326
Journal of Hepatology, 1992; 16:326-331 ©1992 ElsevierScientificPublishers Ireland Ltd. All fights reserved. 0168-8278/92/$05.00
HEPAT01160
Responsiveness to synthetic parathyroid hormone in the portal vein of portal hypertensive rats T.-W. Chao a, P.-C. Yu a, J.-S. K u o b, Peter K.T. Pang c and M a y C.M. Yang" aDepartment of Medical Research, Veterans General HospitaI-Taipei, Taipei. bDepartment of Medical Research, Taichung Veterans General Hospital, Taichung. Taiwan. Republic of China and CDepartment of Physiology. University of Alberta. Edmonton, Canada
(Received2 September 1991)
In addition to its hypotensive action, the parathyroid hormone also decreases portal pressure in portal hypertensive rats. The purpose of this study was to characterize the vascular effects of the parathyroid hormone on the portal vein of portal vein-stenosed rats. When intravenously infused at the rate of i.62 x 10-11 mol/kg per min, the parathyroid hormone lowered portal pressure (15.1 +__0.7 vs. 14.0 + 0.8 mmHg) without affecting systemic blood pressure. With the portal vein isolated, the parathyroid hormone shifted the dose-response curves of KCI and acetylcholine to the right. However, the vasodilator effect of the parathyroid hormone was significantly less in portal hypertensive rats (ECso of KCI increased 129.2% and acetyicholine 199.3%), compared to sham-operated rats (ECso of KC1 increased 158.7% and acetylcholine 270.2%). Similar results were found for the vasodilator action of verapamil (10-9-10 -6 M). On the other hand, the vasodilator effect of forskolin was similar for both groups. These results suggest that decreased responsiveness to the parathyroid hormone may be associated with calcium utilization by vascular smooth muscles. K e y words: Vascular reactivity; Verapamil; Contraction; Relaxation
The parathyroid hormone (PTH) is an endogenous hypotensive peptide. In vivo and in vitro experiments in animals confirm that PTH has vasodilator action on certain vascular beds (I-3). The mechanisms of action of the vasodilator effect of PTH involve both stimulation of adenylate cyclase (4) and inhibition of calcium entry (5,6). In addition to the hypotensive action, it has been reported that PTH decreases portal venous pressure in portal hypertensive rats (7). Several other advantages which favor the use of PTH in the management of portal hypertension have also been discussed. Since vasodilatory substances may lower portal pressure by splanchnic vasoconstriction induced by a sympathetic reflex (8), attempts have been made to investigate whether PTH inhibits the contractile force in venovasculature. In an in vitro experiment with isolated portal veins in normal Sprague-Dawley rats, PTH was found to inhibit both spontaneous and high potassium- and acetyicholineinduced contraction (7). However, since one of the hemodynamic derangements associated with portal hypertension is systemic hypotension (9,10), a hypoten-
sive action may not be desirable in the treatment of portal hypertension. To further characterize the action of PTH in portal hypertension, the present study was first designed to examine the ability of PTH to lower portal pressure without affecting systemic mean blood pressure in portal hypertensive rats induced by partial portal vein ligation, and second, to investigate the venorelaxing effect of PTH in vitro on the isolated portal vein in portal hypertensive rats.
Materials and Methods Portal hypertensive rats
Partial portal vein ligation (PVL) was performed according to the method decribed by Chojkier and Groszmann (!1). Briefly, male Sprague-Dawley rats (200-250 g) were anesthetized with 50 mg/kg i.p. sodium pentobarbital and midline incision was made to expose the portal vein proximal to the bifurcation. A 3-0 silk ligature was made around the portal vein with a piece of PE50 tubing (Clay Adams). The PE tubing was then
Correspondence to." May C.M. Yang, Ph.D., Department of Medical Research,Taipei Veterans General Hospital,Taipei,Taiwan 11217.
327
PARATHYROID HORMONE AND PORTAL VEIN
removed and the abdomen closed. In sham-operated rats, only connective tissue was removed from the portal vein. Ligature was not applied. All surgical procedures were performed under aseptic conditions. Three weeks after surgery, rats were considered ready for experiments if portal pressure was greater than 12 mmHg by iliocolic cannulation. In vivo experiment Experimental details have been reported from our laboratory (7). Six PVL rats were anesthetized and kept on heating pads to maintain body temperature at 38°C. The right femoral artery and femoral vein were cannulated with PE50 tubing to measure mean blood pressure and to administer PTH, respectively. A third cannula was placed in the iliocolic vein for portal pressure measurements. To record pressure changes, each cannula was connected to a polygraph (Gould RS3400) via a strain-gauge transducer. Mean blood pressure was calculated as the sum of the diastolic pressure and one-third of the pulse pressure. A supplement of anesthesia was applied if necessary. When blood pressure had stabilized, the effect of bPTH-(I-34) (Bachem Inc.) (!.62x10 -~1 mol/kg/min) was determined for 15 min at 20 min after the same volume of saline was infused as a control response. In vitro experiment PVL rats were anesthetized and the abdomen opened. The portal vein was carefully removed and placed in aerated Krebs-Henseleit solution containing the following substances (mM): NaCI 115, CaC12 2.1, MgSO4 1.2, NaH2PO4 1.2, NaHCO3 2.5, KCi 5.0 and glucose 11. The removed portal vein was then cut into a helical strip as described previously (6). Both ends of the strip were tied with silk loops. One end was attached to the bottom of a Sawyer-Bartlestone tissue chamber, and the other end was connected to a force displacement transducer (FT03). The tension generated was recorded on a Grass polygraph (7D). The tissue chamber was maintained at 37°C and bubbled with a gas mixture of 95% O2 and 5% CO:. The tissue was equilibrated under resting tension of 0.7 g for 1 h. The readiness of the tissue was indicated by the consistent responses of two consecutive tests with KC1 (40 mM). The first experiment included 7 PVL and 8 shamoperated rats. After equilibration, cumulative concentrations (5-60raM) of KC1 were used to initiate a contraction. The tissue was then rinsed and allowed to recover for 20 min. After the recovery period the same concentration of KCI was used to confirm the reproducibility of the tissue response. The same concentration-
response determination of KCI was repeated 5 min after the presence of 4.9 × 1 0 - 9 M bPTH-(I-34). When the entire concentration-response study was completed, the tissue was rinsed and allowed to recover for 30 min before the next series of doses were given. Similar experiments were performed to determine the concentration-responses of acetylcholine (10 -9-10 -4 M, Sigma). Alternatively, the vasodilator effect of 4.9 x 10 - 9 bPTH-(1-34) was tested when the contraction elicited by a single dose of KCi 40 mM had stabilized at a tonic contractile stage. Tissue stabilization took about 25 min. For each strip the above-described experiments were tested randomly. At the end of the experiments the tissue was challenged again with 4 0 m M KCi. The final response was not significantly different from the initial response. The second experiment included 7 PVL and 7 shamoperated rats. The vasodilator effects of cumulative concentrations of forskolin, 10-7-3 x 10-6 M, and verap a m i l 10 - 9 - 1 0 - 6 M, were tested with vessels contracted by a single dose of KCi 40 mM. Calculations ECso was calculated from the plot of percentage response against log dose. The statistical significance was determined by Student's t-test and, if applicable, paired t-test at p < 0.05. Data were presented as mean _+S.E.
