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Altered prostacyclin synthesis by aortae from hepatic portal vein-constricted rats: evidence for effects on protein kinase C and calcium
Copyright ©Journalof Hepatology 1994
Journal of Hepatology 1994; 21:1017-1022 Prh;ted ht Denmark. All rights reserved Munksgaard. Copenhagen
Journal of Hepatology ISSN 0168-8278
Altered prostacyclin synthesis by aortae from hepatic portal vein-constricted rats: evidence for effects on protein kinase C and calcium Jamie Y. Jeremy I, Dimitri P. Mikhailidis I, Stelios K a r a t a p a n i s 2, David H a r r y 2, Andrew K. Burroughs 2, Neil M c I n t y r e 2, G e r a r d Stansby 3, Michael Jacobs 4 and Aiden M c C o r m i c k 2 University Departnwnts o[" t Chentical Pathology and Hwnan Metabolism, :Medicine, 3Surgery and 4pharnlacology. Royal Free Hospital and School of Medichw, University of London. London, UK
(Received 19 August 1993)
To investigate the mechanisms causing reduced systemic vascular reactivity to vasoconstrictor agents in portal hypertension, we studied receptor- and signal-transduction-linked PGI2 (a vasodilator) synthesis (measured as 6-oxo-PGFl= by radioimmunoassay) in the aorta (ex vivo) of portal vein-constricted rats. PGI2 synthesis was stimulated by adrenaline (via heterogeneous alpha-adrenoceptors), phorbol ester dibutyrate (a protein kinase C activator), arachidonic acid (the substrate for PGI2 synthesis) and the Ca 2+ ionophore A23187 (A23187) and thapsigargin (both of which elevate intracellular Ca 2÷, which in turn elicits the release of arachidonic acid). The release of PGI2 by the aortae of rats with portal hypertension in comparison to sham-operated controls was: 1) enhanced in response to adrenaline, 2) reduced in response to phorbol ester dibutyrate, A23187 and thapsigargin and 3) unchanged in response to arichidonic acid. These data indicate that in aortae from rats with experimental portal hypertension: i) there are no changes in the enzymes involved in PGI2 synthesis (cyclooxygenase, PGI2 synthase), ii) there is a specific increase in adrenoceptor-linked PGI2 synthesis in aortae which may contribute to arterial vasodilation in this experimental model and 3) the diminished response of PGI2 synthesis to A23187, phorbol ester dibutyrate and thapsigargin indicates that there is a generalised attenuation of protein kinase C activator activity and of Ca 2+. Since Ca 2+ is a key component of excitation-contraction coupling and protein kinase C activator has been implicated in mediating this event, attenuation of these systems may also explain, at least in part, the known reduced vasoactivity of aortae from rats with portal hypertension. Whether a similar alteration of these mechanisms occurs in the vasculature of patients with portal hypertension warrants consideration. © Journal of Hepatology. Key words: Portal hypertension; Prostacyclin; Rat aorta
Arterial vasodilation, with increased splanchnic blood flow, is believed to be important in the pathogenesis of portal hypertension (1-4). In both animals and humans with portal hypertension, there are marked changes in systemic haemodynamics characterised by decreased peripheral vascular resistance (5-9). The mechanisms underlying reduced systemic vascular reactivity in portal hypertension are still undefined, although alterations in several systems have been demonstrated in man and experimental models. These include reduced responsiveness to proconstrictor agonists (adrenergic agonists (10,11), 5-hydroxytryptamine (5-HT; 12,13), angiotensin II (14)) and an in-
creased synthesis of the vasodilator, prostacyclin (PGI2; 15-18). It has also been proposed that an increase in the local synthesis of nitric oxide (NO), as a vasodilator, may play a key role in the vascular adaptions to portal hypertension in systemic blood vessels (19-22), although this has recently been contested (23). Some time ago, Murray & Paller (14,24) proposed that there was a post-receptor defect underlying the vascular reactivity changes in human portal hypertension. There is a marked increase in the output of PGI2 by the hepatic portal vein of rats with portal hypertension (1518), and high circulating levels of PGI2 metabolites have
Correspondence to: Dr. P.A. McCormick MD MRCE Liver Unit, St Vincent's Hospital, Elm Park, Dublin 4, Ireland.
1018 been reported in patients with portal hypertension (16). However, the mechanisms controlling vascular PGI2 synthesis, particularly in the systemic vasculature ill portal hypertension, are not clearly defined. Vascular PGI_, production is controlled by a complex interplay between pressor substances and intracellular signal transducers (G proteins, protein kinase C (PKC), inositol trisphosphate and Ca 2+ mobilisation (25)). In order to elucidate the mechanisms that may determine altered vascular PGI_~ synthesis in experimental portal hypertension, the effect of stimulators that act at different sites in the receptor activation-PGl_~ synthesis pathway was investigated in the aorta of the portal hypertensive rat. These were: adrenaline (mediated by heterogeneous aadrenoceptors; (26), phorbol ester dibuytrate a PKC activator; (27,28), thapsigargin (elevates intracellular Ca2+: (29, 30)), Ca 2+ ionophore A23187 (creates artificial Ca 2+ channels (31)) and arachidonic acid (substrate for PGI2 synthesis (32): elevated intracellular Ca 2+ activates phospholipase A2, which liberates arachidonic acid from phospholipid stores (25)). Thus, the above approach may reveal alterations of 'post-receptor' systems that control vasoactivity (viz. PKC, Ca 2+) as was suggested to occur in experimental portal hypertension by Murray & Paller (14,24).
