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Production of prostaglandins I2, E2 and F2α by blood vessels of normotensive and hypertensive, male and female rats
ProstaglandinsLeukotrienesand Medicine25: 71-89,
1986
PRODUCTIONOF PROSTAGLANDINS12, E2 AND F2e BY BLOOD VESSELS OF NORMOTENSIVE AND HYPERTENSIVE,MALE AND FEMALE RATS E. Anne Lennon' and N.L. Poyser Departmentof Pharmacology, Universityof Edinburgh, 1, George Square, Edinburgh,EH8 9JZ, Scotland. (reprintrequeststo N.L. Poyser) ABSTRACT Prostaglandin(PC) 12 (measuredas 6-keto-PGFle)was the major PC synthesizedby aortic homogenatesfrom normotensiveand hypertensive male rats, with lesser quanfitesof PCFa and PGE2. Homogenatesof vena cava from the same rats synthesizedPC12 and PGEZ in similar quantities. PGF2.esynthesisby aortic homogenateswas significantly higher in hypertensivethan in normotensivemale rats. A similar trend was seen in PGF2e synthesisby homogenatesof the vena cava. Separationof the aorta of normotensiveand hypertensive,male rats showed that PC12 was the major PC synthesizedby endothelialcells and smooth muscle, and that PGFa was the major PC synthesizedby adventitia. PC12 synthesisby smooth muscle and PGE;! synthesisby endothelialcells were lower, and PGF2e synthesisby endothelialcells and smooth muscle were higher in hypertensivethan in normotensive,male rats. PC12 was the major PC releasedfrom the aorta of normotensive and hypertensivemale rats, togetherwith lesser quantitiesof PGE2 and PGF2e. Aortic PGF2e output, but not PC12 and PGE2 outputs,was significantlyhigher in hypertensivecomparedto normotensive,male rats.
' Present address:- Departmentof Biochemistryand Nutrition,Nuffield Laboratoriesof ComparativeMedicineInstituteof Zoology,Regent's Park, LONDON,NW1 4RY, England. 71
PGI2 was the major PC synthesized by endothelial cells of normotensive female rats, whereas PGE2 was the major PC synthesized by endothelial cells of hypertensive, female rats. The amounts of PC12 and PCF2, synthesized by the aortic endothelial cells and smooth muscle were significantly lower in hypertensive than in normotensive, female rats. However, the outputs of PGI2, PGF2u and PGE2 from the aorta were significantly higher in hypertensive than in normotensive, female rats. Noradrenaline (NA) increased PG12 output from the mesenteric arterial bed of normotensive and hypertensive, male and female rats. NA also increased PGF2, output from this vascular bed in hypertensive, male rats. Angiotensin II (AII) increased PC12 output from the mesenteric arterial bed of normotensive, male rats but not of hypertensive, male rats. In contrast, AI1 increased the output of PC12 from the mesenteric arterial bed of normotensive and hypertensive, female rats. Overall, differences were found in vascular production of PGs between hypertensive and normotensive rats, of both sexes. However, the differences were not always the same in both sexes, although the vascular output of the vasoconstrictor, PGF2a, was generally higher in hypertensive than in normotensive, male and female rats. INTRODUCTION Arterial blood pressure is regulated by a complex interaction of several controlling mechanisms. It has been proposed that a deficiency in production of vasodilator prostaglandins by blood vessels may lead to the increased peripheral resistance characteristic of essential hypertension (1). However the generation of prostaglandin (PC) 12 and PGE2 by rings, strips or homogenates of aorta in vitro is greater in spontaneously hypertensive rats than in normotensive rats (2,3,4,5). Dusting et al. (6) reported that the vasodepressor effects of arachidoccid were greater and more prolonged in spontaneously hypertensive rats than in normotensive rats, but that vasodepressor responses to PC12 did not differ between the two strains. They concluded that the spontaneously hypertensive rats had an enhanced ability to form vasodilator PCs from exogenous arachidonic acid. In the rat mesenteric arterial bed perfused in vitro, although basal PC12 and PGE2 outputs tended to be higher in hypertensive rats, the increases in PG12 and PGE2 outputs in response to sympathetic nerve stimulation seen in normotensive rats were absent in hypertensive rats (7). Consequently abnormalities in increased output of vasodilator PGs in response to vasoconstrictor stimuli may be more important than basal PG output in affecting blood pressure. Much attention has been focussed in recent years on measuring the output of vasodilator PCs particularly PGI2, from blood vessels, and the output of the vasconstrictor, PGF2a, has been overlooked. An imbalance in the normal production of vasoconstrictor and vasodilator PCs by blood vessels may be a contributory cause of hypertension. Also, most studies have been performed using male rats. Consequently, we have carried out a relatively detailed investigation into the vascular production of PGI2, PGE2 and PGF2a in normotensive and hypertensive, 72
male
and female
rats.
METHODS Male and female wistar rats (normotensive controls) aged 2-3 months and weighing 200-300 g, and male and female New Zealand genetically hypertensive rats of the same age and weighing 180-250 g were used in The animals were housed under controlled the following experiments. lighting conditions (lights on 06.00-20.00 h) and were allowed a Vaginal smears from the female standard diet and water ad libitum. Oestrus was taken rats were taken daily and examined microscopically. as the day of maximum cornification which preceded the day of leucocytic All female rats had shown normal &day cycles before infiltration. being used at 10.00 h on pro-oestrus. PC production Experiment 1. normotensive and hypertensive,
by homogenates male rats.
of aorta
and vena cava from
Systolic blood pressure was recorded in 6 normotensive and 6 method using a Huntingdon hypertensive male rats by the f’tail-cuffll Instruments Rat Blood Pressure Recorder connected to a double-channel The animals were then killed and the servoscribe pen recorder. thoracic aorta and vena cava were removed, Each vessel was rinsed, weighed and homogenized in 10 ml Krebs solution (for composition, see Arachidonic acid (final concentration, 6.6 NM) was added to each 8). homogenate, which was then incubated at 37OC for 60 min and aerated with 5% carbon dioxide, 95X oxygen. The pH of each incubate was then lowered to 4.0 with 1N HCl, and the lipids were extracted by shaking The two ethyl acetate twice with two volumes of ethyl acetate. fractions were combined and evaporated to dryness on a rotary evaporator at 45OC. Each dried extract was re-dissolved in 4 ml ethyl acetate and was stored at -2OOC until the amounts of 6-keto-PGFla, PCE2 and PCFa were measured by radioimmunoassay. Recovery of PGs is high (> 80%) by the extraction method used (9,101, and so the results have not been corrected for procedural losses. Experiment 2. PCFametabolism by homogenates normotensive and hypertensive male rats.
of aorta
from
The thoracic aorta from 4 normotensive and 4 hypertensive rats were removed, rinsed and cut into half. Each half aorta was weighed, homogenized in 5 ml Krebs solution and 5 pCi C3Hl PGF2c, tsp. act 160 Ci/mmol; Amershal International plc, U.K.) was added to each homogenate. In addition, nicotinamide-adenine dinucleotide (NAD+; final concentration 2 mM) and 10 p’g PGF2, were added to one of the aortic halves from each rat. Each homogenate was incubated and the lipids extracted as in Exp.1. The dried extracts were redissolved in 0.2 ml methanol, and were analyzed by thin-layer chromatography (t.1.c.) Using pre-coated silica gel plates (Merck, Darmstadt, W. Germany) and a solvent system of chloroform, methanol, glacial acetic acid and water in the ratio of 90 : 9 : 1 : 0.65; Standard t.1.c. plates onto which had been spotted 10 pg each of PGF2., 15-keto-PGF2,, and 13,14-dihydro-15keto-PGF2 were analyzed similarly. Substances on the standard plates were visualized by exposure to iodine vapour for 30 min. Experimental
73
plates were scanned using a Panex Thin Layer Scanner ATLS-1A to locate the radioactivity. The experimental plates were marked at 0.5 cm intervals, and the silica gel in each zone was scraped off into scintillation vials. After adding 1 ml methanol followed by 10 ml scintillation fluid (10.5 g 2,5-diphenyloxazole in 2.5 1 toluene), the amount of radioactivity present in each vial was counted. The RF values of the radioactive compounds were compared to the RF values of The amount of radioactivity associated with PGF2, and its metabolites. the PG metabolites was correct for the loss of trltium from the carbon at position 15. PGF* metabolism was calculated by expressing the amount of radioactivity associated with the metabolites as a percentage of the total radioactivity on the plate. PC production Experiment 3. normotensive and hypertensive,
in separated layers of aorta male and female rats.
from
A 2.5 cm piece of thoracic aorta from normotensive and hypertensive, male and female rats (6 animals in each group) was removed and separated into 3 layers by the method of Moncada.et al. (11) with Each aorta was rinsed, was opened by cutting some modifications. surface uppermost, was pinned longitudinally and, with its endothelial The endothelial cells were scraped off with a scalpel blade and out. The adventitia and the smooth were suspended in 5 ml Krebs solution. muscle were also separated with a scalpel blade, weighed, and homogenized in 5 ml Krebs solution. After adding arachidonic acid (final concentration 6.6 r_cM)the endothelial cells and tissue homogenates were incubated for 60 min, and the PGs produced were extracted, stored and assayed as in Exp.1. Basal PC output from the aorta Experiment 4. hypertensive, male and female rats.