Results
In vivo experiment At low dose (I.62x10 -1~ mol/kg per min), PTH significantly decreased PVL portal pressure (15.1_+0.7 vs. 14.0_+0.8mmHg, Table I) whereas systolic blood pressure .and heart rate were not affected. Infusion of the same volume of saline did not alter any of the parameters. To further characterize the action of PTH on veno-vasculature, in vitro experiments were performed. In vitro experiment PTH at a concentration of 4.9 x 10-9 M shifted the concentration-response curve of KCI and acetylcholine to the right in the portal vein of both PVL and shamoperated rats (Figs. !, 2). Table 2 indicates that the ECso of KCI and acetylcholine were significantly increased in the presence of PTH compared to results in the absence of PTH. However, compared to sham-operated rats the inhibitory effects of PTH were significantly attenuated
328
T.-W. CHAO et al.
TABLE 1
TABLE 2
Effects of bPTH-(1-34) infusion on the mean systemic blood pressure (MBP, mmHg), portal pressure (PP, mmHg) and heart rate (HR, bpm) of PVL rats
ECsos of KCI and acetylcholine (ACh) on portal veins of PVL and sham-operated rats in the absence (-PTH) and presence (+ PTH) of bPTH- (I-34) (4.9 x l0 -9 M)
Saline MBP PP HR
PTH (1.62 x I0 -ll mol/kg/min)
Before
After
Before
After
974-5 14.24-0.8 2684-22
984-5 14.34-0.8 2734-28
1144-9 15.1 4 - 0 . 7 272+23
1134-12 14.04-0.8" 273+26
ECso Sham
KCI (pM) % ACh (laM) %
*Significantly different from before, by paired t-test, n=6; body wt. 361 4-16 g.
in both KCI and acetylcholine-induced contractions in PVL rats. The vasorelaxing effect of PTH was also less marked in PVL rats. 4.9 x 10 - 9 M PTH relaxed the portal vein of PVL rats by 15.8 +2.7% and of sham-operated rats by 30.5 + 1.8%. Fig. 3 shows no difference between the PVL and shamoperated groups for the effect of forskolin on the portal vein contracted with 40 mM KC1. On the other hand, the action of verapamil was significantly less marked in the portal veins of PVL rats (Fig. 4).
PVL
-PTH
+ PTH
-PTH
+ PTH
16.5+0.9 100 (n=6)
26.24-2.1" 158.74-7.9
17.1+0.7 100 (n=7)
22.04-1.9" 129.24-8.8"*
1.2+0.2 100 (n=8)
3.34-1.1" 270.2+87.6
1.44-0.1 100 (n=7)
2.94-0.4* 199.3+25.7
*Significantly different from the absence of PTH in the same group by Student's paired t-test. **Significantly different from the same treatment in sham-operated group by Student's t-test.
F o r s k o l i n (M) 1~ 7
3~1~7
1~6
3x156
I
,
,
,
-10' o
-20"
-30" -40"
1.4
A
B
11=7
rl=6
-50~ PVL 07.4±O.07gm(n--71 Sham'--...'..~
-.oJ
L0 o
Fig. 3. Relaxation of forskolin on KCI (40 mM)-induced contraction in the isolated portal vein of PVL and sham-operated rats. Tensions produced by KCI before adding forskolin were 0.744-0.07 g and 0.58 +0.10 g for PVL and sham-operated group, respectively.
0.8
o) 0.6 0.4
, ' ~ ' " ' " ~ ~ ~ H . / '-PTH J" *
Control
0.2 0.0
,
0
10
20
30
40
50
60
KCI (mM)
0
~ ,
,
10 20
,
,
,
,
30
40
50
60
Discussion
KCI(mM)
Fig. 1. Effects of bPTH-(I-34)(4.9x10 ~ M)on the concentrationresponse curve of KCl-induced contraction in isolated portal vein of PVL (A) and sham-operated (B) rats. *Significant difference between absence (control and presence of PTH by Student's paired t-test.
1.4 "~
Sham 0.58:t0.10gm (n=6}
The present study demonstrated that the vasodilator effect of PTH was significantly less marked on the portal vein in PVL rats than in sham-operated rats (Table 2).