Materials and Methods
Male Sprague Dawley rats of body weight 250 g were used. Twelve portal vein-constricted rats and 12 shamoperated (control) rats were studied. Portal hypertension was induced surgically by partial ligation of the portal vein (3). The rats were anaesthetised with intramuscular fentanyl (0.04 mg; Hypnorm, Janssen Pharmaceuticals, Belgium) and intraperitoneal diazepam (1 rag; Diazemuls, Dumex Ltd, Bucks UK). The portal vein was isolated and stenosis was created by a single ligature placed around both the portal vein and a blunt-ended 19G needle. Immediate removal of the needle allowed the portal vein to re-expand to the limit imposed by the ligature. In sham-operated animals, the operation consisted of dissection and visual inspection of the portal vein. The rats were allowed to recover and had free access to water and food. After 7 days, animals were anaesthetised with pentobarbitone (90 mg/kg, i.p.) and the portal venous blood pressure was measured following cannulation of the superior mesenteric vein. Thoracic aortae were excised and processed for assessment of PGI2 release as previously described (25-27), Spleens were also excised and weighed.
J.Y. JEREMY et al.
Preparation and incubation o['aortae for assessment o/" PGI_, o'nthesis Having removed adventitia and fatty tissue, the aortae were cut into 2-mm rings with a scalpel blade. Aortic rings from each animal were then placed in Dulbecco's Minimum Essential Medium (pregassed with 95% 0_<5% CO_,) and incubated for 6 h in Minimum Essential Medium at 37°C. with changes of medium every 30 min. to allow prostanoid release elicited by preparative handling to subside (23). Following pre-incubation, tissues were placed ill Minimum Essential Medium (per tube: two aortic rings, in duplicate for each drug dose) containing varying concentrations of adrenaline, Phorbol ester dibutyrate, thapsigargin, arichidonic acid and A23187. All drugs (with tile exception of adrenaline) were made up ill absolute alcohol (10 m~ml) and aliquots taken for dilution to working concentrations ill Minimum Essential Medium. Validation experiments demonstrated that the trace anaounts of alcohol in the final incubations had no effect on PGI2 synthesis. Tissues were then further incubated for 30 min at 37°C and aliquots of supernatant taken for estimation of 6-oxo-PG~,~ concentrations (tile stable spontaneous hydrolysate of PGI_< 32) by radioimmunoassay (as previously described; 26,27). For studies on synthesising capacity, aortae were sequestered into tubes as above, fi'eeze thawed, Minimum Essential Medium was added and the tissues were sonicated (2× 10 s bursts; Soniprep 150: MSE. Sussex, UK) and incubated at 37°C for I h. This procedure effectively destroys plasma membrane integrity and elicits PGI, synthesis via a massive influx of extracellular Ca 2+ and as such is an index of substrate (arachidonic acid) quantity and activity of phospholipase A_~, cyclooxygenase and PGI2 synthase (25). All tissues were weighed and PG release was related to wet weight of tissue.
Calculation o.f results and statistical analysis In the compilation of dose-response curves, basal output of PGI. (unstimulated) was subtracted from the stimulated values (i.e. in response to different concentrations of drugs). Values are expressed as mean -+S.D. of mean. Significance was determined by Student's t-test. Pvalues <0.01 were taken to be significant. Statistically significant differences between responses are indicated by an asterisk on the graphs. The Wilcoxon Rank test was used for statistics on blood pressures and spleen weights.
Materials and drugs Adrenaline bitartrate, arachidonic acid (calcium salt; 99.9% pure), phorbol ester dibutyrate, Ca 2+ ionophore A23187, Dulbecco's Minimum Essential Medium were purchased from Sigma Chemical Co. (Poole, Dorset, UK).
1019
PROSTACYCLIN IN PORTAL HYPERTENSION
Thapsigargin was purchased from Novabiochem UK (Nottingham, UK). Antisera against 6-oxo-PGFt,~ of high serological specificity was purchased from Capell Laboratories (West Chester, PA, USA). [3H]-6-oxo-PGFta (120 Ci/mmol) was purchased from New England Nuclear (Dreieich, Germany) and unlabelled ligand from Up John Co. (Kalamazoo, MI, USA). Results
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Twelve portal vein-constricted rats and 12 sham-operated (control) rats were studied. Portal venous pressure (mmHg [mean-s.e. mean, n= 12]) was significantly elevated in the portal vein-constricted rats compared to controls: 13.3-+0.8 vs 6.3-+0.8 (p<0.01). Spleen weights (g [mean_s.e. mean: n=12]) were also increased in portal vein-constricted rats compared to controls: 1.04---0.07 vs 0.79-+0.03 (not statistically significant). Spontaneous release of PGI2 (ng 6-oxo-PGF~,~/mg tissue/h [mean-+S.D., n = 12]) following freeze-thawing and sonication (an index of substrate and synthesising enzymes [Phospholipase A2, cyclooxygenase and synthase] and substrate availability) was not significantly different between aortae from portal hypertensive rats (8.8-+0.86) and "sham-operated animals (9.2-+0.89). PGI2 release by the aortae from rats with portal hypertension was enhanced in response to adrenaline compared
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to sham-operated animals (Fig. 1). In contrast, PGI2 lease by the aortae from portal hypertensive rats was duced in response to Phorbol ester dibutyrate (Fig. A23187 (Fig. 3) and thapsigargin (Fig. 4) compared
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Fig. 3. Prostacyclin release (as 6-oxo-PGF~a; stimulated by A23187) from aortae of portal vein-constricted rats (0) and sham-operated rats (~1.). Each point=mean-S.D., n= 12. # p<0.01.
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sham-operated controls. There were no differences in aortic PGI,_ output in response to arachidonic acid between rats with portal hypertension and control rats (data not shown). Basal (i.e. unstimulated) concentrations of 6-oxo PGFI,~ (pg/mg tissue/min [mean_S.D: n=12]) released from aortae were: 8+_0.4 (controls) and 9+_0.5 (portal hypertension). These values were not statistically different from each other.
