacyclin, and under optimal conditions, over 80% of PGH2 is converted to prostacyclin by aortic microsomal preparations (Moncada et al., 1976a; Salmon et al., 1978). Furthermore, the isomerase which converts prostaglandin endoperoxides to PGE or PGD is not present in pig aorta (Salmon et al., 1978). This indicates that PG14, rather than PGEB, is the natural product of blood vessel arachidonate cyclooxygenase. The observations that prostaglandins augment vasodilatation and antagonise vasoconstriction have led to the theory that cyclooxygenase activation plays an important part in vascular homeostasis. The demonstration in this paper that prostacyclin is a potent vasodilator is consistent with this proposal and adds force to the concept that the prostaglandin involved in the regulation of blood vessel responses is prostacyclin. Prostaglandins are thought to be inflammatory mediators and some of the most convincing support for this theory is drawn from the evidence that PGEs have potent vasodilator properties. It is argued that the observed release of PGEs in acute inflammation accounts for the characteristic erythema and enhanced vascular permeability. Williams and Peck (1977) have demonstrated that permeabilityincreasing mediators act independently of the cyclooxygenase pathway but that the vasodilator component of an inflammatory response is abolished by cyclooxygenase inhibitors. It seems likely, therefore, that prostacyclin released from inflamed blood vessels accounts for the vasodilatation seen in acute inflammation. REFERENCES ARMSTRONG, J. M., CHAPPLE, D., DUSTING, G. J., HUGHES, R., MONCADA, S., AND VANE, J. R. (1977). Cardiovascular actions of prostacyclin (PGI,) in chloralose anaesthetized dogs. Brif. J. Pharmacol. 61, 136P. BEGENT, N. A., AND BORN, G. V. R. (1970). Growth rate in vivo of platelet thrombi, produced by iontophoresis of ADP as a function of mean blood flow velocity. Nature (London) 227, 926. BUNTING, S., GRYGLEWSKI, R., MONCADA, S., AND VANE, J. R. (1976a). Arterial walls generate from prostaglandin endoperoxides a substance (prostaglandin X) which relaxes strips of mesenteric and coeliac arteries and inhibits platelet aggregation. Prostaglandins 12, 897. BUNTING, S., MONCADA, S., AND VANE, J. R. (1976b). The effects of prostaglandin endoperoxides and thromboxane A2 on strips of rabbit coeliac artery and certain other smooth muscle preparations. Brit. J. Pharmacol. 57, 462P. CARDINAL, D. C., AND HIGGS, G. A. (1978). A photometric device for measuring blood vessel diameter in the microcirculation. J. Physiol. 275, 5P. DAVISON, E. M., FORD-HUTCHINSON, A. W., SMITH, M. J. H., AND WALKER, J. R. (1978). The release of thromboxane B, by rabbit peritoneal polymorphonuclear leucocytes. &it. J. Pharmacol. 63, 407P. DULING, B. R., BERNE, R. M., AND BORN, G. V. R. (1968). Microiontophoretic application of vasoactive agents to the microcirculation of the hamster cheek pouch. Microvasc. Res. 1, 158. DUSTING, G. J., LATTIMER, N., MONCADA, S., AND VANE, J. R. (1977). Prostaglandin X, the vascular metabolite of arachidonic acid responsible for relaxation of bovine coronary artery strips. Brif. J. Pharmncol. 59, 4431.
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G. .I., MONCADA, S., AND VANE, J. R. (1978). Vascular actions of arachidonic acid and its metabolites in perfused mesenteric and femoral beds of the dog. Eur. J. Pharmacol. 49, 65. ECKENFELS, A., AND VANE, J. R. (1972). Prostaglandins, oxygen tension and smooth muscle tone. Brit. J. Pharmacol. 45, 451. GOLDSTEIN, I. M., MALMSTEN, C. L., KAPLAN, H. B., JINDAHL, H., SAMUELSSON, B., AND WEISSMANN, G. (1977). Thromboxane generation by stimulated human granulocytes: Inhibition by glucocorticoids and superoxide dismutase. C/in. Res. 25. 518A. HAMBERG, M., SVENSSON, J., AND SAMUELSSON, B. (1975). Thromboxanes: A new group of biologically active compounds derived from prostaglandin endoperoxides. Proc. Nat. Acad. Sci. USA 72, DUSTING,
2994.