A n=7
B ,,~
n=8
1.2
1:~ 1.0 o
0.8
eontrol/'~¢'~ .1" ,/,/+PTH
o.o"
Co~tro!,,V'"~
0.6 7'
/PTH
~-i 0.4 0.2 0.0 10s 3x10s 107 a=lo7 106 axlo6 105 axlo5 10 s 3x10s 107 3=10 ? 106 3,106 i0 s 3~i0 s 104
A c e t y l c h o l i n e (M)
A c e t y l c h o l i n e (M)
Fig. 2. Effects of bPTH-(I-34) (4.9 x 10-9 M) on the concentration-response curve of acetylcholine-induced contraction in isolated portal vein of PVL (A} and sham-operated (B) rats. *Significant difference between absence (control) and presence of PTH by Student's paired t-test.
PARATHYROID HORMONE AND PORTALVEIN
329
Verapamil (M) i~ g 0
o-
m~ g
t6 8
I
i
3n~ s
i~ 7
s,16~
I
I
I
i~ I
*
"~ -40
i, o-~ -80; PVL 0.86:t0.07gmln=S) -100- Sham 0.57+0.09gin{n=5}
"4ham ""~
Fig. 4. Rclaxation of vcrapamil on KCI (40 mM)-induccd contraction in thc isolatcd portal vein of P V L and sham-opcratcd rats.Tensions produced by KCI bcforc adding vcrapamil wcrc 0.86___0.07 g and 0.57 ±0.09 g for P V L and sham-operated group, respectively.*Significantly diffcrcntfrom thc sham-operatcd group by Student's t-tcst.
The effect of verapamil (a calcium channel antagonist), but not forskolin (an adenylate cyclase activator), was also less marked on the portal vein in PVL rats. These data suggest that the reduced vasodilator effect of PTH in PVL rats may be related to calcium utilization by, but not adenylate cyclase activation of, the vascular smooth muscles. Portal hypertension has been associated with systemic and splanchnic hyperdynamic circulation in both humans (12,13) and experimental animals (10,14). It is now generally accepted that the hyperdynamic circulation involves both increases in blood flow and decreases in vascular resistance. Certain vasoactive substances have been suggested as responsible for the hyperdynamic circulation. These substances - - such as glucagon (15,16), prostaglandins (17) and bile acid (18) - - accumulate in the systemic circulation due to over-production, decreased metabolism and/or portal systemic shunting. Benoit et ai. (9) first suggested that an increase in plasma glucagon levels, a vasodilator, led to decreased vascular resistance in the splanchnic circulation. Later, hyperglucagonism was shown to inhibit the vascular response to such endogenous vasoconstrictors as norepinephrine, angiotensin and vasopressin in the dog (19). In a recent report, Pizcueta et al. (20) found that elevated glucagon levels contributed to decreased systemic vascular sensitivity to norepinephrine. Mesh et al. (21) observed reduced intestinal vascular reactivity to vasopressin in hyperglucagonism of PVL rats. However, unlike the decreased vascular responsiveness in the arterial vasculature in portal hypertensive rats, the responsiveness of venous vasculature is more complicated. Due to the hypertrophy of the veins in portal vein stenosis, responses vary following stimulation by vasoconstrictors or vasodilators. In the present study, despite the similarity in ECsos between the two groups, a greater contractile response to KC1 and acetylcholine was
observed in PVL than in sham-operated rats. Maimqvist and Arner (22) reported an increase in the maximal active force and vessel cross-sectional area in the portal vein of the partial portal vein iigated group. In portal hypertensive rabbits, Jensen et al. (23) reported a marked increase in media thickness in both small mesenteric and esophageal veins. They also failed to observe the differences in the ECsos for angiotension II and norepinephrine. However, in accordance with our results, less vasorelaxation was obtained with fl-adrenergic stimulation and serotonin. Similarly, Kaumann and Groszmann (24) demonstrated less vasorelaxation from catecholamines in the mesenteric and portal veins of PVL rats. In a previous report (7), the portal vein was found to be more sensitive to PTH than the tail artery. It is, therefore, reasonable to speculate that PTH will lower the portal pressure of normal or sham-operated rats. However, with reduced responsiveness to PTH, we were most anxious to examine the ability of PTH to lower portal venous pressure in portal hypertensive rats, and results seem to show that at least part of the lowered portal pressure may be caused by venovasculature dilatation. It is interesting to note that reduced vascular sensitivity may occur due to receptor desensitization. Overactivity of the sympathetic system and elevation of circulating catecholamines have long been documented in portal hypertensives (25-27), and Kiel et al. (28) have suggested that impaired sympathetic activity may play an important role. A receptor down-regulation theory has been proposed (29). By the same token, Kirch et al. (30) reported that plasma PTH levels were increased in cirrhotic patients due to deterioration of liver function. It is not clear whether elevated PTH levels contribute to the systemic hypotension of cirrhotic patients. The increase in circulating levels of PTH may induce receptor desensitization, and a decrease in vascular response. However; this mechanism does not seem likely. The response to endothelin may be decreased, but the circulating levels of endothelin are also decreased in portal hypertensive rats (31). Besides receptor desensitization, there may also be fundamental changes in vascular smooth muscle cells. The results from our studies (4,6) and studies by others (32,33) showed that the mechanisms of action for the vasodilator effect of PTH involve both activation of adenylate cyclase and inhibition of calcium entry. To further characterize which of the pathways was affected in the vasculature of PVL rats, the actions of forskolin (an adenylate activator) and verapamil (a calcium channel antagonist) were tested. The vasorelaxing concentration-response of forskolin was the same in the portal
330
T.-W. CHAO et al.