Discussion
The present study demonstrates firstly that spontaneous release of PGI2 following freeze-thawing and sonication and in response to arachidonate (indices of substrate and synthesing enzymes [phospholipase A2, cyclooxygenase and synthase] and substrate availability (25)) was not significantly different between aortae from portal hypertensive rats and sham-operated animals. This demonstrates that there is no fundamental increase in the activity of PGI2 synthesising enzymes (phospholipase A2, cyclooxygenase, PGI2 synthase) in the aortae of rats with portal hypertension. Thus, in this experimental model, the aorta differs from the portal vein in that PGI2-synthesizing enzymes are increased in portal vein tissue of rats with portal hypertension (15-18). This difference probably reflects the different stresses to which the vessels are subjected in this experimental model. Following ligation of the hepatic portal vein, there is a marked increase in portal venous
blood pressure and distension of the portal vein. Both acute and chronic distension have been shown to increase PGI2 synthesis, not only in blood vessels, (e.g. the hepatic portal vein (15) and human aorta (33) but also in other tissues (e.g. urinary bladder of the diabetic rat (34)). There appears to be a specific enhancement of adrenoceptor-linked PGI2 synthesis in the aortae from rats with portal hypertension which cannot be ascribed to changes in endogenous substrate or PGI2 synthesising enzymes. Nor can it be explained by an increase in PKC activity or Ca 2+ mobilisation as PGI2 release in response to phorbol ester, A23187 and thapsigargin was actually diminished (see below). In a parallel study in the same rat model, phenylephrine-stimulated aortic contractility was significantly reduced in portal hypertensive animals (35). One possible explanation for this disparity is that adrenoceptors may be linked directly to Phospholipase A, via G proteins (effectively by-passing PKC and Ca 2+ mobilisation). Whether the present changes in adrenoceptorlinked PGI2 release reflect specific alterations in a G protein requires further study. The apparent reduction of PKC activity and of activator Ca 2+, as detected by diminished PGI2 release in response to phorbol ester, A23187 and thapsigargin, may explain (at least in part) the well-established diminished contractility of aortae in rats with portal hypertension. Ca 2+ is essential for excitation-contraction coupling in vascular tissue (36). PKC activation (by phorbol esters) also elicits in vitro vasoconstriction of blood vessels and this effect is enhanced by Ca 2+ (37,38)). In addition, virtually every pressor agent hitherto investigated elicits generation of diacyl glycerol and IP3 in vascular tissue (25). Diacyl glycerol activates PKC (which is a Ca2+-dependent enzyme) and [P 3 liberates intracellular stores of Ca 2+, thereby elevating cytosolic Ca -'+ levels (25,28). Since both events are associated with vasoconstriction, it follows that any attenuation of these events would result in a tendency toward vasodilation. In this context, we previously found diminished KCl-stimulated contraction of aortae from portal vein-constricted rats (KCI elicits vasoconstriction via mobilisation of extracellular Ca 2+) compared to controis (35). It is also of interest that phorbol ester inhibits NO synthesis in vascular tissue (39). Thus, a reduction in PKC activity in vessels may increase NO synthesis. It would be of interest to study the converse relationship (i.e. the effect of NO on PKC activity) in vascular tissue from rats with portal hypertension. However, it is known that NO inhibits Ca 2+ mobilization in vascular tissue (40) which in turn may reduce PKC activity, since this enzyme is Ca2+-dependent (28). As with any animal model of pathology, extrapolation to man should be approached with caution. However, the
PROSTACYCLIN IN PORTAL HYPERTENSION
present data are consistent with the results o f a recent study (10), in which the pressor effect of constrictor symp a t h o m i m e t i c s a n d a n g i o t e n s i n II were assessed in patients with cirrhosis. There was a m a r k e d i m p a i r m e n t in pressor effect (irrespective o f the agonist employed) in patients with cirrhosis (10). This was n o t due to a generalised sympathetic desensitisation b u t to i m p a i r m e n t o f responses in peripheral vascular s m o o t h muscle (10). Since, pressor action is mediated by P K C a n d Ca 2+, these d a t a from patients with cirrhosis are in general a g r e e m e n t with o u r results in the rat. Clearly, the a d m i n i s t r a t i o n of phorbol esters, i o n o p h o r e s a n d thapsigargin to patients is not feasible. However, strategies could be devised to investigate whether alterations in P K C a n d Ca 2+ occur in the peripheral vasculature o f patients with portal hypertension. In summary, experimental portal hypertension elicits a specific change o f a d r e n o c e p t o r - l i n k e d PGI2 synthesis in the aorta, which m a y be mediated by alterations of G proteins. Since PGI2 is a vasodilator a n d circulating catecholamines are elevated in portal hypertension (41~,3), this may constitute a m e c h a n i s m by which dilation is enhanced in peripheral vascular tissue. The generalised red u c t i o n in P K C activity a n d Ca -'+. 'availability' m a y also account, at least in part, for the diminished vasoactivity of the a o r t a in portal hypertension. A l t h o u g h the mechanisms that govern the adaptive changes o f the vasculature in response to portal h y p e r t e n s i o n remain u n k n o w n , this p h e n o m e n o n is likely to be multifactorial, involving NO, P G I > P K C , Ca 2÷ a n d G proteins. F u r t h e r m o r e , by using the a p p r o a c h employed in this study, these m e c h a n isms (intrinsic vascular a n d / o r circulating trophic factors) could be dissected out. A n u n d e r s t a n d i n g o f the m e c h a n isms that d e t e r m i n e the vascular changes discussed here may be o f value in designing t r e a t m e n t for patients with d e c o m p e n s a t e d cirrhosis a n d patients with other diseases associated with significant changes in v a s o m o t o r tone.