of normotensive
and
A 2.5 cm piece of thoracic aorta was removed from normotensive and hypertensive male and female rats (6 animals in each group), was placed in a heated (37OC) chamber and was perfused with Krebs solution (5 mUmin) at 37OC pre-gassed with 5% carbon dioxide, 95% oxygen. After an initial settling period of 30 min, perfusate was collected PGs present in the perfusate were during the next 30 min period. extracted, stored and assayed as in Exp.1. Experiment 5. of normotensive
Basal and stimulated PG output from the mesenteric and hypertensive, male and female rats.
bed
male and female rats were killed (4 Normotensive and hypertensive, The abdomen of each rat was opened and the animals per group). The mesenteric bed superior mesenteric artery was quickly cannulated. was removed, as described by McGregor (121, placed in a heated (37OC) chamber and perfused (4 ml/min) with McEwen’s solution (13) at 37OC preAfter an initial settling gassed with 5% carbon dioxide, 95% oxygen. period of 30 min, noradrenaline (NA) and angiotensin II (AI11 (Sigma both at doses of 0.1 ug and 1 ug, Chemical Co. Ltd., Poole, Dorset), Samples of perfusate were given as bolus injections in a random order. were collected for 1 min periods during 2 min immediately before stimulation, during 4 min immediately after stimulation, and at 8 min 74
and 12 min after stimulation. Each mesenteric arterial bed received all 4 treatments at 15 min intervals. The amounts of 6-keto-PGFle, PGE2 and PGF2c in the perfusion fluid were measured by radioimmunoassay without extraction (14). Pressor responses were recorded on a Grass Polygraph Model 7C recorder using a Statham pressure transducer. Details
of radioimmunoassay
PGE2, PGF2, and 6-keto-PGF1, were measured using antisera raised in rabbits in this department and whose cross-reactivities have been reported elsewhere (9,15,16,17). The intra-assay coefficient3 of variation were 11.8% (PGE2), 9.4% (PGFp) and 9.5X (6-keto-PGF-p7. The inter-assay coefficient3 of variation were 12.2% (PCE21, 7.9% (PGF2,) and 6.5% (6-keto-PGFa,). The detection limits per assay tube were 40 pg PGE2, 30 pg PGF2a, and 40 pg 6-keto-PGFla Statistical
tests
Results were analyzed by Student’s t test, which was used in a modified form in the variance3 were unequal by the variance ratio F test, or by the paired t test, as appropriate. RESULTS All results are expresses a3 mean (+ s.e.m., n - 6 in Exps.1, 4, and n = 4 in Exp3.2 and 5, unless otherwise stated). Experiment 1. PC production by homogenate3 of aorta normotensive and hype?%ensive, male rats.
3 and
and vena cava from
The systoltc blood pressure was significantly higher (P < 0.01) in hypertensive rat3 (150.5 L 3.1 mm Hg) than in normotensive rats (104.8 2 4.3 mmHg). 6-Keto-PGF1, was the major PC synthesized by homogenates of the aorta from normotensive and hypertensive rats, together with lesser quantities of PCF2c (Figure 1). The amounts of 6-keto-PGF1, and PGE2 produced were similar in both group3, but PGF2, production by the aorta was significantly higher in the hypertensive group (P < 0.01). 6-Keto-PGF1 production by the vena cava was significantly lower (P < 0.01) than in the aorta in both groups and was similar to PGF2e and PGE2 production by the vena cava in normotensive rat3 and to PGE2 production in hypertensive rats. PGF2, production by the vena cava in hypertensive rats tended to be higher than in normotensive rats but the difference was not statistically significant. F2e metabolism by homogenates Experiment 2. normotensive and hypertensive, male rats.
of aorta
from
Homogenates of aorta incubated with [3H3 PCFaresulted in the formation of metabolites with RF value3 corresponding to 15-keto-PGF2, and 13,14-dihydro-15-keto-PGF2,. The percentage metabolism of PGF2, was low in the absence or presence of MAD+and PGF2, metabolism did not differ significantly between normotensive and hypertensive rat3 (Table 1).
75
4 aJ
9
3 .-t-i +
:
F; cn c
1 0
-A VC V
,A
VC, V
,A
V
VC,
n = 6) amounts of prostaglandins (PCs) Mean (+ s.e.m,, Figure 1. synthesized by homogenates of aorta (A) and vena cava (VC) from normotensive (white columns) and hypertensive (black columns) male rats. ** significantly synthesized
higher by aorta
(P < 0.01) than of normotensive
76
corresponding rats.
amount
of PGFza
F2e Mean (+ s.e.m., n - 4) % metabolism of C3Hl prostaglandin Table 1. by homogenates of aorta from normotensive and hypertensive male rats, in the absence (-1 and presence (+) of nicotinamide-adenine dinucleotide (NAD+; 2mM).
Normotensive
% Metabolism
-NAD+ Total per mg tissue
17.1
L 2.5
+NAD+ - 17.8
0.35 L 0.07
PG production Experiment 3. normotensive and hypertensive, i.
PC production
per unit
Hypertensive
0.40
+ 1.9 + 0.07
-NAD+ 15.2 0.35
L 2.4 + 0.06
in separated layers of aorta male and female rata.
+NAD+ 17.1
L 2.3
0.50 + 0.12
from
length
6-Keto-PGF1e was the major PG synthesized by endothelial cell suspensions and smooth muscle homogenates from the aorta of all 4 groups of rats, except in female hypertensive rats where PGF2e was the major PC1 synthesized by the endothelial cells (Table 2). PGF2u was the major PC synthesized by the adventitia in all groups of rats. The total amounts of 6-keto-PGF1e synthesized by the separated layers were in the order of’ smooth muscle > adventitia > endothelial cells for all rats, except in male hypertensive rats where 6-keto-PGF1, synthesis by the adventitia and endothelial cells were similar. The total amounts of PGF2e and PGE2 synthesized were higher by smooth muscle and adventitia than by endothelial cells in all rats. However, as the endothelial cells constitute about 2% of the tissue present in the aorta, the capacity for synthesizing PC12 (measured as 6-keto-PGF1e), PGE2 and PGF2a is 3- to 30-fold higher in the endothelial cells than in the smooth muscle or adventitia of all groups of rats. Regarding males only, the amounts of 6-keto-PGF1o synthesized by the endothelial cells were similar, but the amounts of 6-keto-PGF1e synthesized by smooth muscle and adventitia were significantly lower (P < 0.05) in hypertensive compared to normotensive rats. PGF2a synthesis was significantly higher (P < 0.05) by the endothelial cells and smooth muscle, and was significantly lower (P < 0.05) by adventitia in hypertensive compared to normotensive, male rats. PGF2e synthesis was significantly lower (P < 0.05) by the endothelial cells and adventitia, but was similar in smooth muscle of hypertensive compared to normotensive male rats. As regards female, the amounts of 6-keto-PGF1e and PGF2osynthesized by endothelial cells, smooth,muscle and adventitia were significantly lower (P < 0.05) in hypertensive compared to normotensive rats. There was no difference in PGE2 synthesis by the 3 tissue types between hypertensive and normotensive, female rats, 77
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synthesis was aignlflcantly Regarding sex differences, 6-keto-PGFle lower (P < 0.05) by the adventitla but did not dlffer by the endothellal cells and smooth muscle of female compared to male normotenslve rats. The amounts of PGFpand PGE2 synthesized by smooth muP)cle, but not by endothelial cell8 or adventitla, uere significantly hfgher (P < 0.05) In farale compared to male, normotenslve rats,although PGF2a. synthesis by the endothellal cells tended to be higher in the former group. The amounts of 6-keto-PGFle and PGFpa, but not of PGE2, synthesized by the endothelial cells were significantly lower (P < 0.05) in the The amount8 of hypertensive compared to normotenslve, female rata. PGE2 but not of 6-keto-PGFI a and PGF20, synthesized by smooth muscle and adventltla were slgnlflcantly higher (P < 0.05) in hypertensive compared to normotenalve, female rata. 11.
