105

Alterations in -Mediated Activation of AZ in DAHL Salt-Sensitive Rats

Yoshio Uehara, Satoru Takada*, Yukari Kawabata, Nobuhito Hirawa**, Junichi Iwai***, Shuntaro Hara****, Ichiro Kudo****, Keizo Inoue****, and Masao Omata

In this study, the subcellular mechanism of impaired prostacyclin (PGI2) synthesis in the vascular sys- tem of Dahl salt-sensitive (Dahl S) rats was investigated. Arachidonate liberation in response to PDGF or bradykinin was decreased in cultured aortic smooth muscle cell (VSMC) from Dahl S rats, compared with Dahl salt-resistant (Dahl R) rats. (PIP2-PLC) activity was lowered and the inositol 1,4,5-triphosphate content was also decreased in the VSMC from Dahl S rats. In fact, cytosolic calcium levels in basal and angiotensin-II stimulated conditions were significantly decreased in VSMC from Dahl S rats, compared with those in Dahl rats. There was no difference, however, in (PLA2) activity in the two strains. Moreover, the PLA2 properties, e.g., its Ca2+ requirement, pH profile, Km value and Vmax, were equal in Dahl S and Dahl R rats. The level of functional PLA2 messenger RNA was found to be greater in the VSMC from Dahl S rats. Similarly, PGI2 synthesis was reduced in Dahl S rats and this was associated with an unaltered PLA2 concentration and decreased PIP2-PLC activity in the arterial wall. Thus, these data indicate that dysfunction of receptor-mediated PLA2 activation is responsible for altered PGI2 synthesis in Dahl S rats. This finding is likely mediated by a decrease in phosphoinositides . (Hypertens Res 1993 ;16 :105-111) Key Words: Dahl salt-sensitive rats, smooth muscle cell, phospholipase A2, phosphoinositide metabol- ism, phospholipase C

The potential role of prostaglandins in the genesis prostaglandin system in Dahl S rats, we have ex- of hypertension has generated considerable research amined PLA2, a key component in prostaglandin interest. Evidence has accumulated which suggests biosynthesis. We report in this study an investiga- that impaired vasodepressor prostaglandin system tion of the enzyme kinetic parameters and altered can contribute to the development of hypertension regulation of PLA2, using cultured aortic smooth in Dahl salt-sensitive (Dahl S) rats. Restoration of muscle cells (VSMC) and the arterial wall from prostaglandin synthesis by linoloate-rich diet can Dahl S rats. lead to attenuation of hypertension in Dahl S rats (1- 4). Few studies, however, have addressed the cause of impaired prostaglandin synthesis at a sub- Materials and Methods cellular level in Dahl S rats. In a recent study, Experiment 1. Craven et al. (5) have reported that a decrease in Cell Culture vasopressin-stimulated prostaglandin synthesis Dahl S and Dahl salt-resistant (Dahi R) rats were occurs in the renomedullary interstitial cells and originally obtained from Brookhaven National that it is associated with a decrease in the receptor- Laboratory, Upton, New York and bred at Tsukuba mediated cytosolic calcium level (5, 6). Since phos- Research Laboratories, Eisai Co., Ltd., Tokyo, pholipase A2 (PLA2) and phospholipase C (PLC) Japan. The rats were fed a low salt (0.3% NaCI) are coupled with receptors and GTP-binding pro- diet after weaning. In this study, 8-week-old Dahl S teins and release free , they may be and Dahl R rats were used. Systolic blood pressure rate limiting for prostaglandin synthesis. was 134 ± 3 (SE) mmHg for Dahl S and 125 ± 2 To understand the abnormal response of the mmHg for Dahl R rats (p < 0.05). The cells were