References 1. Bosch J, Pizcueta MP, Feu E Fernandez M, Garcia-Pagan JC. Pathophysiology of portal hypertension. Gastroenterol Clin N Am 1992; 21: 1-14. 2. Schrier RW, Arroyo V, Bernardi M, Epstein M, Henriksen JH, Rodes J. Peripheral arterial vasodilation hypothesis: a proposal for the initiation of renal sodium and water retention in cirrhosis. Hepatology 1988; 8: 1151-7. 3. VorobioffJ, Bredfeldt JE. Groszmann RJ. Hyperdynamic circulation in portal-hypertensive rat model: a primary factor for maintenance of chronic portal hypertension. Am J Physiol 1983; 244: G52-7. 4. Pizcueta ME De Lacy AM, Kravetz D, Bosch J, Rodes J. Propranolol decreases portal pressure without changing procollateral resistance in cirrhotic rats. Hepatology 1989; 10: 953-7. 5. Murray JE Dawson AM, Sherlock S. Circulatory changes in chronic liver disease. Am J Med 1958; 24: 358-67.
1021 6. Kowalski H, Abelmann WH. Cardiac output at rest in Laennec's cirrhosis. J Clin Invest 1953; 32: 1025-33. 7. Siegel JH, Golgwyn RM, Farrell J. Hyperdynamic states and the physiologic determinants of survival. Arch Surg 1974; 108: 28392. 8. Lee SS. Cardiac abnormalities in liver cirrhosis. West J Med 1989; 1512: 530-5. 9. Bomzon A, Blendis LM. Vascular reactivity in experimental portal hypertension. Am J Physiol 1987; 252: G158-62. 10. MacGilchrist AJ, Sumner D, Reid JL. Impaired pressor reactivity in cirrhosis: evidence for a peripheral vascular defect. Hepatology 1991; 13: 689-94. 11. Pizcueta MP, Casamitjana R, Bosch J, Rodes J. Decreased systemic vascular sensitivity to norepinephrine in portal hypertensive rats. Role of hyperglucagonism. Am J Physiol 1990; 258: G191-5. 12. Kaumann AJ, Morgan JS, Groszmann RC. ICI 169,369 selectively blocks 5-hydroxytryptamine receptors and lowers portal pressure in portal hypertensive rats. Br J Pharmacol 1986; 89: 501-13. 13. Cummings S, Groszmann R, Kaumann A. Hypersensitivity of mesenteric veins to 5-hydroxytryptamine and ketanserin-induced reduction of portal pressure in portal hypertensive rats. Br J Pharmacol 1986; 89: 501-13. 14. Murray BM, Paller MS. Decreased pressor reactivity to angiotensin II in cirrhotic rats: evidence for a post-receptor defect in angiotensin action. Circ Res 1985; 57: 424-31. 15. Hamilton G, Phing RCE Hutton RA, Dandona P, Hobbs KEE The relationship between prostacyclin activity and pressure in the portal vein. Hepatology 1982; 2: 236--42. 16. Ritter JM, Hamilton DP, Barrow SE, Heavey DJ, Hickling NE, Taylor KM, et al. Prostacyclin in the circulation of patients with vascular disorders undergoing surgery. Clin Sci 1986; 71: 743-7. 17. Sitzmann JV, Li SS. Evidence for role of prostacylin as a systemic hormone in portal hypertension. Surgery 1991; 109: 149-53. 18. Guarner C, Soriuno G. Prostaglandins and portal hypertension. Prostagl Leuk Essential Fatty Acids 1993: in press. 19. Vallance P, Moncada S. Hypothesis: induction of nitric oxide synthase in the vasculature underlies the hyperdynamic circulation. Lancet 1991; 337: 776-8. 20. Whittle BJR, Moncada S. Nitric oxide: the elusive mediator of the hyperdynamic circulation of cirrhosis? Hepatology 16: 108992. 21. Pizcueta MPM, Pique JM, Bosch J, Whittle BJR, Moncada S. Nitric oxide and hyperdynamic circulation in portal hypertension. Br J Pharmacol 1992: 105: 184-90. 22. Lee F-A, Albillos A, Colombato LA, Groszmann RJ. The role of nitric oxide in the vascular hyporesponsiveness to methoxamine in portal hypertensive rats. Hepatology 1992; 16: 1043-8. 23. Sogni P, Moreau R, Ohsuga M, Cailmail S, Oberti E Hadengue A, et al. Evidence for normal nitric oxide-mediated vasodilator tone in conscious rats with cirrhosis. Hepatology 1992; 16: 9803. 24. Murray BM, Paller MS. Pressor resistance to vasopressin in sodium depletion and cirrhosis. Am J Physiol 1986; 251: R525-30. 25. Jeremy JY, Mikhailidis DP, Dandona P. Excitatory receptorlinked prostanoid synthesis in mammalian smooth muscle: the role of calcium, protein kinase C and G proteins. Prostagl Leuk Essential Fatty Acids Rev 1988; 34: 215-28. 26. Jeremy JY, Mikhailidis DP, Dandona P. Adrenergic modulation of vascular prostacyclin synthesis. Eur J Pharmacol 1985; 114: 33-40. 27. Jeremy JY, Dandona P. Role of the diacyl glycerol protein kinase C system in mediating adrenoceptor-prostacyclin synthesis coupling in the rat aorta. Eur J Pharmacol 1986; 136:311-6. 28. Nishizuka Y. The role of protein kinase C in cell surface signal transduction and tumor promotion. Nature 1984; 308: 693-8. 29. Levine L, Watanabe M. Amplification of prostaglandin I2 production by thapsigargin. Prostaglandins 1991; 42: 291-302.