PC production
per unit weight
Slgnlflcant differences in 6-keto-PGFle, PGF2a and PGE2 synthesis by amooth muscle between the different group8 when the results are expressed on a unit length baa18 are still present when the result8 are expressed on a unit weight basis, except for PGE2 synthesis by the smooth muscle in female and male, normotenslve rats (Table 2). Additionally PGE2 synthesis by smooth muscle of hypertensive and normotenslve rats now become8 significantly different (P < 0.05). In contrast to smooth muscle, significant difference8 in the amounts of 6-keto-PGF1 a, PGF2a and PGE2 synthesized by adventitia between hypertensive and normotensive, male rats and between hypertensive and normotensive, female rats disappear when the results are expressed on a unit weight basis as opposed to a unit length basis. In addition PGE2 synthesis by the adventltia of normotensive and hypertensive. female rats 1s significantly greater (P < 0.05) in the hypertensive rats when the results are expressed on a unit weight basis. Obviously, the amount of adventltla In the aorta 1s greater in normotensive rats then in hypertensive rats of both sexes. Also the normotensive rats were slightly heavier than the hypertensive rats. However, correcting the results for differences in body weight had no effect on the significant differences observed. Experiment 4. Basal PC output from the aorta hypertensive, male and female rats.
or normotensive
and
6 Keto-PGF1 a was the major PC released from the aorta of noreotenalve and hypertensive male rats, followed by lesser amount8 of PGE2 and PGF2a (Figure 2). PGF2, release was significantly higher (P < 0.01) in hypertensive compared to normotenslve male rats: there was no difference in 6-keto-PGF1, and PGE2 release between the groups. The aorta from female, normotenslve rats released similar quantities of 6keto-PGF1, and PGE2, and smaller quantities of PGF2a. A similar profile was seen in hypertensive, female rats but the release of all 3 PCs was significantly higher (P < 0.05 or < 0.01, as indicated in Figure 2b) in the hypertensive state. The basal release of 6-keto-PGFI, but not of PGF2, and PGE2, was significantly lower (P < 0.05) in female compared to male normotensive rats. The basal release of PCE2, but not
79
a. 8 6 4. **
2, 0.
6-keto -PGFlm
PGF2m
PGE2
cn
s
6 E u4 I-f\ ",2 F 0 -PGFllx (PCs) from a Mean (+ s.e.m., n - 6) outputs of prostaglandlns 2. 2.5 cm length of tEoracic aorta perfused in vitro from normotensive (white columns) and hypertensive (black columns), (aI male rats, and (b) female rats.
Figure
*
Significantly higher than corresponding value (P < 0.051, ** (P < 0.01) for the same PG and same tissue In normotenafve rats of the same sex.
t
significantly different same PG and same tissue
(P < 0.05) from the corresponding value in male rats of the same blood pressure
80
for state.
the
of 6-keto-PGFlc and PGFP, was significantly compared to male hypertensive rats.
higher
Basal and stimulated PC output Experiment 5. arterial bed of normotensive and hypertensive,
in female
(P < 0.05)
from the mesenteric male and female rats.
6-Keto-PGF1 o was the major PC released from the mesenteric vascular bed of male and female normotensive and hypertensive rats, together with lesser quantities of PGF2c and PGE2 (Table 3). Output of all 3 PCs generally declined over the 60 min period of PC measurement, often to non-detectable values (< 0.2 ng/min) for PGF2c and PGE2. PGFPc was more detectable in hypertensive than in normotensive rats, generally particularly the females. Table 3. Mean (+ s.e.m., n = 4 unless otherwise shown) basal (ng/min) of prostaglandins (PCs) from the perfused mesenteric bed of normotensive and hypertensive, male and female rats at beginning (Start) and the finish (End) of the 60 min period of measurement (n.d. =i not detectable, < 0.2 ng/min).
Normotensive
output arterial the PC
Hypertensive
Start
End
Start
End
Male
1.3020.22
0.9820.26
1.9420.22
1.09~0.19
Female
2.7520.51
0.5820.14
2.1520.38
0.78+0.18
Male
n.d.
n.d.
0.4420.66
n.d.
Female
0.61+0.24‘
n.d.
0.6320.23
0.31+0.08
Male
0.54+0.10
0.36~0.03
0.8020.27
0.48’
Female
0.7120.24
0.44.
0.58+0.29’
0.4820.13
6-Keto-PGFl,:
-
t
t t
PGFa:-
PGE2: -
+ significantly
lower
than at start in the same group (P < 0.05) (1 result n.d.1 n-d.1
?? mean + s.e. mean of 3 results ?? mean of 2 results (2 results
NA (0.1 pg and 1 Pg) significantly increased (P < 0.05) 6-keto-PGF,c output from the mesenteric arterial bed of normotensive and hypertensive 81
z
t
2.19+0.40+ L 2.75-o.81+
2.67+0.42+ 2.46+0.42+
2.00+0.41 2.31Z0.5Llf
2.25-0.61f L
1.70+0.21
T
n.d.
0.63+0.37' 0.4110.12
n.d. 0.33+0.09.
0.53' 0.54tO.26
0.36~0.07'
C T
A
o.51+o.181 0.74zo.15
n.d. 0.75+0.22'+
1.0320.33
0.44
n.d. 0.69+0.15m+
POFZCY
0.40+0.09 0.37+0.179
0.49to.16 0.4650.14
o.44to.18. 0.28TO.08
n.d. 0.57L0.19'
C
PCE2
Significantly higher (P < 0.05) than corresponding value (C) before stimulation.
1.12tO.26 1.53z0.56
Norm
Wwr
Female:
1.04~0.20
0.83to.19
1.4tVo.33 1.21z0.28
Female: Norm Hyper
Male: Norm Hyper
1.06+0.11 1.1550.35
C
6-Keto-PCFI,
Male: Norm Hyper
State
?? mean (2 s.e.m.) of 3 values (1 value n.d.) ?? value from 1 animal (3 values n.d.)
+
1.0 pf3
0.1 !Jg
Dose of NA
0.65to.04 0.73~0.10.
0.77-+0.12 0.75'0.21
0.66to.20' 0.6650.26
0.87+0.12 0.94i0.25.
T
Mean (+ s.e.m., n = 4 unless otherwise shown) outputs (ng/min) of prostaglandins (PCs) from the Table 4. mesenteric arterial bed of normotensive (Norm) and hypertensive (Hyper), male and female rats 1 min before administration (n.d. = not detectable, < 0.2 ng/min). (C) and within 2 min (following (T) noradrenaline (NA)
Ei
wg
t m ?? A
1.0 pi3
0.1
1.97+0.81 [email protected]
1.05+0.08 1.4150.21
2.35+0.74t 2.6120.6-/t
3.76+0.65* 1.90~0.23
l-44+0.26
2.1950.45
1.59LO.36
2.39+0.71* 1.58E0.32
1
1.13~0.36
1.5610.33
1.22+0.42
C
6-Keto-PGF1,
0.60. 0.48+0.11
n.d. 0.63.
0.75 A 0.48+0.04
n.d. 0.42'
C
T
0.84* 0.98+0.38
n.d. 0.60.
0.70 A 0.64+0.10
n.d. 0.32 A
POF2,
0.47+0.20m 0.7650.36m
0.51~0.07 0.39.
0.72. 0.6820.29
0.63+0.08 0.97G
C
Significantly higher (P < 0.05) than corresponding value (C) before Stimulation. mean (+ s.e.m.) of 3 values (1 value n.d.1 mean 07 2 values (2 values n.d.1 value from 1 animal (3 values n.d.1
Female: Norm Hyper
Per
Male: Norm
Female: Norm Hyper
Male: Norm Hyper
Dose of AI1 State T
0.74+0.37. 0.85T0.40m
1.11+0.11 0.601
0.51. 0.46+0.06 _
0.73+0.21 o. 803
PGE2
Table 5. Mean (+ s.e.m., n = 4 unless otherwise shown) outputs (ng/min) of prostaglandins (PCs) from the mesenteric arterial bed of normotensive (Norm) and hypertensive (Hyper), male and female rats 1 min before (C) and within 2 min (following (T) angiotensin II (AII) administration (n.d. = not detectable, < 0.2 ng/min).