veins of b o t h
the
PVL
and
sham-operated
groups,
h y p e r t e n s i o n d o e s n o t c a u s e h y p e r t r o p h y of the p o r t a l
s u g g e s t i n g t h a t the a c t i v i t y of a d e n y l a t e cyclase m a y n o t
vein.
be c h a n g e d in P V L rats. Since the a c t i o n of v e r a p a m i l was r e d u c e d in the p o r t a l vein of P V L rats, c h a n g e s in
p o r t a l h y p e r t e n s i o n n e e d to be e x p l o r e d . It will be of
c a l c i u m u t i l i z a t i o n m a y be related to the e n t r y of e x t r a c e l l u l a r c a l c i u m . In s t u d y i n g the c o n t r a c t i l e p r o p e r t i e s of h y p e r t r o p h y o f the s m o o t h m u s c l e in the rat p o r t a l
T h e m e c h a n i s m s of d e c r e a s e d v a s c u l a r r e a c t i v i t y in i n t e r e s t to s t u d y h o w c a l c i u m u t i l i z a t i o n is affected in this a n i m a l m o d e l a n d h o w this a l t e r a t i o n is a s s o c i a t e d with hyperglucagonism.
vein, M a l m q v i s t a n d A r n e r (22) o b s e r v e d a d e c r e a s e d maximal shortening velocity and suggested alterations in the e x c i t a t i o n - c o n t r a c t i o n c o u p l i n g . N e v e r t h e l e s s , Y o s h i m u r a et al. (34) r e p o r t e d t h a t the effects o f D - 6 0 0 did n o t differ in the p o r t a l veins of c a r b o n t e t r a c h l o r i d e i n d u c e d p o r t a l h y p e r t e n s i v e rats a n d n o r m a l W i s t a r rats.
Acknowledgement T h i s s t u d y was s u p p o r t e d by a r e s e a r c h g r a n t f r o m
T h e d i s c r e p a n c y m a y be e x p l a i n e d by a difference in a n i m a l m o d e l s , since c a r b o n t e t r a c h l o r i d e - i n d u c e d p o r t a l
the N a t i o n a l Sciences C o u n c i l ( N S C 7 9 - 0 4 1 2 - B 0 7 5 - 6 6 ) to
References
15 Keller U, Sonnenberg CE, Burckhardt D, Perruchoud A. Evidence for an augmented glucagon dependence of hepatic glucose production in cirrhosis of the liver. J Clin Endocrinol Metab 1982: 54:96 I-8. 16 Benoit JN, Zimmerman B, Premen AJ, Go VLW, Granger DN. Role of glucagon in the splanchnic hyperemia of chronic portal hypertension. Am J Physiol 1986; 251: G674-7. 17 Pique JP, Leung FW, Kitahora T, Sarfeh IJ, Tarnawski A, Guth PH. Gastric mucosal blood flow and acid secretion in portal hypertensive rats. Gastroenterology 1988; 95: 727-33. 18 Benoit JN, Granger DN. Intestinal microvascular adaptation to chronic portal hypertension in the rat. Gastroenterology 1988; 94: 471-6. 19 Richardson PDI, Withrington PG. The inhibition by glucagon of the vasoconstrictor actions of noradrenaline, angiotensin and vasopressin on the hepatic arterial vascular bed of the dog. Br J Pharmacol 1976; 57: 93-102. 20 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. 21 Mesh CL, Joh T, Korthuis RJ, Granger DN, Benoit JN. Intestinal vascular sensitivity to vasopressin in portal hypertensive rats. Gastroenterology 1991; 100: 916-21. 22 Malmqvist U, Arner A. Contractile properties during development of hypertrophy of the smooth muscle in the rat portal vein. Acta Physiol Scand 1988: 133: 49-61. 23 Jensen LS, Juhl CO, Mulvany MJ. Mechanical, morphological and pharmacological properties of oesophageal varices and small mesenteric veins in portal hypertensive rabbits. Acta Physiol Scand 1987: 130: 649-56. 24 Kaumann AJ, Groszmann RJ. Catecholamines relax portal and mesenteric veins from normal and portal hypertensive rats. Am J Physiol 1989: 257:G977-81. 25 Ring-Larsen H. Hesse B, Henriksen JH, Christensen NJ. Sympathetic nervous activity and renal and systemic hemodynamics in cirrhosis: plasma norepinephrine concentration, hepatic extraction, and renal release. Hepatology 1982: 2: 304-10. 26 Rodriguez-Puyol D, Alsasua A, Santos JC, Blanchart A, LopezNovoa JM. Plasma catecholamines and urinary excretion of their main metabolites in three models of portal hypertension. Clin Physiol Biochem 1988: 6: 301-9. 27 Gaudin C, Ruget G, Braillon A, Selz F, Cuche JL, Lebrec D. Portal and arterial free and conjugated noradrenaline in two models of portal hypertension in rats. Life Sci 1989; 45: 1333-9. 28 Kiel JW, Pitts V, Benoit JN, Granger DN, Shepherd AP. Reduced
1 Pang PKT, Tenner TE Jr, Yee JA, Yang M, Janssen HF. Hypotensive action of parathyroid hormone preparations on rats and dogs. Proc Natl Acad Sci USA 1980: 77: 675-8. 2 Pang PKT, Yang MCM, Tenner TE Jr, Chang JK, Shimizu M. Hypotensive action of synthetic fragments of parathyroid hormone. J Pharmacol Exp Ther 1981; 216:567 71. 3 Wang HG, Drugge ED, Yen YC, Blumenthal MRB, Pang PKT. Effects of synthetic parathyroid hormone on hemodynamics and reginal blood flow. Eur J Pharmacol 1984: 97: 209-15. 4 Helwig J J, Yang MCM, Bollack C, Judes C, Pang PKT. Structureactivity relationship of parathyroid hormone: relative sensitivity of rabbit renal microvessel and tubule adenylate cyclases to oxidized PTH and PTH inhibitors. Eur J Pharmacol 1987: 140: 247-57. 5 Pang PKT. Yang MCM, Sham JSK. Parathyroid hormone and calcium entry blockade in a vascular tissue. Life Sci 1988: 42: 1395-1400. 6 Yang MCM. Kuo JS, Pang PKT. Mechanisms of the vascular action of parathyroid hormone. J Pharmacol Exp Ther 1990: 252: 840-4. 7 Yang MCM, Pang PKT, Lay CS, et al. Effect of parathyroid hormone on portal pressure of portal hypertensive rats. Liver 1990:
10:11-6. 8 Kroeger RJ, Groszmann RJ. The effect of the combination of nitroglycerin and propranolol on splanchnic and systemic hemodynamics in a portal hypertensive rat model. Hepatology 1985: 5: 425-30. 9 Benoit JN, Barrowman JA, Harper SL, Kvietys PR, Granger DN. Role of humoral factors in the intestinal hyperemia associated with chronic portal hypertension. Am J Physiol 1984: 247: G486-93. 10 Vorobioff J. 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. 11 Chojkier M, Groszmann RJ. Measurement of portal-systemic shunting in the rat by using r-labeled microspheres. Am J Physiol 1981: 140: G371-5. 12 Kontos HH, Shapiro W, Mauck HP. Patterson JL Jr. General and regional circulatory alterations in cirrhosis of the liver. Am J Med 1964: 37: 526-35. 13 Henderson JM, Ibrahim SZ, Millikan WJ Jr, Santi M, Warren WD. Cimetidine does not reduce liver blood flow in cirrhosis. Hepatology 1983: 3: 919-22. 14 Blanchet L, Lebrec D. Changes in splanchnic blood flow in portal hypertensive rats. Eur J Clin Invest. 1982: 12: 327-30.
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PARATHYROID HORMONE AND PORTAL VEIN
29
30
31 32
vascular sensitivity to norepinephrine in portal-hypertensive rats. Am J Physiol 1985; 248: GI92-5. Reichen J. Liver function and pharmacological considerations in pathogenesis and treatment of portal hypertension. Hepatology 1990; 11: 1066-79. Kirch W, Hofig M, Ledendecker T, Schmidt-Gayk H. Parathyroid hormone and cirrhosis of the liver. J Clin Endocrinol Metab 1990; 71: 1561-66. Yu PC, Kuo JS, Lin HC, Yang MCM. Effects of endothelin in portal hypertensive rats. Clin Sci 1992; in press. Nickol AG. Increased cyclic AMP in cultured vascular smooth
331 muscle cell and relaxation of aortic strips by parathyroid hormone. Eur J Pharmacol 1985; 116: 137-44. 33 Pang PKT, Wang R, Shan J, Karpinski E, Benishin CG. Specific inhibition of long-lasting, L-type calcium channels by synthetic parathyroid hormone. Proc Natl Acad Sci USA 1990; 87: 623-7. 34 Yoshimura T, Arita M, Kobayashi M. Characteristics of contractile response of isolated portal veins from chronic portal hypertensive rats under altered levels of external K ÷, Ca 2 ÷, and norepinephrine concentrations: a comparison with normal Wistar rats. Jpn J Physiol 1988; 38: 459-78.
Journal of Hepatology, 1992; 16:326-331 ©1992 ElsevierScientificPublishers Ireland Ltd. All fights reserved. 0168-8278/92/$05.00
HEPAT01160
Responsiveness to synthetic parathyroid hormone in the portal vein of portal hypertensive rats T.-W. Chao a, P.-C. Yu a, J.-S. K u o b, Peter K.T. Pang c and M a y C.M. Yang" aDepartment of Medical Research, Veterans General HospitaI-Taipei, Taipei. bDepartment of Medical Research, Taichung Veterans General Hospital, Taichung. Taiwan. Republic of China and CDepartment of Physiology. University of Alberta. Edmonton, Canada
(Received2 September 1991)
In addition to its hypotensive action, the parathyroid hormone also decreases portal pressure in portal hypertensive rats. The purpose of this study was to characterize the vascular effects of the parathyroid hormone on the portal vein of portal vein-stenosed rats. When intravenously infused at the rate of i.62 x 10-11 mol/kg per min, the parathyroid hormone lowered portal pressure (15.1 +__0.7 vs. 14.0 + 0.8 mmHg) without affecting systemic blood pressure. With the portal vein isolated, the parathyroid hormone shifted the dose-response curves of KCI and acetylcholine to the right. However, the vasodilator effect of the parathyroid hormone was significantly less in portal hypertensive rats (ECso of KCI increased 129.2% and acetyicholine 199.3%), compared to sham-operated rats (ECso of KC1 increased 158.7% and acetylcholine 270.2%). Similar results were found for the vasodilator action of verapamil (10-9-10 -6 M). On the other hand, the vasodilator effect of forskolin was similar for both groups. These results suggest that decreased responsiveness to the parathyroid hormone may be associated with calcium utilization by vascular smooth muscles. K e y words: Vascular reactivity; Verapamil; Contraction; Relaxation
The parathyroid hormone (PTH) is an endogenous hypotensive peptide. In vivo and in vitro experiments in animals confirm that PTH has vasodilator action on certain vascular beds (I-3). The mechanisms of action of the vasodilator effect of PTH involve both stimulation of adenylate cyclase (4) and inhibition of calcium entry (5,6). In addition to the hypotensive action, it has been reported that PTH decreases portal venous pressure in portal hypertensive rats (7). Several other advantages which favor the use of PTH in the management of portal hypertension have also been discussed. Since vasodilatory substances may lower portal pressure by splanchnic vasoconstriction induced by a sympathetic reflex (8), attempts have been made to investigate whether PTH inhibits the contractile force in venovasculature. In an in vitro experiment with isolated portal veins in normal Sprague-Dawley rats, PTH was found to inhibit both spontaneous and high potassium- and acetyicholineinduced contraction (7). However, since one of the hemodynamic derangements associated with portal hypertension is systemic hypotension (9,10), a hypoten-
sive action may not be desirable in the treatment of portal hypertension. To further characterize the action of PTH in portal hypertension, the present study was first designed to examine the ability of PTH to lower portal pressure without affecting systemic mean blood pressure in portal hypertensive rats induced by partial portal vein ligation, and second, to investigate the venorelaxing effect of PTH in vitro on the isolated portal vein in portal hypertensive rats.