M., SVENSSON, J., WAKABAYASHI, T., AND SAMUELSSON, B. (1974). Isolation and structure of two prostaglandin endoperoxides that cause platelet aggregation. Proc. Nat. Acad. Sci. USA 71, 345. HIGGS, G. A., BUNTING, S., MONCADA, S., AND VANE, J. R. (1976). Polymorphonuclear leukocytes produce thromboxane A,-like activity during phagocytosis. Prostaglandins 12, 749. HIGGS, E. A., HIGGS, G. A., MONCADA, S., AND VANE, J. R. (1978a). Prostacyclin (PGI,) inhibits the formation of platelet thrombi in arterioles and venules of the hamster cheek pouch. Brit. J. HAMBERG,
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MONCADA, S., AND VANE, J. R. (1977). Prostacyclin (PGI,) inhibits the formation of platelet thrombi induced by adenosine diphosphate (ADP) in vivo. Brit. J. Pharmacol. 61, 137P. HIGGS, G. A., MONCADA, S., AND VANE, J. R. (1978b). Prostacyclin as a potent dilator of arterioles in the hamster cheek pouch. J. Physiol. 275, 30P. JOHNSON, R. A., MORTON, D. R., KINNER, J. H., GORMAN, R. R., MCGUIRE, J. C., SUN, F. F., WHITTAKER, N., BUNTING, S., SALMON, J. A., MONCADA, S., AND VANE, J. R. (1976). The chemical structure of prostaglandin X (prostacyclin). Prostaglandins 12, 915. KALEY, G., AND WEINER, R. (1968). Microcirculatory studies with prostaglandin E,. In “Prostaglandin Symposium of the Worcester Foundation for Experimental Biology” (P. W. Ramwell and J. E. Shaw, eds.), p. 321. Wiley-Interscience, New York. LEWIS, G. P., WESTWICK, J., AND WILLIAMS, T. J. (1977). Microvascular responses produced by the prostaglandin endoperoxide PGG, in vivo. Brit. J. Pharmacol. 59, 442P. MESSINA, E. J., RODENBURG, J., SLOMIANY, B. L., ROBERTS, A. M., HINTZE, T. H., AND KALEY, G. (1977). Microcirculatory effects of arachidonic acid and an authentic prostaglandin endoperoxide (PGH,). The Physiologist, 20, 63. MESSINA, E. J., WEINER, R., AND KALEY, G. (1974). Microcirculatory effects of prostaglandins E,, El and A, in the rat mesentery and cremaster muscle. Microvasc. Res. 8, 77. MESSINA, E. J., WEINER, R., AND KALEY, G. (1976). Prostaglandins and local circulatory control. HIGGS,
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S., GRYGLEWSKI, R., BUNTING, S., AND VANE, J. R. (1976a). An enzyme isolated from arteries transforms prostaglandin endoperoxides to an unstable substance that inhibits platelet aggregation. Nature (London) 263, 663. MONCADA, S., GRYGLEWSKI, R., BUNTING, S., AND VANE, J. R. (1976b). A lipid peroxide inhibits the enzyme in blood vessel microsomes that generates from prostaglandin endoperoxides the substance (prostaglandin X) which prevents platelet aggregation. Prostaglandins 12, 715. MONCADA, S., HIGGS, E. A., AND VANE, J. R. (1977). Human arterial and venous tissues generate prostacyclin (prostaglandin X), a potent inhibitor of platelet aggregation. Lancer 1, 18. NEEDLEMAN, P., MONCADA, S., BUNTING, S., VANE, J. R., HAMBERG, M., AND SAMUELSSON, B. (1976). Identification of an enzyme in platelet microsomes which generates thromboxane A, from prostaglandin endoperoxides. Nature (London) 261, 558. SALMON, J. A., SMITH, D. R., FLOWER, R. J., MONCADA, S., AND VANE, J. R. (1978). Further studies on the enzymatic conversion of prostaglandin endoperoxide into prostacyclin by porcine aorta microsomes. Biochim. Biophys. Acta 523, 250. SIGGINS, G. R. (1972). Prostaglandins and the microvascular system: Physiological and histochemical correlations. In “Prostaglandins in Cellular Biology” (P. W. Ramwell and B. B. Phaniss, eds.), p. 451. Plenum, New York/London. UBATUBA, F., MONCADA, S., AND VANE, J. R. (1979). The effect of prostacyclin (PGI,) on platelet behaviour, thrombus formation in vivo and bleeding time. Thromb. Haemorrh. 41, 425. MONCADA,
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WELCH, K. M. A., KNOWLES, L., AND SPIRA, P. (1974). Local effects of prostaglandins on cat pial arteries. Eur. J. Pharmacol. 25, 155. WESTWICK, J., AND LEWIS, G. P. (1976). Reversal of noradrenaline induced constriction of hamster cheek pouch arterioles by prostaglandins and their metabolites. In “IXth European Conference of Microcirculation, Antwerp”; Bibl. Anat. 16, 466. WILLIAMS, T. J., AND PECK, M. J. (1977). Role of prostaglandin-mediated vasodilatation in inflammation. Nature (London) 270, 530. WHITTAKER, N. (1977). A synthesis of prostacyclin sodium salt. Tetrahedron Left. 32, 2805.