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Age-dependent increase in the formation of prostaglandin I2 by intact and homogenised aortae from the developing spontaneously hypertensive rat
177 Biochimica
et Biophysics
o Elsevier/North-Holland
Acta, 574 (1979) 177-181 Biomedical Press
BBA Report BBA 51252
AGE-DEPENDENT INCREASE IN THE FORMATION OF PROSTAGLANDIN IZ BY INTACT AND HOMOGENISED AORTAE FROM THE DEVELOPING SPONTANEOUSLY HYPERTENSIVE RAT
C.R. PACE-ASCIAK Research Institute, Ontario M5G 1X8
(Received Key
words:
and M.C. CARRARA The Hospital (Canada)
March 12th, Prostaglandin
for Sick
Children,
555
University
Avenue,
Toronto,
1979) Zz; Aorta;
Hypertension;
(Rat)
Summary The prostaglandin IZ biosynthetic capacity of aortae from spontaneously hypertensive rats of various ages (1, 2, 3, 4, and 5 months) was investigated. An age-dependent increase in enzyme activity was observed reaching maximum by three months of age which correlated well with the age- related increase in the systolic blood pressure. These results support our notion that the enhanced aortic synthesis of the potent vasodepressor prostaglandin IZ by the spontaneously hypertensive rat likely represents an adaptive mechanism for the attenuation of the sustained elevation in blood pressure in this animal model.
Prostaglandin IZ is a potent vasodepressor substance which survives passage through the lungs [l-3]. While it is about twice as active as prostaglandin E, in lowering the arterial blood pressure of the normotensive rat, it is 3-4 times more active in the spontaneously hypertensive rat [l] . This enhanced responsiveness of the spontaneously hypertensive rat towards prostaglandin IZ extends also to the enzymic capacity in aortic tissue which produces prostaglandin I2 not only to a larger extent but also more specifically than aorta from normotensive rats [4]. These observations have led us to propose a systemic antihypertensive role for this unique bicyclic prostaglandin [ 1,4,5] . We speculated that the enhanced vascular synthesis of prostaglandin I2 during hypertension might be an adaptive response to the elevations in blood pressure. Hence the present study was carried out to determine whether a correlation in time existed between the increase in aortic synthesis of prostaglandin IZ with the onset of hypertension in the developing spontaneously hypertensive rat.
178
The prostaglandin I* released by intact aorta incubated in 2 ml Tris-HCl buffer (pH 8) (10 min, 37°C 100% oxygen) was quantitated by its ability to depress the rat arterial blood pressure. For this purpose a normal Inactin anaesthetised adult rat was set up with polyethylene cannulas (PE50) inserted into the jugular vein (for sample injection) and carotid artery (for blood pressure monitoring). Arterial blood pressure was monitored on a Brush 2200 recorder via a Statham miniature arterial pressure transducer. Prior to sample injection, a dose response curve was generated by injecting 5, 10, 20, and 40 ng synthetic prostaglandin I, respectively in 0.1 ml Tris buffer. This enabled quantitation of the released vasodepressor substance in prostaglandin Iz equivalents. Injection of sample by the intravenous route eliminated the response being attributed to prostaglandin Ez since this would be metabolised by the lungs at the small doses investigated [l].After bioassay, the tissue was withdrawn from the incubation medium and 10 ml ethanol were added to the remaining solution (1.9 ml). A mixture of 3,3,4,4_tetradeutero prostaglandin E, and 6-keto prostaglandin F,, (1 fig each) was added together with a small amount of high specific activity prostaglandin E, (200 000 cpm). The samples were taken to dryness in vacua and the residue was partitioned between diethyl ether and dilute HCl (pH 3). After neutral washing the diethyl ether phase was taken to complete dryness and the residue was converted into the methyl ester (by ethereal diazomethane), 0-methyloxime (15 ~1 MOX reagent Pierce Chemical Co., 23°C overnight) and trimethylsilyl ether (15 ~1, TRI-SIL Z, Pierce Chemical Co., 5 min, 60°C). Assay by mass fragmentography was carried out by monitoring fragments