male and female rats (Table 4). NA (0.1 kg and 1 pg) tended also to increase PCE2 output in all 4 groups of rats, but none of the increases were significant. NA (0.1 and 1.0 kg) had little apparent effect on PGF2aoutput from the mesenteric arterial bed of normotensive, male and female rats. In contrast, NA significantly increased (P < 0.05) PGF2a output in male, hypertensive rats, and tended to increase PGF2a output in female, hypertensive rats. AI1 (0.1 and 1.0 pg) significantly increased (P < 0.05) 6-keto-PGFla output in male normotensive rats but not in male, hypertensive rats AI1 (0.1 pg) had no effect on 6-keto-PCFla output in (Table 5). female, normotensive or hypertensive rats. However, AI1 at the higher dose (1.0 vg) significantly increased (P < 0.05) 6-keto-PGFla output in female, normotensive and hypertensive rats. The effects of AI1 on PGF2a and PGE2 outputs were inconclusive since many of the outputs were below the detection limit. Notwithstanding, AI1 (0.1 kg) appeared to have little effect on the outputs of PGF2a and PGE2 in any of the 4 groups while AI1 (1.0 kg) significantly increased (P < 0.05) PGE2 output in male, normotensive rats (Table 5). The % changes in perfusion pressure of the mesenteric vascular bed fOllOWing treatment with NA and AI1 (0.1 and 1.0 kg) are shown in Table 6. NA (1.0 kg) and AI1 (1.0 kg) produced a significantly greater (P < 0.05) % increase in perfusion pressure in hypertensive than in normotensive, male rats. This significant difference was not present in female rats. Table 6. n = 4) % changes in perfusion pressure of Mean (+ s.e.m., the mesenteric vascular bed of normotensive (Norm) and hypertensive (Hyper) rats following in vitro treatment with noradrenaline and angiotensin. Noradrenaline
Male: Norm Female Female: Norm Hyper
Angiotensin
II
O*l IJg
1.0 Vg
0.1 kg
1.0 !Jg
62.3+34.7 153.0z43.5
174.4+ 4.4 261.053’33.5t
20.a+ 9.8 58.6220.2
150.4233.2 136.6242.6
14.7+ 6.36 31.6Z16.1
40.8+ 4.7 52.2z26.2
tsignificantly higher same dose (P < 0.05)
than normotensive
(AII)
33.0+ 8.0 61 .8-25.8
group of same sex receiving
DISCUSSION PG12 (measured as 6-keto-PGFla)
was the major PC synthesized 84
by
homogenates of the aorta from normotensive and hypertensive, male rats, whereas homogenates of the vena cava synthesized PC12 and PGF2cr in There was no difference in PG12 synthesis by similar quantities. homogenates of the aorta between hypertensive and normotensive, male rats, in spite of the lower capacity of the aortic smooth muscle of There was no difference in hypertensive male rats to synthesize PG12. PC12 output from the aorta of hypertensive compared to normotensive, PG12 male rats which is in contrast to other studies (2,3,4,5). synthesis by homogenates of the vena cava did no differ between Similarly PGE2 synthesis by hypertensive and normotensive, male rats. homogenates of the vena cava or aorta did not differ between hypertensive and normotenaive male rats, in spite of a lower capacity of aortic endothelial cells to synthesize PGE2 in the hypertensive state. There was no difference in PGE2 output from the aorta between the two blood pressure states in male rats. PGF2c, production by homogenates of the aorta was significantly The difference higher in hypertensive than in normotensive, male rats. was not due to changes in PGF2a metabolism, and may be related to greater ability of the aortic endothelial cells and smooth muscle to synthesize PGF2, in hypertensive, male rats. Also, this increased PGF2a synthesizing capacity may be the reason for the higher basal output of PGF2, from the aorta of hypertensive compared with normotensive male There was a tendency for the vena cava also to have a higher rats. synthesizing capacity for PGF2. in the hypertensive state, in male rats. One could speculate from these observations that the aetiology of hypertension is not connected with a decrease in output’ of vasodilator PCs by blood vessels, but is rather connected with an increase in output of the vasoconstrictor, PGFp, due to a higher PGF2d synthesizing capacity of the vasculature in the hypertensive state. However, the findings in female rats may not support this suggestion. The PC12 and PGE2 synthesizing capacities of the endothelial cells and smooth muscle were significantly lower in hypertensive than in normotensive, female rats, yet the outputs of PG12, PGF2a and PGE2 from the aorta were higher in the hypertensive state. Admittedly, PGF2e output is increased in female, hypertensive rats as in male, hypertensive rats, but it might be expected that the increase in outputs of PG12 and PGE2 may compensate for this increase in PGF2a output, unless PGF2a can act on the vascular smooth muscle unhindered by PC12 and PGE2. Interestingly, the higher output of PC12 from the aorta of hypertensive, albeit female rats is in agreement with previous studies The basal output of PG12 from the aorta was lower in the (2,3,4,5). female than in the male, normotensive rats. NA stimulated PC12 output and, to a lesser extent, PGE2 output from the mesenteric arterial bed of normotensive and hypertensive, male rats, which is in agreement with previous studies (7.18). The present study has shown that NA will stimulate also the release of PG12 and, to a lesser extent, the release of PGE2 from normotensive and hypertensive, female rats. In the rat mesenteric arterial bed and hind limb, PGE2 potentiates and PC12 attenuates the pressor response to NA while, on the splenic artery, PGE2 and PC12 both attenuate the effect of NA (19). effect of PGE2 may be specific to the rat However, the potentiating
85
since, in rabbits, PGE2 attenuates the pressor response of NA in the mesenteric arterial bed (20). Also topical application of PGE2 to the rat mesentery in vivo attenuates the pressor response of NA (21). However inhibition of PG synthesis reduces the pressor response of the rat mesenteric arterial bed perfused in vitro to NA to sympathetic nerve stimulation indicating that endogenous PGs are normally necessary for the full effect of NA to become manifest (20.22). There was no difference in the effects of exogenous NA on PC12 and PGE2, outputs from the mesenteric arterial bed between the hypertensive and normotensive states in rats of either sex. However, NA significantly increased the output of PGF2, from the mesenteric arterial bed of hypertensive, male rats and tended to increase PGF2, output from hypertensive, female rats, without any apparent effect of PCF2, release in normotensive male and female rats. Also, PGF2e was more readily detectable in the perfusate of the mesenteric arterial bed of hypertensive than of normotensive rats of either sex. However, whether increased basal and NA-stimulated PGF2, output is connected with hypertension requires further investigation. The pressor response to NA (1.0 pg) in the mesenteric arterial bed was significantly higher in hypertensive than in normotensive male but not female rats. AI1 increased the outputs of PC12 and, to a lesser extent, PGE2 from the mesenteric arterial bed of normotensive, male rats which is in agreement with previous studies (18,23,24). In contrast, AI1 failed to increase PC12 output from the mesenteric arterial bed of hypertensive, male rats. The effects of AI1 on PCE2 output were inconclusive. Studies in rabbits and sheep have indicated that the pressor effect of AI1 is normally attenuated by PGs produced endogenously in response to Consequently, hypertension could be due at AI1 treatment (25,261. least in part, to a lack of vascular PC synthesis in response to AII. The pressor response to AI1 in the mesenteric arterial bed was higher in However, the results in hypertensive than in normotensive, male rats. the female rats do not agree with this suggestion since AI1 (1.0 pg) stimulated PC12 output in female normotensive and hypertensive rats unless sex differences exist in the cause of hypertension. Certainly, the pressor response to AI1 (1.0 pg) did not differ significantly between hypertensive and normotensive, female rats. Overall, this study has highlighted some differences in vascular PC In addition, it production between hypertensive and normotensive rats. has shown that there are important differences between the sexes. Consequently, hypotheses on connections between hypertension and vascular PG synthesis should not be based on studied in males alone. The vascular output of the vasoconstrictor, PGF2c, was generally higher in hypertensive than in normotensive, male and female rats. This agrees with Hoffman et al. (271, who found that the increase in blood pressure of salt-load rats induced by a dietary linoleate deficiency was associated with an increase in the PCF2e to PGE2 ratio of production in the aorta and kidney. As well as being a vasoconstrictor, PGF2u potentiates responses produced by sympathetic nerve stimulation, especially in vascular smooth muscle (281, which would also increase vascular tone and blood pressure. However, whether the increase in vascular production of PGF2c is connected with the aetiology of hypertension requires further study. In this context, in a previous
86
study (291, PGF2e output from blood vessels of old, male and female rats was increased coapared to young rats. These old rats were also Again, whether the hypertension was the result of or the hypertensive. cause of increased vascular PGF2e output, or whether there was any connection between these two parameters at all, is requiring further study. ACKNOWLEDGEMENTS This study was supported by a grant from the Scottish Home and Health Department and a MRCpostgraduate studentship to E.A.L. Authentic PGs were kindly supplied by the Upjohn Company, Kalamaioo, Michigan, USA. REFERENCES 1.
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AC, Higgs EA, Vane JR. Differential formation (PGX or PGI2) by layers of the arterial wall. of the anti-thrombotic properties of vascular Thromb. Res. 11: 323, 1977.
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E, Poyser NL. Effects of nerve stimulation and of administration of noradrenaline or potassium chloride upon the release of prostaglandins 12, E2 and F2 from the perfused mesenteric arterial bed of the rabbit. Br. J. Pharmac. 72: 89, 1981.
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KK, Emslie HA, Henderson development of antisera to analysis using solid phase methods. Br. J. Pharmac.
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Development and use of a radioimmunoassay for NL. p.44 in Prostaglandins, measuring 6-oxo-prostaglandin Fla. Prostacyclin and Thromboxanes Measurement. (J Boeynaems, Herman eds) Martinus Nijhoff, The Hague, 1980.
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LK, Rutherford R, Simon L. The prostaglandins B2 and F2a and their and double antibody radioimmunoassay 55: 503, 1975.
Differential stimulation of prostaglandin and NL. thromboxane synthesizing capacities in guinea-pig uterus ovary. Prostaglandins, Leuk. & Med. 10: 167, 1983.
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S, Gutkowa J, Garcia R, Genest J. Effect of angiotensin-II and norepinephrine on release of prostaglandins E2 and I2 by the perfused mesenteric rat artery. Prostaglandins 24: 105, 1982.
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Effects of prostaglandins T, Suzuki H. Saruta T. E2 and 12 and arachidonic acid on vascular reactivity to norepinephrine in isolated rat mesenteric artery, hind limb splenic artery. Prostaglandins & Med. 4: 21, 1980.