1022 30. Thastrup O. Role of Ca 2+ (+)-ATPase in the regulation of cellular Ca-'" signalling, as studied with the selective microsomal Ca -'+ (+)-ATPase inhibitor, thapsigargin. Agents Actions 1990; 29: 815. 31. Reed PW, Lardy HA. A23187: a divalent cation ionophore. J Biol Chem 1972; 247: 6970-8. 32. Moncada S, Vane JR. Prostacyclin formation and effects. In: Roberts SM, Scheinman E eds. Chemistry, Biochemistry and Pharmacology of Prostacyclin. Oxford: Pergamon Press, 1978; 25873. 33. Tsang V, Jeremy JY, Mikhailidis DE Walesby RK. Wright JC, Dandona E Release of prostacyclin from the human aorta. Cardiovasc Res 1988; 22: 489-93. 34. Jeremy JY, Thompson CS, Barradas MA. Mikailidis DE Dandona E The effect of streptozotocin-induced diabetes on prostacyclin synthesis by the rat bladder. J Urol 1986; 135: 1290-2. 35. Karatapanis S, McCormick A, Jacobs M, Jeremy JY, Stansby G, Collins E et al. Role of nitric oxide in the altered systemic vascular reactivity in portal vein constricted rat. J Hepatol 1992: 16: suppl. 1: $34. 36. Somlyo AV, Somlyo AP. Electromechanical coupling and pharmacomechanical coupling in vascular smooth muscle. J Pharmacol Exp Ther 1968; 159: 129-39. 37. Danthaluri NR, Deth RC. Phorbol ester-induced contraction of
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Journal of Hepatology 1994; 21:1017-1022 Prh;ted ht Denmark. All rights reserved Munksgaard. Copenhagen
Journal of Hepatology ISSN 0168-8278
Altered prostacyclin synthesis by aortae from hepatic portal vein-constricted rats: evidence for effects on protein kinase C and calcium Jamie Y. Jeremy I, Dimitri P. Mikhailidis I, Stelios K a r a t a p a n i s 2, David H a r r y 2, Andrew K. Burroughs 2, Neil M c I n t y r e 2, G e r a r d Stansby 3, Michael Jacobs 4 and Aiden M c C o r m i c k 2 University Departnwnts o[" t Chentical Pathology and Hwnan Metabolism, :Medicine, 3Surgery and 4pharnlacology. Royal Free Hospital and School of Medichw, University of London. London, UK
(Received 19 August 1993)
To investigate the mechanisms causing reduced systemic vascular reactivity to vasoconstrictor agents in portal hypertension, we studied receptor- and signal-transduction-linked PGI2 (a vasodilator) synthesis (measured as 6-oxo-PGFl= by radioimmunoassay) in the aorta (ex vivo) of portal vein-constricted rats. PGI2 synthesis was stimulated by adrenaline (via heterogeneous alpha-adrenoceptors), phorbol ester dibutyrate (a protein kinase C activator), arachidonic acid (the substrate for PGI2 synthesis) and the Ca 2+ ionophore A23187 (A23187) and thapsigargin (both of which elevate intracellular Ca 2÷, which in turn elicits the release of arachidonic acid). The release of PGI2 by the aortae of rats with portal hypertension in comparison to sham-operated controls was: 1) enhanced in response to adrenaline, 2) reduced in response to phorbol ester dibutyrate, A23187 and thapsigargin and 3) unchanged in response to arichidonic acid. These data indicate that in aortae from rats with experimental portal hypertension: i) there are no changes in the enzymes involved in PGI2 synthesis (cyclooxygenase, PGI2 synthase), ii) there is a specific increase in adrenoceptor-linked PGI2 synthesis in aortae which may contribute to arterial vasodilation in this experimental model and 3) the diminished response of PGI2 synthesis to A23187, phorbol ester dibutyrate and thapsigargin indicates that there is a generalised attenuation of protein kinase C activator activity and of Ca 2+. Since Ca 2+ is a key component of excitation-contraction coupling and protein kinase C activator has been implicated in mediating this event, attenuation of these systems may also explain, at least in part, the known reduced vasoactivity of aortae from rats with portal hypertension. Whether a similar alteration of these mechanisms occurs in the vasculature of patients with portal hypertension warrants consideration. © Journal of Hepatology. Key words: Portal hypertension; Prostacyclin; Rat aorta
Arterial vasodilation, with increased splanchnic blood flow, is believed to be important in the pathogenesis of portal hypertension (1-4). In both animals and humans with portal hypertension, there are marked changes in systemic haemodynamics characterised by decreased peripheral vascular resistance (5-9). The mechanisms underlying reduced systemic vascular reactivity in portal hypertension are still undefined, although alterations in several systems have been demonstrated in man and experimental models. These include reduced responsiveness to proconstrictor agonists (adrenergic agonists (10,11), 5-hydroxytryptamine (5-HT; 12,13), angiotensin II (14)) and an in-
creased synthesis of the vasodilator, prostacyclin (PGI2; 15-18). It has also been proposed that an increase in the local synthesis of nitric oxide (NO), as a vasodilator, may play a key role in the vascular adaptions to portal hypertension in systemic blood vessels (19-22), although this has recently been contested (23). Some time ago, Murray & Paller (14,24) proposed that there was a post-receptor defect underlying the vascular reactivity changes in human portal hypertension. There is a marked increase in the output of PGI2 by the hepatic portal vein of rats with portal hypertension (1518), and high circulating levels of PGI2 metabolites have
Correspondence to: Dr. P.A. McCormick MD MRCE Liver Unit, St Vincent's Hospital, Elm Park, Dublin 4, Ireland.