at m/e 508 and 512 for the endogenous and deuterium-labelled carrier respectively, as reported previously [ 41. In additional experiments [3Hs] arachidonic acid (NEN, spec. act. 72 Ci/mmol) was incubated (10 min, 37°C 100% oxygen) with homogenates of aorta prepared in 2 ml 0.05 M KH2P04/NaOH (pH 7.4). Incubations were terminated with ethanol (10 ml); after filtration, the samples were taken to complete dryness in vacua. An aliquot (60/500 ~1 chloroform/methanol (l:l, v/v) was spotted on two silica gel G plates (Brinkmann, 5 X 20 cm); one plate was developed with chloroform/methanol/acetic acid/water (90:9: 1:0.65, v/v) and the other with the organic phase of ethyl acetate/2,2,4trimethylpentane/acetic acid/water (110: 50:20: 100, v/v). After development the plates were scanned on a Panax TLC radioscanner; zones corresponding to radioactive peaks were subsequently scraped into scintillation vials followed by 1 ml water/methanol (1: 1, v/v) and 10 ml Biofluor. Radioactivity in vials was measured in a Beckman LS-255 scintillation counter. When homogenates of aorta from hypertensive rats aged 1 and 5 months were incubated with tritium-labelled arachidonic acid, a great difference was observed in their ability to oxidise arachidonic acid into tritiated prostaglandins (Fig. 1). While the adult tissue is directed principally towards prostaglandin I, formation (detected here as its stable hydration product, 6-keto prostaglandin F,,) very little formation of this or any other prostaglandin was observed with the aorta from the l-month-old rat. That this product was 6keto prostaglandin F1, and not prostaglandin Ez was confirmed in these experiments by analysing the extract on TLC plates developed in System 2
179
SYSTEM
1
0
00
654
00
.
654
321
321
SYSTEM 2
65
4
32
1
00
0
00
65
4
32
.
1
Fig. 1. Thin layer radiochromatograms of ethanol extracts of homogenates of aorta from spontaneously hypertensive rats aged 1 month (left) and 5 months (right) incubated with tritiated arachidonic acid (1.5 X 10’ cpm) for 10 min at 37’C in 100% oxygen. Aliquots of the extract (60/500 ~1) were spotted on two plates and developed either with System 1 or System 2 (see text) Reference compounds are: 6. arachidonic acid; 5, ll-hydroxystearic acid; 4. prostaglandin A, ; 3, prostaglandin E, ; 2, Prostanglandin Fza. Samples were spotted at position 1. 6-Keto prostaglandin F,, standard comigrates with prostaglandin E, in System 1 but is resolved in System 2 migrating at Rf slightly more polar than prostaglandin F,,.
which resolves 6-keto prostaglandin F1, from prostaglandin Ez . Additional confirmation of this was obtained in other experiments where the products were assayed by gas chromatography-mass spectrometry (see below) as well as electron capture gas chromatography (unpublished) where complete resolution between prostaglandin Ez and 6-keto prostaglandin F,, is obtained. The progress of development of the aortic prostaglandin Iz -forming capacity as a function of age is shown in Fig. 2. The lower panels show experiments in which prostaglandin Iz released from intact incubated aorta has been quantitated directly by bioassay on the rat blood pressure as well as by mass spectrometry (in the same sample) after its conversion into 6-keto prostaglandin F,, . Also shown is the conversion of tritiated arachidonic acid into 6-keto prostaglandin F,, by aortic homogenates. As shown an exceedingly good correlation exists between all three methods of assay indicating that prostaglandin Iz formation increases with age reaching a maximum by 3
180
1
2
3 Age
4
5
(months)
Fig. 2. Correlation between age-related development of an elevation in systolic blood pressure and prostaglandin I, synthesis and release in aorta (intact and homogenised) of the spontaneously hypertens&e rat. Left scales denote measurements of prostaglandin I, and 6-keto prostaglandin F,, released by the intact aorta with the rat blood pressure bioassay and mass spectrometry, respectively. The right scale refers to experiments with homogenates where the conversion of tritiated arachidonic acid into 6-keto prostaglandin F,, was measured. Values represent mean f S.E.