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Malik
KU, Ryan P, McCiff JC. Modification by prostaglandins and E2, indomethacin, and arachidonic acid of the vasoconstrictor responses of the isolated perfused rabbit
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Prostaglandin action, release and inactivation by rat 22. Coupar IM. solated perfused mesenteric blood vessels. Br. J. Pharmac. 68: 757, 1980. 23.
Dusting CJ, Mullins EM, Nolan RD. Prostacyclin (PC121 release accompanying angiotensin conversion in rat mesenteric vasculature. Eur. J. Pharmac. 70: 129, 1981.
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Phosphilipase inhibition and the RD, Dusting GJ, Martin TJ. mechanism of angiotensin-induced prostacyclin release from rat mesenteric vasculature. Biochem. Pharmac. 30: 2121, 1981.
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DS, Coghlan JP, Denton DA, Graham WF, Humphrey TJ, Scoggins A, Whitworth JA. In vivo modification of angiotensin II -pressor responsiveness in sheep by indomethacin. Clin. Exp. Pharmac. Physiol. 8: 33, 1981.
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Blumberg AL, Denny SE, Marshall GR, Needleman P. Blood vesselhormone interactions: angiotensin, bradykinin, and prostaglandins. Amer. J. Physiol. 232: H305, 1977.
27.
Hoffman P, Taube Ch, Ponicke K, Zehl U, Beitz J, Forster L, Orbetzova V, Dadivova F. Alterations in renal prostaglandin E and F formation with blood pressure salt loaded rats after dietary linoleate deficiency. Int. Pharmacodyn. 259: 40, 1982.
28.
Brody ,JM, Kadowitz PJ. autonomic nervous
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1986
PRODUCTIONOF PROSTAGLANDINS12, E2 AND F2e BY BLOOD VESSELS OF NORMOTENSIVE AND HYPERTENSIVE,MALE AND FEMALE RATS E. Anne Lennon' and N.L. Poyser Departmentof Pharmacology, Universityof Edinburgh, 1, George Square, Edinburgh,EH8 9JZ, Scotland. (reprintrequeststo N.L. Poyser) ABSTRACT Prostaglandin(PC) 12 (measuredas 6-keto-PGFle)was the major PC synthesizedby aortic homogenatesfrom normotensiveand hypertensive male rats, with lesser quanfitesof PCFa and PGE2. Homogenatesof vena cava from the same rats synthesizedPC12 and PGEZ in similar quantities. PGF2.esynthesisby aortic homogenateswas significantly higher in hypertensivethan in normotensivemale rats. A similar trend was seen in PGF2e synthesisby homogenatesof the vena cava. Separationof the aorta of normotensiveand hypertensive,male rats showed that PC12 was the major PC synthesizedby endothelialcells and smooth muscle, and that PGFa was the major PC synthesizedby adventitia. PC12 synthesisby smooth muscle and PGE;! synthesisby endothelialcells were lower, and PGF2e synthesisby endothelialcells and smooth muscle were higher in hypertensivethan in normotensive,male rats. PC12 was the major PC releasedfrom the aorta of normotensive and hypertensivemale rats, togetherwith lesser quantitiesof PGE2 and PGF2e. Aortic PGF2e output, but not PC12 and PGE2 outputs,was significantlyhigher in hypertensivecomparedto normotensive,male rats.
' Present address:- Departmentof Biochemistryand Nutrition,Nuffield Laboratoriesof ComparativeMedicineInstituteof Zoology,Regent's Park, LONDON,NW1 4RY, England. 71
PGI2 was the major PC synthesized by endothelial cells of normotensive female rats, whereas PGE2 was the major PC synthesized by endothelial cells of hypertensive, female rats. The amounts of PC12 and PCF2, synthesized by the aortic endothelial cells and smooth muscle were significantly lower in hypertensive than in normotensive, female rats. However, the outputs of PGI2, PGF2u and PGE2 from the aorta were significantly higher in hypertensive than in normotensive, female rats. Noradrenaline (NA) increased PG12 output from the mesenteric arterial bed of normotensive and hypertensive, male and female rats. NA also increased PGF2, output from this vascular bed in hypertensive, male rats. Angiotensin II (AII) increased PC12 output from the mesenteric arterial bed of normotensive, male rats but not of hypertensive, male rats. In contrast, AI1 increased the output of PC12 from the mesenteric arterial bed of normotensive and hypertensive, female rats. Overall, differences were found in vascular production of PGs between hypertensive and normotensive rats, of both sexes. However, the differences were not always the same in both sexes, although the vascular output of the vasoconstrictor, PGF2a, was generally higher in hypertensive than in normotensive, male and female rats. INTRODUCTION Arterial blood pressure is regulated by a complex interaction of several controlling mechanisms. It has been proposed that a deficiency in production of vasodilator prostaglandins by blood vessels may lead to the increased peripheral resistance characteristic of essential hypertension (1). However the generation of prostaglandin (PC) 12 and PGE2 by rings, strips or homogenates of aorta in vitro is greater in spontaneously hypertensive rats than in normotensive rats (2,3,4,5). Dusting et al. (6) reported that the vasodepressor effects of arachidoccid were greater and more prolonged in spontaneously hypertensive rats than in normotensive rats, but that vasodepressor responses to PC12 did not differ between the two strains. They concluded that the spontaneously hypertensive rats had an enhanced ability to form vasodilator PCs from exogenous arachidonic acid. In the rat mesenteric arterial bed perfused in vitro, although basal PC12 and PGE2 outputs tended to be higher in hypertensive rats, the increases in PG12 and PGE2 outputs in response to sympathetic nerve stimulation seen in normotensive rats were absent in hypertensive rats (7). Consequently abnormalities in increased output of vasodilator PGs in response to vasoconstrictor stimuli may be more important than basal PG output in affecting blood pressure. Much attention has been focussed in recent years on measuring the output of vasodilator PCs particularly PGI2, from blood vessels, and the output of the vasconstrictor, PGF2a, has been overlooked. An imbalance in the normal production of vasoconstrictor and vasodilator PCs by blood vessels may be a contributory cause of hypertension. Also, most studies have been performed using male rats. Consequently, we have carried out a relatively detailed investigation into the vascular production of PGI2, PGE2 and PGF2a in normotensive and hypertensive, 72
male
and female
rats.
METHODS Male and female wistar rats (normotensive controls) aged 2-3 months and weighing 200-300 g, and male and female New Zealand genetically hypertensive rats of the same age and weighing 180-250 g were used in The animals were housed under controlled the following experiments. lighting conditions (lights on 06.00-20.00 h) and were allowed a Vaginal smears from the female standard diet and water ad libitum. Oestrus was taken rats were taken daily and examined microscopically. as the day of maximum cornification which preceded the day of leucocytic All female rats had shown normal &day cycles before infiltration. being used at 10.00 h on pro-oestrus. PC production Experiment 1. normotensive and hypertensive,
by homogenates male rats.
of aorta
and vena cava from
Systolic blood pressure was recorded in 6 normotensive and 6 method using a Huntingdon hypertensive male rats by the f’tail-cuffll Instruments Rat Blood Pressure Recorder connected to a double-channel The animals were then killed and the servoscribe pen recorder. thoracic aorta and vena cava were removed, Each vessel was rinsed, weighed and homogenized in 10 ml Krebs solution (for composition, see Arachidonic acid (final concentration, 6.6 NM) was added to each 8). homogenate, which was then incubated at 37OC for 60 min and aerated with 5% carbon dioxide, 95X oxygen. The pH of each incubate was then lowered to 4.0 with 1N HCl, and the lipids were extracted by shaking The two ethyl acetate twice with two volumes of ethyl acetate. fractions were combined and evaporated to dryness on a rotary evaporator at 45OC. Each dried extract was re-dissolved in 4 ml ethyl acetate and was stored at -2OOC until the amounts of 6-keto-PGFla, PCE2 and PCFa were measured by radioimmunoassay. Recovery of PGs is high (> 80%) by the extraction method used (9,101, and so the results have not been corrected for procedural losses. Experiment 2. PCFametabolism by homogenates normotensive and hypertensive male rats.