Materials and Methods Portal hypertensive rats
Partial portal vein ligation (PVL) was performed according to the method decribed by Chojkier and Groszmann (!1). Briefly, male Sprague-Dawley rats (200-250 g) were anesthetized with 50 mg/kg i.p. sodium pentobarbital and midline incision was made to expose the portal vein proximal to the bifurcation. A 3-0 silk ligature was made around the portal vein with a piece of PE50 tubing (Clay Adams). The PE tubing was then
Correspondence to." May C.M. Yang, Ph.D., Department of Medical Research,Taipei Veterans General Hospital,Taipei,Taiwan 11217.
327
PARATHYROID HORMONE AND PORTAL VEIN
removed and the abdomen closed. In sham-operated rats, only connective tissue was removed from the portal vein. Ligature was not applied. All surgical procedures were performed under aseptic conditions. Three weeks after surgery, rats were considered ready for experiments if portal pressure was greater than 12 mmHg by iliocolic cannulation. In vivo experiment Experimental details have been reported from our laboratory (7). Six PVL rats were anesthetized and kept on heating pads to maintain body temperature at 38°C. The right femoral artery and femoral vein were cannulated with PE50 tubing to measure mean blood pressure and to administer PTH, respectively. A third cannula was placed in the iliocolic vein for portal pressure measurements. To record pressure changes, each cannula was connected to a polygraph (Gould RS3400) via a strain-gauge transducer. Mean blood pressure was calculated as the sum of the diastolic pressure and one-third of the pulse pressure. A supplement of anesthesia was applied if necessary. When blood pressure had stabilized, the effect of bPTH-(I-34) (Bachem Inc.) (!.62x10 -~1 mol/kg/min) was determined for 15 min at 20 min after the same volume of saline was infused as a control response. In vitro experiment PVL rats were anesthetized and the abdomen opened. The portal vein was carefully removed and placed in aerated Krebs-Henseleit solution containing the following substances (mM): NaCI 115, CaC12 2.1, MgSO4 1.2, NaH2PO4 1.2, NaHCO3 2.5, KCi 5.0 and glucose 11. The removed portal vein was then cut into a helical strip as described previously (6). Both ends of the strip were tied with silk loops. One end was attached to the bottom of a Sawyer-Bartlestone tissue chamber, and the other end was connected to a force displacement transducer (FT03). The tension generated was recorded on a Grass polygraph (7D). The tissue chamber was maintained at 37°C and bubbled with a gas mixture of 95% O2 and 5% CO:. The tissue was equilibrated under resting tension of 0.7 g for 1 h. The readiness of the tissue was indicated by the consistent responses of two consecutive tests with KC1 (40 mM). The first experiment included 7 PVL and 8 shamoperated rats. After equilibration, cumulative concentrations (5-60raM) of KC1 were used to initiate a contraction. The tissue was then rinsed and allowed to recover for 20 min. After the recovery period the same concentration of KCI was used to confirm the reproducibility of the tissue response. The same concentration-
response determination of KCI was repeated 5 min after the presence of 4.9 × 1 0 - 9 M bPTH-(I-34). When the entire concentration-response study was completed, the tissue was rinsed and allowed to recover for 30 min before the next series of doses were given. Similar experiments were performed to determine the concentration-responses of acetylcholine (10 -9-10 -4 M, Sigma). Alternatively, the vasodilator effect of 4.9 x 10 - 9 bPTH-(1-34) was tested when the contraction elicited by a single dose of KCi 40 mM had stabilized at a tonic contractile stage. Tissue stabilization took about 25 min. For each strip the above-described experiments were tested randomly. At the end of the experiments the tissue was challenged again with 4 0 m M KCi. The final response was not significantly different from the initial response. The second experiment included 7 PVL and 7 shamoperated rats. The vasodilator effects of cumulative concentrations of forskolin, 10-7-3 x 10-6 M, and verap a m i l 10 - 9 - 1 0 - 6 M, were tested with vessels contracted by a single dose of KCi 40 mM. Calculations ECso was calculated from the plot of percentage response against log dose. The statistical significance was determined by Student's t-test and, if applicable, paired t-test at p < 0.05. Data were presented as mean _+S.E.
Results
In vivo experiment At low dose (I.62x10 -1~ mol/kg per min), PTH significantly decreased PVL portal pressure (15.1_+0.7 vs. 14.0_+0.8mmHg, Table I) whereas systolic blood pressure .and heart rate were not affected. Infusion of the same volume of saline did not alter any of the parameters. To further characterize the action of PTH on veno-vasculature, in vitro experiments were performed. In vitro experiment PTH at a concentration of 4.9 x 10-9 M shifted the concentration-response curve of KCI and acetylcholine to the right in the portal vein of both PVL and shamoperated rats (Figs. !, 2). Table 2 indicates that the ECso of KCI and acetylcholine were significantly increased in the presence of PTH compared to results in the absence of PTH. However, compared to sham-operated rats the inhibitory effects of PTH were significantly attenuated
328
T.-W. CHAO et al.