From the 2nd Department of Medicine, University of Tokyo, *Division of Hypertension and Cardiorenal Disease, Dok- kyo University School of Medicine, Mibu, Tochigi, **Kantou-Teishin Hospital, Shinagawa, Tokyo, Japan, ***Brookhaven National Laboratory, Upton, New York, USA, ****Department of Health Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Japan. This study was supported in part by grants from the Ministry of Education, Science, and Culture of Japan (No.03670438, No.04670516) and grants from Sankyo Life Science Research Foundation. Address for Reprints: Yoshio Uehara, M.D., The 2nd Department of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan. Received August 18, 1992; accepted in revised form April 15, 1993. 106 Hypertens Res Vol. 16, No. 2 (1993) cultured by the explant method (7, 8). Strips from layer chromatography.The radioactivityof arachi- the descending thoracic aorta were placed in 120- donate was measured by a liquid scintillationcoun- mm polystyrene dishes and cultured in Dulbecco's ter (10). modified Eagle medium (MEM) containing 20% To determine phosphatidylcholine(PC)-hydrolyz- (vlv) fetal bovine serum (FBS), penicillin (100 ing activity(phospholipase A2 activity,PLA2), 10 g units/ml) and streptomycin (100 ,ag/ml). The cells supernatant of the homogenizedVSMC was incu- were maintained at 37°C under a humidified atmos- bated in 0.1 M glycinebuffer at pH 8.0 containing phere of 95% air and 5% CO2. Media were 0.2 M NaCI, 2 mM CaCl2and 1.25 mM deoxycholic changed to MEM with 0.01 units/ml platelet derived acid sodium (11, 12). The reaction was initiated growth factor (PDGF) (Transformation Research, under Vmax conditionsby adding 3 X 104 dpm of Inc., Framingham, Massachusetts, U.S.A.). This 1-stearoyl-2-(1-3H) arachidonyl L-3-PC substrate amount of PDGF is equivalent to 10% FBS as de- (0.5-1 Ci/mol) and terminated after 30 minutes us- termined by (3H)thymidine uptake. The experi- ing Folch's solvent. Hydrolyzed (3H)arachidonate ments were performed using VSMC at the 2nd pas- was separated by silicicacid thin layer chromatogra- sage. phy and the amount of radioactivitywas determined The cells cultured in this experiment have been by a liquidscintillation counter. well characterized in our previous studies (8, 9). PhosphoinositideTurnover They showed a typical "hills and valleys" growth Phospholipase C activity (PLC) was assessed as pattern on phase contrast micrograph and a-actin phosphatidyl-inositol4, 5-bisphosphatehydrolyzing molecules were demonstrated by immunohistochem- activity (PIP2-P C) . The assay mixture consistedof ical analysis (8). The cells were distinct from fibrob- 50 ,al of the i04 g enzyme solution and 200 ,al of lasts in morphological appearance and in biochemic- 50 mM HEPES buffer (pH 7.0) including 0.1 M al properties. Thus, the cells exhibited morphologic- NaCI, 5 mM CaCl2and 1.25 mM deoxycholicacid al and biochemical characteristics consistent with sodium (11, 12). The reaction was started under vascular smooth muscle cell origin. Vmax conditionsions by adding the substrate, L-3-phos4 Determination of Prostaglandin Synthesis phatidyl (2-3H)inositol 4, 5-bisphosphate(3 X 10 To measure the biosynthesis of prostacyclin (PGI2), dpm, 0.5-1 Ci/mol), and terminated after 30 mi- a major product of arachidonate metabolism in nutes with Folch'ssolvent. The amount of (2-3H)1P3 VSMC (8, 9), confluent VSMC at the 2nd passage in the aqueous phase was determinedusing a liquid were washed with Dulbecco's phosphate-buffered scintillationcounter. saline solution (D-PBS) and then cultured with a To assess phosphoinositide(PI) metabolism, we given concentration of either PDGF, bradykinin or measured the intracellularcontent of inositol 1, 4, the calcium ionophore, A23187. 5-trisphosphate(1P3), a byproduct of PIP2. Briefly, The amount of prostaglandin released into the IP3 was extracted from VSMC with an ice-chilled media for one hour was determined by direct 20% perchloric acid solution. After being neutral- radioimmunoassay as described in previous studies ized by 10 N KOH, the acid-solublefraction was (2, 8). Briefly, PGI2 was measured as stable 6-keto- used for IP3 determination.IP3 was measured using PGF1a. The assay mixture consisted of 0.1 ml of an adrenal protein binding assay method (1P3Assay (3H)prostaglandin (10 dpm)(120-180 Ci/mmol), 0.1 Systemkit, AmershamJapan, Tokyo, Japan). ml of a diluted sample or standard solution, and 0.1 Assay of ProstacyclinSynthase ml of a diluted antibody solution. The assay mixture For assay of PGI2 synthase, the 104gsupernatant