of aorta
from
The thoracic aorta from 4 normotensive and 4 hypertensive rats were removed, rinsed and cut into half. Each half aorta was weighed, homogenized in 5 ml Krebs solution and 5 pCi C3Hl PGF2c, tsp. act 160 Ci/mmol; Amershal International plc, U.K.) was added to each homogenate. In addition, nicotinamide-adenine dinucleotide (NAD+; final concentration 2 mM) and 10 p’g PGF2, were added to one of the aortic halves from each rat. Each homogenate was incubated and the lipids extracted as in Exp.1. The dried extracts were redissolved in 0.2 ml methanol, and were analyzed by thin-layer chromatography (t.1.c.) Using pre-coated silica gel plates (Merck, Darmstadt, W. Germany) and a solvent system of chloroform, methanol, glacial acetic acid and water in the ratio of 90 : 9 : 1 : 0.65; Standard t.1.c. plates onto which had been spotted 10 pg each of PGF2., 15-keto-PGF2,, and 13,14-dihydro-15keto-PGF2 were analyzed similarly. Substances on the standard plates were visualized by exposure to iodine vapour for 30 min. Experimental
73
plates were scanned using a Panex Thin Layer Scanner ATLS-1A to locate the radioactivity. The experimental plates were marked at 0.5 cm intervals, and the silica gel in each zone was scraped off into scintillation vials. After adding 1 ml methanol followed by 10 ml scintillation fluid (10.5 g 2,5-diphenyloxazole in 2.5 1 toluene), the amount of radioactivity present in each vial was counted. The RF values of the radioactive compounds were compared to the RF values of The amount of radioactivity associated with PGF2, and its metabolites. the PG metabolites was correct for the loss of trltium from the carbon at position 15. PGF* metabolism was calculated by expressing the amount of radioactivity associated with the metabolites as a percentage of the total radioactivity on the plate. PC production Experiment 3. normotensive and hypertensive,
in separated layers of aorta male and female rats.
from
A 2.5 cm piece of thoracic aorta from normotensive and hypertensive, male and female rats (6 animals in each group) was removed and separated into 3 layers by the method of Moncada.et al. (11) with Each aorta was rinsed, was opened by cutting some modifications. surface uppermost, was pinned longitudinally and, with its endothelial The endothelial cells were scraped off with a scalpel blade and out. The adventitia and the smooth were suspended in 5 ml Krebs solution. muscle were also separated with a scalpel blade, weighed, and homogenized in 5 ml Krebs solution. After adding arachidonic acid (final concentration 6.6 r_cM)the endothelial cells and tissue homogenates were incubated for 60 min, and the PGs produced were extracted, stored and assayed as in Exp.1. Basal PC output from the aorta Experiment 4. hypertensive, male and female rats.
of normotensive
and
A 2.5 cm piece of thoracic aorta was removed from normotensive and hypertensive male and female rats (6 animals in each group), was placed in a heated (37OC) chamber and was perfused with Krebs solution (5 mUmin) at 37OC pre-gassed with 5% carbon dioxide, 95% oxygen. After an initial settling period of 30 min, perfusate was collected PGs present in the perfusate were during the next 30 min period. extracted, stored and assayed as in Exp.1. Experiment 5. of normotensive
Basal and stimulated PG output from the mesenteric and hypertensive, male and female rats.
bed
male and female rats were killed (4 Normotensive and hypertensive, The abdomen of each rat was opened and the animals per group). The mesenteric bed superior mesenteric artery was quickly cannulated. was removed, as described by McGregor (121, placed in a heated (37OC) chamber and perfused (4 ml/min) with McEwen’s solution (13) at 37OC preAfter an initial settling gassed with 5% carbon dioxide, 95% oxygen. period of 30 min, noradrenaline (NA) and angiotensin II (AI11 (Sigma both at doses of 0.1 ug and 1 ug, Chemical Co. Ltd., Poole, Dorset), Samples of perfusate were given as bolus injections in a random order. were collected for 1 min periods during 2 min immediately before stimulation, during 4 min immediately after stimulation, and at 8 min 74
and 12 min after stimulation. Each mesenteric arterial bed received all 4 treatments at 15 min intervals. The amounts of 6-keto-PGFle, PGE2 and PGF2c in the perfusion fluid were measured by radioimmunoassay without extraction (14). Pressor responses were recorded on a Grass Polygraph Model 7C recorder using a Statham pressure transducer. Details
of radioimmunoassay
PGE2, PGF2, and 6-keto-PGF1, were measured using antisera raised in rabbits in this department and whose cross-reactivities have been reported elsewhere (9,15,16,17). The intra-assay coefficient3 of variation were 11.8% (PGE2), 9.4% (PGFp) and 9.5X (6-keto-PGF-p7. The inter-assay coefficient3 of variation were 12.2% (PCE21, 7.9% (PGF2,) and 6.5% (6-keto-PGFa,). The detection limits per assay tube were 40 pg PGE2, 30 pg PGF2a, and 40 pg 6-keto-PGFla Statistical
tests
Results were analyzed by Student’s t test, which was used in a modified form in the variance3 were unequal by the variance ratio F test, or by the paired t test, as appropriate. RESULTS All results are expresses a3 mean (+ s.e.m., n - 6 in Exps.1, 4, and n = 4 in Exp3.2 and 5, unless otherwise stated). Experiment 1. PC production by homogenate3 of aorta normotensive and hype?%ensive, male rats.
3 and
and vena cava from
The systoltc blood pressure was significantly higher (P < 0.01) in hypertensive rat3 (150.5 L 3.1 mm Hg) than in normotensive rats (104.8 2 4.3 mmHg). 6-Keto-PGF1, was the major PC synthesized by homogenates of the aorta from normotensive and hypertensive rats, together with lesser quantities of PCF2c (Figure 1). The amounts of 6-keto-PGF1, and PGE2 produced were similar in both group3, but PGF2, production by the aorta was significantly higher in the hypertensive group (P < 0.01). 6-Keto-PGF1 production by the vena cava was significantly lower (P < 0.01) than in the aorta in both groups and was similar to PGF2e and PGE2 production by the vena cava in normotensive rat3 and to PGE2 production in hypertensive rats. PGF2, production by the vena cava in hypertensive rats tended to be higher than in normotensive rats but the difference was not statistically significant. F2e metabolism by homogenates Experiment 2. normotensive and hypertensive, male rats.
of aorta
from
Homogenates of aorta incubated with [3H3 PCFaresulted in the formation of metabolites with RF value3 corresponding to 15-keto-PGF2, and 13,14-dihydro-15-keto-PGF2,. The percentage metabolism of PGF2, was low in the absence or presence of MAD+and PGF2, metabolism did not differ significantly between normotensive and hypertensive rat3 (Table 1).
75
4 aJ
9
3 .-t-i +
:
F; cn c
1 0
-A VC V
,A
VC, V
,A
V
VC,
n = 6) amounts of prostaglandins (PCs) Mean (+ s.e.m,, Figure 1. synthesized by homogenates of aorta (A) and vena cava (VC) from normotensive (white columns) and hypertensive (black columns) male rats. ** significantly synthesized
higher by aorta
(P < 0.01) than of normotensive
76
corresponding rats.
amount
of PGFza
F2e Mean (+ s.e.m., n - 4) % metabolism of C3Hl prostaglandin Table 1. by homogenates of aorta from normotensive and hypertensive male rats, in the absence (-1 and presence (+) of nicotinamide-adenine dinucleotide (NAD+; 2mM).
Normotensive
% Metabolism
-NAD+ Total per mg tissue
17.1
L 2.5
+NAD+ - 17.8
0.35 L 0.07
PG production Experiment 3. normotensive and hypertensive, i.
PC production
per unit
Hypertensive
0.40
+ 1.9 + 0.07
-NAD+ 15.2 0.35
L 2.4 + 0.06
in separated layers of aorta male and female rata.
+NAD+ 17.1
L 2.3
0.50 + 0.12
from
length
6-Keto-PGF1e was the major PG synthesized by endothelial cell suspensions and smooth muscle homogenates from the aorta of all 4 groups of rats, except in female hypertensive rats where PGF2e was the major PC1 synthesized by the endothelial cells (Table 2). PGF2u was the major PC synthesized by the adventitia in all groups of rats. The total amounts of 6-keto-PGF1e synthesized by the separated layers were in the order of’ smooth muscle > adventitia > endothelial cells for all rats, except in male hypertensive rats where 6-keto-PGF1, synthesis by the adventitia and endothelial cells were similar. The total amounts of PGF2e and PGE2 synthesized were higher by smooth muscle and adventitia than by endothelial cells in all rats. However, as the endothelial cells constitute about 2% of the tissue present in the aorta, the capacity for synthesizing PC12 (measured as 6-keto-PGF1e), PGE2 and PGF2a is 3- to 30-fold higher in the endothelial cells than in the smooth muscle or adventitia of all groups of rats. Regarding males only, the amounts of 6-keto-PGF1o synthesized by the endothelial cells were similar, but the amounts of 6-keto-PGF1e synthesized by smooth muscle and adventitia were significantly lower (P < 0.05) in hypertensive compared to normotensive rats. PGF2a synthesis was significantly higher (P < 0.05) by the endothelial cells and smooth muscle, and was significantly lower (P < 0.05) by adventitia in hypertensive compared to normotensive, male rats. PGF2e synthesis was significantly lower (P < 0.05) by the endothelial cells and adventitia, but was similar in smooth muscle of hypertensive compared to normotensive male rats. As regards female, the amounts of 6-keto-PGF1e and PGF2osynthesized by endothelial cells, smooth,muscle and adventitia were significantly lower (P < 0.05) in hypertensive compared to normotensive rats. There was no difference in PGE2 synthesis by the 3 tissue types between hypertensive and normotensive, female rats, 77
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synthesis was aignlflcantly Regarding sex differences, 6-keto-PGFle lower (P < 0.05) by the adventitla but did not dlffer by the endothellal cells and smooth muscle of female compared to male normotenslve rats. The amounts of PGFpand PGE2 synthesized by smooth muP)cle, but not by endothelial cell8 or adventitla, uere significantly hfgher (P < 0.05) In farale compared to male, normotenslve rats,although PGF2a. synthesis by the endothellal cells tended to be higher in the former group. The amounts of 6-keto-PGFle and PGFpa, but not of PGE2, synthesized by the endothelial cells were significantly lower (P < 0.05) in the The amount8 of hypertensive compared to normotenslve, female rata. PGE2 but not of 6-keto-PGFI a and PGF20, synthesized by smooth muscle and adventltla were slgnlflcantly higher (P < 0.05) in hypertensive compared to normotenalve, female rata. 11.