1018 been reported in patients with portal hypertension (16). However, the mechanisms controlling vascular PGI2 synthesis, particularly in the systemic vasculature ill portal hypertension, are not clearly defined. Vascular PGI_, production is controlled by a complex interplay between pressor substances and intracellular signal transducers (G proteins, protein kinase C (PKC), inositol trisphosphate and Ca 2+ mobilisation (25)). In order to elucidate the mechanisms that may determine altered vascular PGI_~ synthesis in experimental portal hypertension, the effect of stimulators that act at different sites in the receptor activation-PGl_~ synthesis pathway was investigated in the aorta of the portal hypertensive rat. These were: adrenaline (mediated by heterogeneous aadrenoceptors; (26), phorbol ester dibuytrate a PKC activator; (27,28), thapsigargin (elevates intracellular Ca2+: (29, 30)), Ca 2+ ionophore A23187 (creates artificial Ca 2+ channels (31)) and arachidonic acid (substrate for PGI2 synthesis (32): elevated intracellular Ca 2+ activates phospholipase A2, which liberates arachidonic acid from phospholipid stores (25)). Thus, the above approach may reveal alterations of 'post-receptor' systems that control vasoactivity (viz. PKC, Ca 2+) as was suggested to occur in experimental portal hypertension by Murray & Paller (14,24).
Materials and Methods
Male Sprague Dawley rats of body weight 250 g were used. Twelve portal vein-constricted rats and 12 shamoperated (control) rats were studied. Portal hypertension was induced surgically by partial ligation of the portal vein (3). The rats were anaesthetised with intramuscular fentanyl (0.04 mg; Hypnorm, Janssen Pharmaceuticals, Belgium) and intraperitoneal diazepam (1 rag; Diazemuls, Dumex Ltd, Bucks UK). The portal vein was isolated and stenosis was created by a single ligature placed around both the portal vein and a blunt-ended 19G needle. Immediate removal of the needle allowed the portal vein to re-expand to the limit imposed by the ligature. In sham-operated animals, the operation consisted of dissection and visual inspection of the portal vein. The rats were allowed to recover and had free access to water and food. After 7 days, animals were anaesthetised with pentobarbitone (90 mg/kg, i.p.) and the portal venous blood pressure was measured following cannulation of the superior mesenteric vein. Thoracic aortae were excised and processed for assessment of PGI2 release as previously described (25-27), Spleens were also excised and weighed.
J.Y. JEREMY et al.
Preparation and incubation o['aortae for assessment o/" PGI_, o'nthesis Having removed adventitia and fatty tissue, the aortae were cut into 2-mm rings with a scalpel blade. Aortic rings from each animal were then placed in Dulbecco's Minimum Essential Medium (pregassed with 95% 0_<5% CO_,) and incubated for 6 h in Minimum Essential Medium at 37°C. with changes of medium every 30 min. to allow prostanoid release elicited by preparative handling to subside (23). Following pre-incubation, tissues were placed ill Minimum Essential Medium (per tube: two aortic rings, in duplicate for each drug dose) containing varying concentrations of adrenaline, Phorbol ester dibutyrate, thapsigargin, arichidonic acid and A23187. All drugs (with tile exception of adrenaline) were made up ill absolute alcohol (10 m~ml) and aliquots taken for dilution to working concentrations ill Minimum Essential Medium. Validation experiments demonstrated that the trace anaounts of alcohol in the final incubations had no effect on PGI2 synthesis. Tissues were then further incubated for 30 min at 37°C and aliquots of supernatant taken for estimation of 6-oxo-PG~,~ concentrations (tile stable spontaneous hydrolysate of PGI_< 32) by radioimmunoassay (as previously described; 26,27). For studies on synthesising capacity, aortae were sequestered into tubes as above, fi'eeze thawed, Minimum Essential Medium was added and the tissues were sonicated (2× 10 s bursts; Soniprep 150: MSE. Sussex, UK) and incubated at 37°C for I h. This procedure effectively destroys plasma membrane integrity and elicits PGI, synthesis via a massive influx of extracellular Ca 2+ and as such is an index of substrate (arachidonic acid) quantity and activity of phospholipase A_~, cyclooxygenase and PGI2 synthase (25). All tissues were weighed and PG release was related to wet weight of tissue.
Calculation o.f results and statistical analysis In the compilation of dose-response curves, basal output of PGI. (unstimulated) was subtracted from the stimulated values (i.e. in response to different concentrations of drugs). Values are expressed as mean -+S.D. of mean. Significance was determined by Student's t-test. Pvalues <0.01 were taken to be significant. Statistically significant differences between responses are indicated by an asterisk on the graphs. The Wilcoxon Rank test was used for statistics on blood pressures and spleen weights.
Materials and drugs Adrenaline bitartrate, arachidonic acid (calcium salt; 99.9% pure), phorbol ester dibutyrate, Ca 2+ ionophore A23187, Dulbecco's Minimum Essential Medium were purchased from Sigma Chemical Co. (Poole, Dorset, UK).