months. The top panel shows data of systolic blood pressures of the hypertensive rats used in the study. Little difference in blood pressure exists at 1 month of age between the hypertensive and normotensive rats (not shown). Increased blood pressure in the hypertensive rats becomes obvious by the second month and reaches maximum ‘adult’ pressures by 3 months. Comparison of the profiles shown in Fig. 2 (top vs. bottom panels) indicates an exceedingly good correlation between synthesis of the vasodepressor prostaglandin I2 and the elevation in blood pressure. This data suggests that prostaglandin I2 synthesis might be an adaptive homeostatic response to attenuate the sustained elevations in blood pressure. If this hypothesis is correct it should be possible to elevate further the blood pressure of hypertensive rats through the use of specific blockers of prostaglandin I2 synthesis (when such blockers become available). Conversely it should be possible to reduce or abolish hypertension through the use of agents which activate further prostaglandin I2 synthesis and release. In this respect, stable analogs of prostaglandin I2 should prove beneficial in antihypertensive therapy. This work was supported by a grant to C.R.P.-A. from the Canadian Ontario Heart Foundation (No. 4-6). All unlabeled and deuterium-labeled standards were generously provided by Dr. Udo Axen, The Upjohn Company, Kalamazoo, MI. Authentic prostaglandin I2 was kindly provided by Dr. K.C.
181
Nicolaou, Department of Chemistry, University of Pennsylvania, Philadelphia, PA. The mass fragmentographic assay was performed with the assistance of Mr. Lajos Marai on a Varian MAT CH-5 coupled gas chromatograph-mass spectrometer, an MRC regional facility at the Best Institute, University of Toronto, Toronto. References 1 2 3 4 5
Pace-As&k, CR.. Carrara. M.C. and Nicolaou, K.C. (1978) Prostaglandins 15. 999-1003 Armstrong. J.M., Lattimer, N., Moncada, S. and Vane, J.R. (1978) Br. J. Pharmacol. 62, 125-130 Bolger, P.M., Eisner, G.M., Ramwell. P.W., Slotkoff, L.M. and Corey. E.J. (1978) Nature 271, 467469 Pace-Asciak, C.R., Carrara, M.C., Rangaraj, G. and Nicolaou, K.C. (1978) Prostaglandins 15, 1005-1012 Pace-Asciak, C.R. and Carrara, M.C. (1978) Prostaglandins 15, 704
et Biophysics
o Elsevier/North-Holland
Acta, 574 (1979) 177-181 Biomedical Press
BBA Report BBA 51252
AGE-DEPENDENT INCREASE IN THE FORMATION OF PROSTAGLANDIN IZ BY INTACT AND HOMOGENISED AORTAE FROM THE DEVELOPING SPONTANEOUSLY HYPERTENSIVE RAT
C.R. PACE-ASCIAK Research Institute, Ontario M5G 1X8
(Received Key
words:
and M.C. CARRARA The Hospital (Canada)
March 12th, Prostaglandin
for Sick
Children,
555
University
Avenue,
Toronto,
1979) Zz; Aorta;
Hypertension;
(Rat)
Summary The prostaglandin IZ biosynthetic capacity of aortae from spontaneously hypertensive rats of various ages (1, 2, 3, 4, and 5 months) was investigated. An age-dependent increase in enzyme activity was observed reaching maximum by three months of age which correlated well with the age- related increase in the systolic blood pressure. These results support our notion that the enhanced aortic synthesis of the potent vasodepressor prostaglandin IZ by the spontaneously hypertensive rat likely represents an adaptive mechanism for the attenuation of the sustained elevation in blood pressure in this animal model.
Prostaglandin IZ is a potent vasodepressor substance which survives passage through the lungs [l-3]. While it is about twice as active as prostaglandin E, in lowering the arterial blood pressure of the normotensive rat, it is 3-4 times more active in the spontaneously hypertensive rat [l] . This enhanced responsiveness of the spontaneously hypertensive rat towards prostaglandin IZ extends also to the enzymic capacity in aortic tissue which produces prostaglandin I2 not only to a larger extent but also more specifically than aorta from normotensive rats [4]. These observations have led us to propose a systemic antihypertensive role for this unique bicyclic prostaglandin [ 1,4,5] . We speculated that the enhanced vascular synthesis of prostaglandin I2 during hypertension might be an adaptive response to the elevations in blood pressure. Hence the present study was carried out to determine whether a correlation in time existed between the increase in aortic synthesis of prostaglandin IZ with the onset of hypertension in the developing spontaneously hypertensive rat.