was incubated for 24 hours at 4°C. 0.1 ml of was incubatedin 25 mM Tris-Cl(pH 7.6) containing dextran-coated charcoal solution (2.5% charcoal 100 mM NaCI (12). The conversionfrom PGH2 to and 0.25% dextran) was added to the assay mixture prostacyclinwas initiated by adding 0.5 ,ag PGH2, and the unabsorbed (3H)prostaglandin (bound the substrate of prostacyclinsynthase. The reaction form) was determined using an automatic liquid was terminated with 50 mM FeCl2 after 3 minutes scintillation counter. and synthesizedPGI2 was directly radioimmunoas- Antisera were obtained using prostaglandin-cou- sayed as 6-keto-PGF1a.Non-specific conversion by pled thyroglobulin and Freund's complete adjuvant the boiled enzymesolution was less than 2% of the as the immunogen. Anti-6-keto-PGF1a serum cross- generated PGI2(12). reacted 1% with PGE2, 2% with PGF2«, 0.08% MessengerRNA of PhospholipaseA2 with PGD2, 0.01% with TXB2 and less than 0.001% Total ribonucleicacid (RNA), includingribosomal, with arachidonic acid. transfer and essenger RNA (mRNA), was ex- Assay of Phospholipase A2 tracted from 108VSMC according to the single-step To determine the arachidonate-liberating activity in method of RNA isolationusing guanidium thiocyan- VSMC, confluent VSMC were incubated with ate, phenol and chloroform (13), mRNA was iso- (3H)arachidonate (1.0 pCi) for one hour. The la- lated from the crude, total RNA with a 2% (w/v) belled VSMC were washed with D-PBS containing oligo(dT)-latexsuspension (Daiichi Chemical Co., 10-5 M indomethacin and then cultured for one Tokyo, Japan). Using mRNA and rabbit reticulo- hour with a test compound and 10-5 M indometha- cyte lysate (In Vitro Protein SynthesisSystem, Nip- cin. The amount of radiolabelled arachidonate re- pon Gene Co., Tokyo, Japan), cell-free protein leased into the medium was determined by extrac- synthesiswas performed a3 30°Cfor one hour (14). tion with Folch's solvent (chloroform : methanol = In this assay condition, (S)methionine was time- 2 : 1, v/ v) and by separation using silicic acid thin dependentlyincorporated into acid-insolubleprotein Uehara et al: Phospholipase and Dahl Rats 107 fraction for one hour. Moreover, this assay system when compared with Dahl R rats. Moreover, the was found to be a mRNA-dependent event because response of PGI2 synthesis to PDGF or bradykinin the amount of mRNA added to this assay mixture was significantly blunted in the VSMC from Dahl S was well correlated to the radioactive protein rats. Interestingly, however, when PGI2 synthesis formation (r = 0.96 (n = 4) , p < 0.05) . Phospholipase was maximally stimulated by a 10 M dose of A2 activity produced in this cell free system was de- A23187, the PGI2 synthesis by the VSMC from termined as described in the text. The amount of Dahl S rats was 55% greater than that observed in phospholipase A2 mRNA is expressed as phospholi- Dahl R rats. pase A2 activity synthesized under these assay con- Arachidonate Liberation ditions. Exogenous (3H)arachidonate was incorporated into Cytosolic Calcium Analysis the VSMC over one hour in a time-dependent Cytosolic calcium levels were determined using fashion. There was no difference in the amount of fura-2/AM (Dojin Chemical Laboratory, Ltd., radioactivity incorporated into VSMC between Dahl Tokyo, Japan) and a Nihon Bunko calcium auto- R and Dah6S rats (6.54± 0.18vs. 6.48 ± 0.16 X analyzer, model CAF-100 (Nihon Bunko Industry, 10 dpm/10 cells/hour(n -12)). Both PDGF and Ltd. , Tokyo, Japan) (15, 16). Briefly, 5 M of fura- b3adykinin significantly enhanced the liberationof 2/AM resolved in MEM was loaded on dispersed ( H)arachidonateby the VSMCfrom Dahl R rats ( VSMC for 30 minutes at 37°C. The cells were spun Fig. 1b). However,this normalresponse to PDGF at 1400g for 5 minutes and resuspended in calcium- or bradykininwas completelyabolished in Dahl S free 10 mM hepes buffer at pH 7.4 containing 129 rats. In contrast,the stimulationby 105M A23187 mM NaCI, 8.9 mM NaHCO3, 0.8 mM KH2PO4, 0.8 led to a significantincrease in the arachidonateli- mM