1019
PROSTACYCLIN IN PORTAL HYPERTENSION
Thapsigargin was purchased from Novabiochem UK (Nottingham, UK). Antisera against 6-oxo-PGFt,~ of high serological specificity was purchased from Capell Laboratories (West Chester, PA, USA). [3H]-6-oxo-PGFta (120 Ci/mmol) was purchased from New England Nuclear (Dreieich, Germany) and unlabelled ligand from Up John Co. (Kalamazoo, MI, USA). Results
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Twelve portal vein-constricted rats and 12 sham-operated (control) rats were studied. Portal venous pressure (mmHg [mean-s.e. mean, n= 12]) was significantly elevated in the portal vein-constricted rats compared to controls: 13.3-+0.8 vs 6.3-+0.8 (p<0.01). Spleen weights (g [mean_s.e. mean: n=12]) were also increased in portal vein-constricted rats compared to controls: 1.04---0.07 vs 0.79-+0.03 (not statistically significant). Spontaneous release of PGI2 (ng 6-oxo-PGF~,~/mg tissue/h [mean-+S.D., n = 12]) following freeze-thawing and sonication (an index of substrate and synthesising enzymes [Phospholipase A2, cyclooxygenase and synthase] and substrate availability) was not significantly different between aortae from portal hypertensive rats (8.8-+0.86) and "sham-operated animals (9.2-+0.89). PGI2 release by the aortae from rats with portal hypertension was enhanced in response to adrenaline compared
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to sham-operated animals (Fig. 1). In contrast, PGI2 lease by the aortae from portal hypertensive rats was duced in response to Phorbol ester dibutyrate (Fig. A23187 (Fig. 3) and thapsigargin (Fig. 4) compared
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sham-operated controls. There were no differences in aortic PGI,_ output in response to arachidonic acid between rats with portal hypertension and control rats (data not shown). Basal (i.e. unstimulated) concentrations of 6-oxo PGFI,~ (pg/mg tissue/min [mean_S.D: n=12]) released from aortae were: 8+_0.4 (controls) and 9+_0.5 (portal hypertension). These values were not statistically different from each other.
Discussion
The present study demonstrates firstly that spontaneous release of PGI2 following freeze-thawing and sonication and in response to arachidonate (indices of substrate and synthesing enzymes [phospholipase A2, cyclooxygenase and synthase] and substrate availability (25)) was not significantly different between aortae from portal hypertensive rats and sham-operated animals. This demonstrates that there is no fundamental increase in the activity of PGI2 synthesising enzymes (phospholipase A2, cyclooxygenase, PGI2 synthase) in the aortae of rats with portal hypertension. Thus, in this experimental model, the aorta differs from the portal vein in that PGI2-synthesizing enzymes are increased in portal vein tissue of rats with portal hypertension (15-18). This difference probably reflects the different stresses to which the vessels are subjected in this experimental model. Following ligation of the hepatic portal vein, there is a marked increase in portal venous
blood pressure and distension of the portal vein. Both acute and chronic distension have been shown to increase PGI2 synthesis, not only in blood vessels, (e.g. the hepatic portal vein (15) and human aorta (33) but also in other tissues (e.g. urinary bladder of the diabetic rat (34)). There appears to be a specific enhancement of adrenoceptor-linked PGI2 synthesis in the aortae from rats with portal hypertension which cannot be ascribed to changes in endogenous substrate or PGI2 synthesising enzymes. Nor can it be explained by an increase in PKC activity or Ca 2+ mobilisation as PGI2 release in response to phorbol ester, A23187 and thapsigargin was actually diminished (see below). In a parallel study in the same rat model, phenylephrine-stimulated aortic contractility was significantly reduced in portal hypertensive animals (35). One possible explanation for this disparity is that adrenoceptors may be linked directly to Phospholipase A, via G proteins (effectively by-passing PKC and Ca 2+ mobilisation). Whether the present changes in adrenoceptorlinked PGI2 release reflect specific alterations in a G protein requires further study. The apparent reduction of PKC activity and of activator Ca 2+, as detected by diminished PGI2 release in response to phorbol ester, A23187 and thapsigargin, may explain (at least in part) the well-established diminished contractility of aortae in rats with portal hypertension. Ca 2+ is essential for excitation-contraction coupling in vascular tissue (36). PKC activation (by phorbol esters) also elicits in vitro vasoconstriction of blood vessels and this effect is enhanced by Ca 2+ (37,38)). In addition, virtually every pressor agent hitherto investigated elicits generation of diacyl glycerol and IP3 in vascular tissue (25). Diacyl glycerol activates PKC (which is a Ca2+-dependent enzyme) and [P 3 liberates intracellular stores of Ca 2+, thereby elevating cytosolic Ca -'+ levels (25,28). Since both events are associated with vasoconstriction, it follows that any attenuation of these events would result in a tendency toward vasodilation. In this context, we previously found diminished KCl-stimulated contraction of aortae from portal vein-constricted rats (KCI elicits vasoconstriction via mobilisation of extracellular Ca 2+) compared to controis (35). It is also of interest that phorbol ester inhibits NO synthesis in vascular tissue (39). Thus, a reduction in PKC activity in vessels may increase NO synthesis. It would be of interest to study the converse relationship (i.e. the effect of NO on PKC activity) in vascular tissue from rats with portal hypertension. However, it is known that NO inhibits Ca 2+ mobilization in vascular tissue (40) which in turn may reduce PKC activity, since this enzyme is Ca2+-dependent (28). As with any animal model of pathology, extrapolation to man should be approached with caution. However, the
PROSTACYCLIN IN PORTAL HYPERTENSION
present data are consistent with the results o f a recent study (10), in which the pressor effect of constrictor symp a t h o m i m e t i c s a n d a n g i o t e n s i n II were assessed in patients with cirrhosis. There was a m a r k e d i m p a i r m e n t in pressor effect (irrespective o f the agonist employed) in patients with cirrhosis (10). This was n o t due to a generalised sympathetic desensitisation b u t to i m p a i r m e n t o f responses in peripheral vascular s m o o t h muscle (10). Since, pressor action is mediated by P K C a n d Ca 2+, these d a t a from patients with cirrhosis are in general a g r e e m e n t with o u r results in the rat. Clearly, the a d m i n i s t r a t i o n of phorbol esters, i o n o p h o r e s a n d thapsigargin to patients is not feasible. However, strategies could be devised to investigate whether alterations in P K C a n d Ca 2+ occur in the peripheral vasculature o f patients with portal hypertension. In summary, experimental portal hypertension elicits a specific change o f a d r e n o c e p t o r - l i n k e d PGI2 synthesis in the aorta, which m a y be mediated by alterations of G proteins. Since PGI2 is a vasodilator a n d circulating catecholamines are elevated in portal hypertension (41~,3), this may constitute a m e c h a n i s m by which dilation is enhanced in peripheral vascular tissue. The generalised red u c t i o n in P K C activity a n d Ca -'+. 'availability' m a y also account, at least in part, for the diminished vasoactivity of the a o r t a in portal hypertension. A l t h o u g h the mechanisms that govern the adaptive changes o f the vasculature in response to portal h y p e r t e n s i o n remain u n k n o w n , this p h e n o m e n o n is likely to be multifactorial, involving NO, P G I > P K C , Ca 2÷ a n d G proteins. F u r t h e r m o r e , by using the a p p r o a c h employed in this study, these m e c h a n isms (intrinsic vascular a n d / o r circulating trophic factors) could be dissected out. A n u n d e r s t a n d i n g o f the m e c h a n isms that d e t e r m i n e the vascular changes discussed here may be o f value in designing t r e a t m e n t for patients with d e c o m p e n s a t e d cirrhosis a n d patients with other diseases associated with significant changes in v a s o m o t o r tone.