TABLE 1
TABLE 2
Effects of bPTH-(1-34) infusion on the mean systemic blood pressure (MBP, mmHg), portal pressure (PP, mmHg) and heart rate (HR, bpm) of PVL rats
ECsos of KCI and acetylcholine (ACh) on portal veins of PVL and sham-operated rats in the absence (-PTH) and presence (+ PTH) of bPTH- (I-34) (4.9 x l0 -9 M)
Saline MBP PP HR
PTH (1.62 x I0 -ll mol/kg/min)
Before
After
Before
After
974-5 14.24-0.8 2684-22
984-5 14.34-0.8 2734-28
1144-9 15.1 4 - 0 . 7 272+23
1134-12 14.04-0.8" 273+26
ECso Sham
KCI (pM) % ACh (laM) %
*Significantly different from before, by paired t-test, n=6; body wt. 361 4-16 g.
in both KCI and acetylcholine-induced contractions in PVL rats. The vasorelaxing effect of PTH was also less marked in PVL rats. 4.9 x 10 - 9 M PTH relaxed the portal vein of PVL rats by 15.8 +2.7% and of sham-operated rats by 30.5 + 1.8%. Fig. 3 shows no difference between the PVL and shamoperated groups for the effect of forskolin on the portal vein contracted with 40 mM KC1. On the other hand, the action of verapamil was significantly less marked in the portal veins of PVL rats (Fig. 4).
PVL
-PTH
+ PTH
-PTH
+ PTH
16.5+0.9 100 (n=6)
26.24-2.1" 158.74-7.9
17.1+0.7 100 (n=7)
22.04-1.9" 129.24-8.8"*
1.2+0.2 100 (n=8)
3.34-1.1" 270.2+87.6
1.44-0.1 100 (n=7)
2.94-0.4* 199.3+25.7
*Significantly different from the absence of PTH in the same group by Student's paired t-test. **Significantly different from the same treatment in sham-operated group by Student's t-test.
F o r s k o l i n (M) 1~ 7
3~1~7
1~6
3x156
I
,
,
,
-10' o
-20"
-30" -40"
1.4
A
B
11=7
rl=6
-50~ PVL 07.4±O.07gm(n--71 Sham'--...'..~
-.oJ
L0 o
Fig. 3. Relaxation of forskolin on KCI (40 mM)-induced contraction in the isolated portal vein of PVL and sham-operated rats. Tensions produced by KCI before adding forskolin were 0.744-0.07 g and 0.58 +0.10 g for PVL and sham-operated group, respectively.
0.8
o) 0.6 0.4
, ' ~ ' " ' " ~ ~ ~ H . / '-PTH J" *
Control
0.2 0.0
,
0
10
20
30
40
50
60
KCI (mM)
0
~ ,
,
10 20
,
,
,
,
30
40
50
60
Discussion
KCI(mM)
Fig. 1. Effects of bPTH-(I-34)(4.9x10 ~ M)on the concentrationresponse curve of KCl-induced contraction in isolated portal vein of PVL (A) and sham-operated (B) rats. *Significant difference between absence (control and presence of PTH by Student's paired t-test.
1.4 "~
Sham 0.58:t0.10gm (n=6}
The present study demonstrated that the vasodilator effect of PTH was significantly less marked on the portal vein in PVL rats than in sham-operated rats (Table 2).
A n=7
B ,,~
n=8
1.2
1:~ 1.0 o
0.8
eontrol/'~¢'~ .1" ,/,/+PTH
o.o"
Co~tro!,,V'"~
0.6 7'
/PTH
~-i 0.4 0.2 0.0 10s 3x10s 107 a=lo7 106 axlo6 105 axlo5 10 s 3x10s 107 3=10 ? 106 3,106 i0 s 3~i0 s 104
A c e t y l c h o l i n e (M)
A c e t y l c h o l i n e (M)
Fig. 2. Effects of bPTH-(I-34) (4.9 x 10-9 M) on the concentration-response curve of acetylcholine-induced contraction in isolated portal vein of PVL (A} and sham-operated (B) rats. *Significant difference between absence (control) and presence of PTH by Student's paired t-test.
PARATHYROID HORMONE AND PORTALVEIN
329
Verapamil (M) i~ g 0
o-
m~ g
t6 8
I
i
3n~ s
i~ 7
s,16~
I
I
I
i~ I
*
"~ -40
i, o-~ -80; PVL 0.86:t0.07gmln=S) -100- Sham 0.57+0.09gin{n=5}
"4ham ""~
Fig. 4. Rclaxation of vcrapamil on KCI (40 mM)-induccd contraction in thc isolatcd portal vein of P V L and sham-opcratcd rats.Tensions produced by KCI bcforc adding vcrapamil wcrc 0.86___0.07 g and 0.57 ±0.09 g for P V L and sham-operated group, respectively.*Significantly diffcrcntfrom thc sham-operatcd group by Student's t-tcst.