178
The prostaglandin I* released by intact aorta incubated in 2 ml Tris-HCl buffer (pH 8) (10 min, 37°C 100% oxygen) was quantitated by its ability to depress the rat arterial blood pressure. For this purpose a normal Inactin anaesthetised adult rat was set up with polyethylene cannulas (PE50) inserted into the jugular vein (for sample injection) and carotid artery (for blood pressure monitoring). Arterial blood pressure was monitored on a Brush 2200 recorder via a Statham miniature arterial pressure transducer. Prior to sample injection, a dose response curve was generated by injecting 5, 10, 20, and 40 ng synthetic prostaglandin I, respectively in 0.1 ml Tris buffer. This enabled quantitation of the released vasodepressor substance in prostaglandin Iz equivalents. Injection of sample by the intravenous route eliminated the response being attributed to prostaglandin Ez since this would be metabolised by the lungs at the small doses investigated [l].After bioassay, the tissue was withdrawn from the incubation medium and 10 ml ethanol were added to the remaining solution (1.9 ml). A mixture of 3,3,4,4_tetradeutero prostaglandin E, and 6-keto prostaglandin F,, (1 fig each) was added together with a small amount of high specific activity prostaglandin E, (200 000 cpm). The samples were taken to dryness in vacua and the residue was partitioned between diethyl ether and dilute HCl (pH 3). After neutral washing the diethyl ether phase was taken to complete dryness and the residue was converted into the methyl ester (by ethereal diazomethane), 0-methyloxime (15 ~1 MOX reagent Pierce Chemical Co., 23°C overnight) and trimethylsilyl ether (15 ~1, TRI-SIL Z, Pierce Chemical Co., 5 min, 60°C). Assay by mass fragmentography was carried out by monitoring fragments at m/e 508 and 512 for the endogenous and deuterium-labelled carrier respectively, as reported previously [ 41. In additional experiments [3Hs] arachidonic acid (NEN, spec. act. 72 Ci/mmol) was incubated (10 min, 37°C 100% oxygen) with homogenates of aorta prepared in 2 ml 0.05 M KH2P04/NaOH (pH 7.4). Incubations were terminated with ethanol (10 ml); after filtration, the samples were taken to complete dryness in vacua. An aliquot (60/500 ~1 chloroform/methanol (l:l, v/v) was spotted on two silica gel G plates (Brinkmann, 5 X 20 cm); one plate was developed with chloroform/methanol/acetic acid/water (90:9: 1:0.65, v/v) and the other with the organic phase of ethyl acetate/2,2,4trimethylpentane/acetic acid/water (110: 50:20: 100, v/v). After development the plates were scanned on a Panax TLC radioscanner; zones corresponding to radioactive peaks were subsequently scraped into scintillation vials followed by 1 ml water/methanol (1: 1, v/v) and 10 ml Biofluor. Radioactivity in vials was measured in a Beckman LS-255 scintillation counter. When homogenates of aorta from hypertensive rats aged 1 and 5 months were incubated with tritium-labelled arachidonic acid, a great difference was observed in their ability to oxidise arachidonic acid into tritiated prostaglandins (Fig. 1). While the adult tissue is directed principally towards prostaglandin I, formation (detected here as its stable hydration product, 6-keto prostaglandin F,,) very little formation of this or any other prostaglandin was observed with the aorta from the l-month-old rat. That this product was 6keto prostaglandin F1, and not prostaglandin Ez was confirmed in these experiments by analysing the extract on TLC plates developed in System 2
179
SYSTEM
1
0
00
654
00
.
654
321
321
SYSTEM 2
65
4
32
1
00
0
00
65
4
32
.