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1021 6. Kowalski H, Abelmann WH. Cardiac output at rest in Laennec's cirrhosis. J Clin Invest 1953; 32: 1025-33. 7. Siegel JH, Golgwyn RM, Farrell J. Hyperdynamic states and the physiologic determinants of survival. Arch Surg 1974; 108: 28392. 8. Lee SS. Cardiac abnormalities in liver cirrhosis. West J Med 1989; 1512: 530-5. 9. Bomzon A, Blendis LM. Vascular reactivity in experimental portal hypertension. Am J Physiol 1987; 252: G158-62. 10. MacGilchrist AJ, Sumner D, Reid JL. Impaired pressor reactivity in cirrhosis: evidence for a peripheral vascular defect. Hepatology 1991; 13: 689-94. 11. Pizcueta MP, Casamitjana R, Bosch J, Rodes J. Decreased systemic vascular sensitivity to norepinephrine in portal hypertensive rats. Role of hyperglucagonism. Am J Physiol 1990; 258: G191-5. 12. Kaumann AJ, Morgan JS, Groszmann RC. ICI 169,369 selectively blocks 5-hydroxytryptamine receptors and lowers portal pressure in portal hypertensive rats. Br J Pharmacol 1986; 89: 501-13. 13. Cummings S, Groszmann R, Kaumann A. Hypersensitivity of mesenteric veins to 5-hydroxytryptamine and ketanserin-induced reduction of portal pressure in portal hypertensive rats. Br J Pharmacol 1986; 89: 501-13. 14. Murray BM, Paller MS. Decreased pressor reactivity to angiotensin II in cirrhotic rats: evidence for a post-receptor defect in angiotensin action. Circ Res 1985; 57: 424-31. 15. Hamilton G, Phing RCE Hutton RA, Dandona P, Hobbs KEE The relationship between prostacyclin activity and pressure in the portal vein. Hepatology 1982; 2: 236--42. 16. Ritter JM, Hamilton DP, Barrow SE, Heavey DJ, Hickling NE, Taylor KM, et al. Prostacyclin in the circulation of patients with vascular disorders undergoing surgery. Clin Sci 1986; 71: 743-7. 17. Sitzmann JV, Li SS. Evidence for role of prostacylin as a systemic hormone in portal hypertension. Surgery 1991; 109: 149-53. 18. Guarner C, Soriuno G. Prostaglandins and portal hypertension. Prostagl Leuk Essential Fatty Acids 1993: in press. 19. Vallance P, Moncada S. Hypothesis: induction of nitric oxide synthase in the vasculature underlies the hyperdynamic circulation. Lancet 1991; 337: 776-8. 20. Whittle BJR, Moncada S. Nitric oxide: the elusive mediator of the hyperdynamic circulation of cirrhosis? Hepatology 16: 108992. 21. Pizcueta MPM, Pique JM, Bosch J, Whittle BJR, Moncada S. Nitric oxide and hyperdynamic circulation in portal hypertension. Br J Pharmacol 1992: 105: 184-90. 22. Lee F-A, Albillos A, Colombato LA, Groszmann RJ. The role of nitric oxide in the vascular hyporesponsiveness to methoxamine in portal hypertensive rats. Hepatology 1992; 16: 1043-8. 23. Sogni P, Moreau R, Ohsuga M, Cailmail S, Oberti E Hadengue A, et al. Evidence for normal nitric oxide-mediated vasodilator tone in conscious rats with cirrhosis. Hepatology 1992; 16: 9803. 24. Murray BM, Paller MS. Pressor resistance to vasopressin in sodium depletion and cirrhosis. Am J Physiol 1986; 251: R525-30. 25. Jeremy JY, Mikhailidis DP, Dandona P. Excitatory receptorlinked prostanoid synthesis in mammalian smooth muscle: the role of calcium, protein kinase C and G proteins. Prostagl Leuk Essential Fatty Acids Rev 1988; 34: 215-28. 26. Jeremy JY, Mikhailidis DP, Dandona P. Adrenergic modulation of vascular prostacyclin synthesis. Eur J Pharmacol 1985; 114: 33-40. 27. Jeremy JY, Dandona P. Role of the diacyl glycerol protein kinase C system in mediating adrenoceptor-prostacyclin synthesis coupling in the rat aorta. Eur J Pharmacol 1986; 136:311-6. 28. Nishizuka Y. The role of protein kinase C in cell surface signal transduction and tumor promotion. Nature 1984; 308: 693-8. 29. Levine L, Watanabe M. Amplification of prostaglandin I2 production by thapsigargin. Prostaglandins 1991; 42: 291-302.
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