PC production
per unit weight
Slgnlflcant differences in 6-keto-PGFle, PGF2a and PGE2 synthesis by amooth muscle between the different group8 when the results are expressed on a unit length baa18 are still present when the result8 are expressed on a unit weight basis, except for PGE2 synthesis by the smooth muscle in female and male, normotenslve rats (Table 2). Additionally PGE2 synthesis by smooth muscle of hypertensive and normotenslve rats now become8 significantly different (P < 0.05). In contrast to smooth muscle, significant difference8 in the amounts of 6-keto-PGF1 a, PGF2a and PGE2 synthesized by adventitia between hypertensive and normotensive, male rats and between hypertensive and normotensive, female rats disappear when the results are expressed on a unit weight basis as opposed to a unit length basis. In addition PGE2 synthesis by the adventltia of normotensive and hypertensive. female rats 1s significantly greater (P < 0.05) in the hypertensive rats when the results are expressed on a unit weight basis. Obviously, the amount of adventltla In the aorta 1s greater in normotensive rats then in hypertensive rats of both sexes. Also the normotensive rats were slightly heavier than the hypertensive rats. However, correcting the results for differences in body weight had no effect on the significant differences observed. Experiment 4. Basal PC output from the aorta hypertensive, male and female rats.
or normotensive
and
6 Keto-PGF1 a was the major PC released from the aorta of noreotenalve and hypertensive male rats, followed by lesser amount8 of PGE2 and PGF2a (Figure 2). PGF2, release was significantly higher (P < 0.01) in hypertensive compared to normotenslve male rats: there was no difference in 6-keto-PGF1, and PGE2 release between the groups. The aorta from female, normotenslve rats released similar quantities of 6keto-PGF1, and PGE2, and smaller quantities of PGF2a. A similar profile was seen in hypertensive, female rats but the release of all 3 PCs was significantly higher (P < 0.05 or < 0.01, as indicated in Figure 2b) in the hypertensive state. The basal release of 6-keto-PGFI, but not of PGF2, and PGE2, was significantly lower (P < 0.05) in female compared to male normotensive rats. The basal release of PCE2, but not
79
a. 8 6 4. **
2, 0.
6-keto -PGFlm
PGF2m
PGE2
cn
s
6 E u4 I-f\ ",2 F 0 -PGFllx (PCs) from a Mean (+ s.e.m., n - 6) outputs of prostaglandlns 2. 2.5 cm length of tEoracic aorta perfused in vitro from normotensive (white columns) and hypertensive (black columns), (aI male rats, and (b) female rats.
Figure
*
Significantly higher than corresponding value (P < 0.051, ** (P < 0.01) for the same PG and same tissue In normotenafve rats of the same sex.
t
significantly different same PG and same tissue
(P < 0.05) from the corresponding value in male rats of the same blood pressure
80
for state.
the
of 6-keto-PGFlc and PGFP, was significantly compared to male hypertensive rats.
higher
Basal and stimulated PC output Experiment 5. arterial bed of normotensive and hypertensive,
in female
(P < 0.05)
from the mesenteric male and female rats.
6-Keto-PGF1 o was the major PC released from the mesenteric vascular bed of male and female normotensive and hypertensive rats, together with lesser quantities of PGF2c and PGE2 (Table 3). Output of all 3 PCs generally declined over the 60 min period of PC measurement, often to non-detectable values (< 0.2 ng/min) for PGF2c and PGE2. PGFPc was more detectable in hypertensive than in normotensive rats, generally particularly the females. Table 3. Mean (+ s.e.m., n = 4 unless otherwise shown) basal (ng/min) of prostaglandins (PCs) from the perfused mesenteric bed of normotensive and hypertensive, male and female rats at beginning (Start) and the finish (End) of the 60 min period of measurement (n.d. =i not detectable, < 0.2 ng/min).
Normotensive
output arterial the PC
Hypertensive
Start
End
Start
End
Male
1.3020.22
0.9820.26
1.9420.22
1.09~0.19
Female
2.7520.51
0.5820.14
2.1520.38
0.78+0.18
Male
n.d.
n.d.
0.4420.66
n.d.
Female
0.61+0.24‘
n.d.
0.6320.23
0.31+0.08
Male
0.54+0.10
0.36~0.03
0.8020.27
0.48’
Female
0.7120.24
0.44.
0.58+0.29’
0.4820.13
6-Keto-PGFl,:
-
t
t t
PGFa:-
PGE2: -
+ significantly
lower
than at start in the same group (P < 0.05) (1 result n.d.1 n-d.1
?? mean + s.e. mean of 3 results ?? mean of 2 results (2 results
NA (0.1 pg and 1 Pg) significantly increased (P < 0.05) 6-keto-PGF,c output from the mesenteric arterial bed of normotensive and hypertensive 81
z
t
2.19+0.40+ L 2.75-o.81+
2.67+0.42+ 2.46+0.42+
2.00+0.41 2.31Z0.5Llf
2.25-0.61f L
1.70+0.21
T
n.d.
0.63+0.37' 0.4110.12
n.d. 0.33+0.09.
0.53' 0.54tO.26
0.36~0.07'
C T
A
o.51+o.181 0.74zo.15
n.d. 0.75+0.22'+
1.0320.33
0.44
n.d. 0.69+0.15m+
POFZCY
0.40+0.09 0.37+0.179
0.49to.16 0.4650.14
o.44to.18. 0.28TO.08
n.d. 0.57L0.19'
C
PCE2
Significantly higher (P < 0.05) than corresponding value (C) before stimulation.
1.12tO.26 1.53z0.56
Norm
Wwr
Female:
1.04~0.20
0.83to.19
1.4tVo.33 1.21z0.28
Female: Norm Hyper
Male: Norm Hyper
1.06+0.11 1.1550.35
C
6-Keto-PCFI,
Male: Norm Hyper
State
?? mean (2 s.e.m.) of 3 values (1 value n.d.) ?? value from 1 animal (3 values n.d.)
+
1.0 pf3
0.1 !Jg
Dose of NA
0.65to.04 0.73~0.10.
0.77-+0.12 0.75'0.21
0.66to.20' 0.6650.26
0.87+0.12 0.94i0.25.
T
Mean (+ s.e.m., n = 4 unless otherwise shown) outputs (ng/min) of prostaglandins (PCs) from the Table 4. mesenteric arterial bed of normotensive (Norm) and hypertensive (Hyper), male and female rats 1 min before administration (n.d. = not detectable, < 0.2 ng/min). (C) and within 2 min (following (T) noradrenaline (NA)
Ei
wg
t m ?? A
1.0 pi3
0.1
1.97+0.81 [email protected]
1.05+0.08 1.4150.21
2.35+0.74t 2.6120.6-/t
3.76+0.65* 1.90~0.23
l-44+0.26
2.1950.45
1.59LO.36
2.39+0.71* 1.58E0.32
1
1.13~0.36
1.5610.33
1.22+0.42
C
6-Keto-PGF1,
0.60. 0.48+0.11
n.d. 0.63.
0.75 A 0.48+0.04
n.d. 0.42'
C
T
0.84* 0.98+0.38
n.d. 0.60.
0.70 A 0.64+0.10
n.d. 0.32 A
POF2,
0.47+0.20m 0.7650.36m
0.51~0.07 0.39.