The effect of verapamil (a calcium channel antagonist), but not forskolin (an adenylate cyclase activator), was also less marked on the portal vein in PVL rats. These data suggest that the reduced vasodilator effect of PTH in PVL rats may be related to calcium utilization by, but not adenylate cyclase activation of, the vascular smooth muscles. Portal hypertension has been associated with systemic and splanchnic hyperdynamic circulation in both humans (12,13) and experimental animals (10,14). It is now generally accepted that the hyperdynamic circulation involves both increases in blood flow and decreases in vascular resistance. Certain vasoactive substances have been suggested as responsible for the hyperdynamic circulation. These substances - - such as glucagon (15,16), prostaglandins (17) and bile acid (18) - - accumulate in the systemic circulation due to over-production, decreased metabolism and/or portal systemic shunting. Benoit et ai. (9) first suggested that an increase in plasma glucagon levels, a vasodilator, led to decreased vascular resistance in the splanchnic circulation. Later, hyperglucagonism was shown to inhibit the vascular response to such endogenous vasoconstrictors as norepinephrine, angiotensin and vasopressin in the dog (19). In a recent report, Pizcueta et al. (20) found that elevated glucagon levels contributed to decreased systemic vascular sensitivity to norepinephrine. Mesh et al. (21) observed reduced intestinal vascular reactivity to vasopressin in hyperglucagonism of PVL rats. However, unlike the decreased vascular responsiveness in the arterial vasculature in portal hypertensive rats, the responsiveness of venous vasculature is more complicated. Due to the hypertrophy of the veins in portal vein stenosis, responses vary following stimulation by vasoconstrictors or vasodilators. In the present study, despite the similarity in ECsos between the two groups, a greater contractile response to KC1 and acetylcholine was
observed in PVL than in sham-operated rats. Maimqvist and Arner (22) reported an increase in the maximal active force and vessel cross-sectional area in the portal vein of the partial portal vein iigated group. In portal hypertensive rabbits, Jensen et al. (23) reported a marked increase in media thickness in both small mesenteric and esophageal veins. They also failed to observe the differences in the ECsos for angiotension II and norepinephrine. However, in accordance with our results, less vasorelaxation was obtained with fl-adrenergic stimulation and serotonin. Similarly, Kaumann and Groszmann (24) demonstrated less vasorelaxation from catecholamines in the mesenteric and portal veins of PVL rats. In a previous report (7), the portal vein was found to be more sensitive to PTH than the tail artery. It is, therefore, reasonable to speculate that PTH will lower the portal pressure of normal or sham-operated rats. However, with reduced responsiveness to PTH, we were most anxious to examine the ability of PTH to lower portal venous pressure in portal hypertensive rats, and results seem to show that at least part of the lowered portal pressure may be caused by venovasculature dilatation. It is interesting to note that reduced vascular sensitivity may occur due to receptor desensitization. Overactivity of the sympathetic system and elevation of circulating catecholamines have long been documented in portal hypertensives (25-27), and Kiel et al. (28) have suggested that impaired sympathetic activity may play an important role. A receptor down-regulation theory has been proposed (29). By the same token, Kirch et al. (30) reported that plasma PTH levels were increased in cirrhotic patients due to deterioration of liver function. It is not clear whether elevated PTH levels contribute to the systemic hypotension of cirrhotic patients. The increase in circulating levels of PTH may induce receptor desensitization, and a decrease in vascular response. However; this mechanism does not seem likely. The response to endothelin may be decreased, but the circulating levels of endothelin are also decreased in portal hypertensive rats (31). Besides receptor desensitization, there may also be fundamental changes in vascular smooth muscle cells. The results from our studies (4,6) and studies by others (32,33) showed that the mechanisms of action for the vasodilator effect of PTH involve both activation of adenylate cyclase and inhibition of calcium entry. To further characterize which of the pathways was affected in the vasculature of PVL rats, the actions of forskolin (an adenylate activator) and verapamil (a calcium channel antagonist) were tested. The vasorelaxing concentration-response of forskolin was the same in the portal
330
T.-W. CHAO et al.
veins of b o t h
the
PVL
and
sham-operated
groups,
h y p e r t e n s i o n d o e s n o t c a u s e h y p e r t r o p h y of the p o r t a l
s u g g e s t i n g t h a t the a c t i v i t y of a d e n y l a t e cyclase m a y n o t
vein.
be c h a n g e d in P V L rats. Since the a c t i o n of v e r a p a m i l was r e d u c e d in the p o r t a l vein of P V L rats, c h a n g e s in
p o r t a l h y p e r t e n s i o n n e e d to be e x p l o r e d . It will be of
c a l c i u m u t i l i z a t i o n m a y be related to the e n t r y of e x t r a c e l l u l a r c a l c i u m . In s t u d y i n g the c o n t r a c t i l e p r o p e r t i e s of h y p e r t r o p h y o f the s m o o t h m u s c l e in the rat p o r t a l
T h e m e c h a n i s m s of d e c r e a s e d v a s c u l a r r e a c t i v i t y in i n t e r e s t to s t u d y h o w c a l c i u m u t i l i z a t i o n is affected in this a n i m a l m o d e l a n d h o w this a l t e r a t i o n is a s s o c i a t e d with hyperglucagonism.
vein, M a l m q v i s t a n d A r n e r (22) o b s e r v e d a d e c r e a s e d maximal shortening velocity and suggested alterations in the e x c i t a t i o n - c o n t r a c t i o n c o u p l i n g . N e v e r t h e l e s s , Y o s h i m u r a et al. (34) r e p o r t e d t h a t the effects o f D - 6 0 0 did n o t differ in the p o r t a l veins of c a r b o n t e t r a c h l o r i d e i n d u c e d p o r t a l h y p e r t e n s i v e rats a n d n o r m a l W i s t a r rats.
Acknowledgement T h i s s t u d y was s u p p o r t e d by a r e s e a r c h g r a n t f r o m
T h e d i s c r e p a n c y m a y be e x p l a i n e d by a difference in a n i m a l m o d e l s , since c a r b o n t e t r a c h l o r i d e - i n d u c e d p o r t a l
the N a t i o n a l Sciences C o u n c i l ( N S C 7 9 - 0 4 1 2 - B 0 7 5 - 6 6 ) to
References
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31 32
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