1
Fig. 1. Thin layer radiochromatograms of ethanol extracts of homogenates of aorta from spontaneously hypertensive rats aged 1 month (left) and 5 months (right) incubated with tritiated arachidonic acid (1.5 X 10’ cpm) for 10 min at 37’C in 100% oxygen. Aliquots of the extract (60/500 ~1) were spotted on two plates and developed either with System 1 or System 2 (see text) Reference compounds are: 6. arachidonic acid; 5, ll-hydroxystearic acid; 4. prostaglandin A, ; 3, prostaglandin E, ; 2, Prostanglandin Fza. Samples were spotted at position 1. 6-Keto prostaglandin F,, standard comigrates with prostaglandin E, in System 1 but is resolved in System 2 migrating at Rf slightly more polar than prostaglandin F,,.
which resolves 6-keto prostaglandin F1, from prostaglandin Ez . Additional confirmation of this was obtained in other experiments where the products were assayed by gas chromatography-mass spectrometry (see below) as well as electron capture gas chromatography (unpublished) where complete resolution between prostaglandin Ez and 6-keto prostaglandin F,, is obtained. The progress of development of the aortic prostaglandin Iz -forming capacity as a function of age is shown in Fig. 2. The lower panels show experiments in which prostaglandin Iz released from intact incubated aorta has been quantitated directly by bioassay on the rat blood pressure as well as by mass spectrometry (in the same sample) after its conversion into 6-keto prostaglandin F,, . Also shown is the conversion of tritiated arachidonic acid into 6-keto prostaglandin F,, by aortic homogenates. As shown an exceedingly good correlation exists between all three methods of assay indicating that prostaglandin Iz formation increases with age reaching a maximum by 3
180
1
2
3 Age
4
5
(months)
Fig. 2. Correlation between age-related development of an elevation in systolic blood pressure and prostaglandin I, synthesis and release in aorta (intact and homogenised) of the spontaneously hypertens&e rat. Left scales denote measurements of prostaglandin I, and 6-keto prostaglandin F,, released by the intact aorta with the rat blood pressure bioassay and mass spectrometry, respectively. The right scale refers to experiments with homogenates where the conversion of tritiated arachidonic acid into 6-keto prostaglandin F,, was measured. Values represent mean f S.E.
months. The top panel shows data of systolic blood pressures of the hypertensive rats used in the study. Little difference in blood pressure exists at 1 month of age between the hypertensive and normotensive rats (not shown). Increased blood pressure in the hypertensive rats becomes obvious by the second month and reaches maximum ‘adult’ pressures by 3 months. Comparison of the profiles shown in Fig. 2 (top vs. bottom panels) indicates an exceedingly good correlation between synthesis of the vasodepressor prostaglandin I2 and the elevation in blood pressure. This data suggests that prostaglandin I2 synthesis might be an adaptive homeostatic response to attenuate the sustained elevations in blood pressure. If this hypothesis is correct it should be possible to elevate further the blood pressure of hypertensive rats through the use of specific blockers of prostaglandin I2 synthesis (when such blockers become available). Conversely it should be possible to reduce or abolish hypertension through the use of agents which activate further prostaglandin I2 synthesis and release. In this respect, stable analogs of prostaglandin I2 should prove beneficial in antihypertensive therapy. This work was supported by a grant to C.R.P.-A. from the Canadian Ontario Heart Foundation (No. 4-6). All unlabeled and deuterium-labeled standards were generously provided by Dr. Udo Axen, The Upjohn Company, Kalamazoo, MI. Authentic prostaglandin I2 was kindly provided by Dr. K.C.
181
Nicolaou, Department of Chemistry, University of Pennsylvania, Philadelphia, PA. The mass fragmentographic assay was performed with the assistance of Mr. Lajos Marai on a Varian MAT CH-5 coupled gas chromatograph-mass spectrometer, an MRC regional facility at the Best Institute, University of Toronto, Toronto. References 1 2 3 4 5
Pace-As&k, CR.. Carrara. M.C. and Nicolaou, K.C. (1978) Prostaglandins 15. 999-1003 Armstrong. J.M., Lattimer, N., Moncada, S. and Vane, J.R. (1978) Br. J. Pharmacol. 62, 125-130 Bolger, P.M., Eisner, G.M., Ramwell. P.W., Slotkoff, L.M. and Corey. E.J. (1978) Nature 271, 467469 Pace-Asciak, C.R., Carrara, M.C., Rangaraj, G. and Nicolaou, K.C. (1978) Prostaglandins 15, 1005-1012 Pace-Asciak, C.R. and Carrara, M.C. (1978) Prostaglandins 15, 704