0.72. 0.6820.29
0.63+0.08 0.97G
C
Significantly higher (P < 0.05) than corresponding value (C) before Stimulation. mean (+ s.e.m.) of 3 values (1 value n.d.1 mean 07 2 values (2 values n.d.1 value from 1 animal (3 values n.d.1
Female: Norm Hyper
Per
Male: Norm
Female: Norm Hyper
Male: Norm Hyper
Dose of AI1 State T
0.74+0.37. 0.85T0.40m
1.11+0.11 0.601
0.51. 0.46+0.06 _
0.73+0.21 o. 803
PGE2
Table 5. Mean (+ s.e.m., n = 4 unless otherwise shown) outputs (ng/min) of prostaglandins (PCs) from the mesenteric arterial bed of normotensive (Norm) and hypertensive (Hyper), male and female rats 1 min before (C) and within 2 min (following (T) angiotensin II (AII) administration (n.d. = not detectable, < 0.2 ng/min).
male and female rats (Table 4). NA (0.1 kg and 1 pg) tended also to increase PCE2 output in all 4 groups of rats, but none of the increases were significant. NA (0.1 and 1.0 kg) had little apparent effect on PGF2aoutput from the mesenteric arterial bed of normotensive, male and female rats. In contrast, NA significantly increased (P < 0.05) PGF2a output in male, hypertensive rats, and tended to increase PGF2a output in female, hypertensive rats. AI1 (0.1 and 1.0 pg) significantly increased (P < 0.05) 6-keto-PGFla output in male normotensive rats but not in male, hypertensive rats AI1 (0.1 pg) had no effect on 6-keto-PCFla output in (Table 5). female, normotensive or hypertensive rats. However, AI1 at the higher dose (1.0 vg) significantly increased (P < 0.05) 6-keto-PGFla output in female, normotensive and hypertensive rats. The effects of AI1 on PGF2a and PGE2 outputs were inconclusive since many of the outputs were below the detection limit. Notwithstanding, AI1 (0.1 kg) appeared to have little effect on the outputs of PGF2a and PGE2 in any of the 4 groups while AI1 (1.0 kg) significantly increased (P < 0.05) PGE2 output in male, normotensive rats (Table 5). The % changes in perfusion pressure of the mesenteric vascular bed fOllOWing treatment with NA and AI1 (0.1 and 1.0 kg) are shown in Table 6. NA (1.0 kg) and AI1 (1.0 kg) produced a significantly greater (P < 0.05) % increase in perfusion pressure in hypertensive than in normotensive, male rats. This significant difference was not present in female rats. Table 6. n = 4) % changes in perfusion pressure of Mean (+ s.e.m., the mesenteric vascular bed of normotensive (Norm) and hypertensive (Hyper) rats following in vitro treatment with noradrenaline and angiotensin. Noradrenaline
Male: Norm Female Female: Norm Hyper
Angiotensin
II
O*l IJg
1.0 Vg
0.1 kg
1.0 !Jg
62.3+34.7 153.0z43.5
174.4+ 4.4 261.053’33.5t
20.a+ 9.8 58.6220.2
150.4233.2 136.6242.6
14.7+ 6.36 31.6Z16.1
40.8+ 4.7 52.2z26.2
tsignificantly higher same dose (P < 0.05)
than normotensive
(AII)
33.0+ 8.0 61 .8-25.8
group of same sex receiving
DISCUSSION PG12 (measured as 6-keto-PGFla)
was the major PC synthesized 84
by
homogenates of the aorta from normotensive and hypertensive, male rats, whereas homogenates of the vena cava synthesized PC12 and PGF2cr in There was no difference in PG12 synthesis by similar quantities. homogenates of the aorta between hypertensive and normotensive, male rats, in spite of the lower capacity of the aortic smooth muscle of There was no difference in hypertensive male rats to synthesize PG12. PC12 output from the aorta of hypertensive compared to normotensive, PG12 male rats which is in contrast to other studies (2,3,4,5). synthesis by homogenates of the vena cava did no differ between Similarly PGE2 synthesis by hypertensive and normotensive, male rats. homogenates of the vena cava or aorta did not differ between hypertensive and normotenaive male rats, in spite of a lower capacity of aortic endothelial cells to synthesize PGE2 in the hypertensive state. There was no difference in PGE2 output from the aorta between the two blood pressure states in male rats. PGF2c, production by homogenates of the aorta was significantly The difference higher in hypertensive than in normotensive, male rats. was not due to changes in PGF2a metabolism, and may be related to greater ability of the aortic endothelial cells and smooth muscle to synthesize PGF2, in hypertensive, male rats. Also, this increased PGF2a synthesizing capacity may be the reason for the higher basal output of PGF2, from the aorta of hypertensive compared with normotensive male There was a tendency for the vena cava also to have a higher rats. synthesizing capacity for PGF2. in the hypertensive state, in male rats. One could speculate from these observations that the aetiology of hypertension is not connected with a decrease in output’ of vasodilator PCs by blood vessels, but is rather connected with an increase in output of the vasoconstrictor, PGFp, due to a higher PGF2d synthesizing capacity of the vasculature in the hypertensive state. However, the findings in female rats may not support this suggestion. The PC12 and PGE2 synthesizing capacities of the endothelial cells and smooth muscle were significantly lower in hypertensive than in normotensive, female rats, yet the outputs of PG12, PGF2a and PGE2 from the aorta were higher in the hypertensive state. Admittedly, PGF2e output is increased in female, hypertensive rats as in male, hypertensive rats, but it might be expected that the increase in outputs of PG12 and PGE2 may compensate for this increase in PGF2a output, unless PGF2a can act on the vascular smooth muscle unhindered by PC12 and PGE2. Interestingly, the higher output of PC12 from the aorta of hypertensive, albeit female rats is in agreement with previous studies The basal output of PG12 from the aorta was lower in the (2,3,4,5). female than in the male, normotensive rats. NA stimulated PC12 output and, to a lesser extent, PGE2 output from the mesenteric arterial bed of normotensive and hypertensive, male rats, which is in agreement with previous studies (7.18). The present study has shown that NA will stimulate also the release of PG12 and, to a lesser extent, the release of PGE2 from normotensive and hypertensive, female rats. In the rat mesenteric arterial bed and hind limb, PGE2 potentiates and PC12 attenuates the pressor response to NA while, on the splenic artery, PGE2 and PC12 both attenuate the effect of NA (19). effect of PGE2 may be specific to the rat However, the potentiating
85
since, in rabbits, PGE2 attenuates the pressor response of NA in the mesenteric arterial bed (20). Also topical application of PGE2 to the rat mesentery in vivo attenuates the pressor response of NA (21). However inhibition of PG synthesis reduces the pressor response of the rat mesenteric arterial bed perfused in vitro to NA to sympathetic nerve stimulation indicating that endogenous PGs are normally necessary for the full effect of NA to become manifest (20.22). There was no difference in the effects of exogenous NA on PC12 and PGE2, outputs from the mesenteric arterial bed between the hypertensive and normotensive states in rats of either sex. However, NA significantly increased the output of PGF2, from the mesenteric arterial bed of hypertensive, male rats and tended to increase PGF2, output from hypertensive, female rats, without any apparent effect of PCF2, release in normotensive male and female rats. Also, PGF2e was more readily detectable in the perfusate of the mesenteric arterial bed of hypertensive than of normotensive rats of either sex. However, whether increased basal and NA-stimulated PGF2, output is connected with hypertension requires further investigation. The pressor response to NA (1.0 pg) in the mesenteric arterial bed was significantly higher in hypertensive than in normotensive male but not female rats. AI1 increased the outputs of PC12 and, to a lesser extent, PGE2 from the mesenteric arterial bed of normotensive, male rats which is in agreement with previous studies (18,23,24). In contrast, AI1 failed to increase PC12 output from the mesenteric arterial bed of hypertensive, male rats. The effects of AI1 on PCE2 output were inconclusive. Studies in rabbits and sheep have indicated that the pressor effect of AI1 is normally attenuated by PGs produced endogenously in response to Consequently, hypertension could be due at AI1 treatment (25,261. least in part, to a lack of vascular PC synthesis in response to AII. The pressor response to AI1 in the mesenteric arterial bed was higher in However, the results in hypertensive than in normotensive, male rats. the female rats do not agree with this suggestion since AI1 (1.0 pg) stimulated PC12 output in female normotensive and hypertensive rats unless sex differences exist in the cause of hypertension. Certainly, the pressor response to AI1 (1.0 pg) did not differ significantly between hypertensive and normotensive, female rats. Overall, this study has highlighted some differences in vascular PC In addition, it production between hypertensive and normotensive rats. has shown that there are important differences between the sexes. Consequently, hypotheses on connections between hypertension and vascular PG synthesis should not be based on studied in males alone. The vascular output of the vasoconstrictor, PGF2c, was generally higher in hypertensive than in normotensive, male and female rats. This agrees with Hoffman et al. (271, who found that the increase in blood pressure of salt-load rats induced by a dietary linoleate deficiency was associated with an increase in the PCF2e to PGE2 ratio of production in the aorta and kidney. As well as being a vasoconstrictor, PGF2u potentiates responses produced by sympathetic nerve stimulation, especially in vascular smooth muscle (281, which would also increase vascular tone and blood pressure. However, whether the increase in vascular production of PGF2c is connected with the aetiology of hypertension requires further study. In this context, in a previous
86
study (291, PGF2e output from blood vessels of old, male and female rats was increased coapared to young rats. These old rats were also Again, whether the hypertension was the result of or the hypertensive. cause of increased vascular PGF2e output, or whether there was any connection between these two parameters at all, is requiring further study. ACKNOWLEDGEMENTS This study was supported by a grant from the Scottish Home and Health Department and a MRCpostgraduate studentship to E.A.L. Authentic PGs were kindly supplied by the Upjohn Company, Kalamaioo, Michigan, USA. REFERENCES 1.
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B
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