MgCl2 and 5.6 mM dextrose. The cell density berationin the VSMCfrom Dahl S rats. was adjusted to 10 cells/ml. Extracellular calcium Analysesof concentration was adjusted to 1 mM at 5 minutes Table 1 showsthe basal levelsof PLA2and PIP2- before assay. Cytosolic fura-2 was alternately ex- PLC activitiesin the VSMCfrom DahlS and Dahl cited with ultraviolet waves at 340 and 380 nm and R rats. The PLA2activity was decreased in Dahl S ratio of the emission at 510 nm was determined. rats althoughthe differencewas not statisticallysig- Cytosolic calcium levels were measured in a basal nificant.In contrast,the PIP2-PLCactivity was sig- condition and after 106M angiotensin II stimula- nificantlylower by 20% in the VSMCfrom Dahl S tion. rats whencompared with Dahl R rats. The reduc- tion of PIP2-PLCactivity was reflectedby a signi- Experiment II. ficant decreasein the intracellularIP3 concentra- We investigated prostacyclin synthesis and its reg- tion. On the other hand, there was no differencein ulation in the arterial walls of Dahl S and Dahl R PGI2synthase activity between the two strains. rats. Eight-week-old rats were fed a low salt (0.3% Characterizationof PLA2activity in the VSMC NaCI) diet after weaning. The descending thoracic wasalso performed.There was no differencein the aortae were obtained under pentobarbital anesthe- calciumrequirement (Fig. 2a) or pH profile(Fig. sia (25 mg/kg). A part of the vessel was incubated 2b). Moreover, Linewever-Burkplot analysis in D-PBS at 37°C for 30 min and prostaglandin re- showedthat the Km valueof PLA2was essentially leased into the medium was radioimmunoassayed as equivalentin Dahl S rats and in Dahl R rats (0.337 described above (10, 12). A portion of the vessel for Dahl S vs 0.325 M for Dahl R rats). In addi- wasalso homogenizedby a Polytronblender and tion, Vmaxwas 0.337 nmoles/30 min for Dahl S and spun at 104g for 30 min. In the supernatant, PLA2, 0.325nmoles/30 minutes for Dahl R rats. These PIP2-PLC and PGI2 activities were determined (11, data indicatethat PLA2enzyme kinetic parameters 12). Enzyme activities were determined as described are quite similarin DahlS and DahlR rats. in the text above. CytosolicCalcium Levels Reagents Cytosoliccalcium levels are shownin Table2. The Reagents were all of analytical grade. Radioactive basal level was significantlydecreased by 26% in materials were purchased from Amersham Japan, VSMCfrom normotensiveDahl S rats, compared Tokyo, Japan. and related compound with hosefrom control Dahl R rats. AngiotensinII were a gift from Ono Pharmaceutical Co., Ltd., (10_6 M) enhancedthe cytosoliccalcium level in Osaka. VSMCboth from Dahl S and Dahl R rats; howev- Statistical Analysis er, the level was significantlylowered by 32% in Values are expressed as mean ± SE. Differences VSMCfrom Dahl S rats whencompared with those were assessed by Student's t test, one way analysis fromDahl R rats. of variance or two way analysis of variance. MessengerRNA Expression In an additionalexperiment, we assessedPLA2 mRNAlevels (Table 3). Therewas no differencein Results the amountof total RNAextracted from 108 VSMC Experiment 1. betweenDahl S and Dahl R rats. Interestingly, Prostacyclin Synthesis however,the PLA2activity synthesized by mRNA Figure 1a illustrates the alterations in PGI2 synth- from Dahl S rats was significantlygreater than the esis by the VSMC from Dahl rats. Basal PGI2 activityproduced by mRNA from Dahl R rats. synthesis was decreased significantly in Dahl S rats Thesedata suggestthat the functionalPLA2 mRNA 108 Hypertens Res Vol. 16, No. 2 (1993)

Fig. 1. Prostacyclin generation and arachidonate liberation in cultured vascular smooth muscle cells from Dahl rats. (a) Upper graph shows PGI2 synthesis in the VSMC of Dahl S and Dahl R rats in response to various stimuli. Differences were analyzed by one way analysis of variance. F values were 30.1 for Dahl R (p < 0.0001) and 21.3 for Dahl S (p < 0.0001) in PDGF, 72.3 for Dahl R (p < 0.0001) and 74.5 for Dahl S (p < 0.0001) in bradykinin and 19.5 for Dahl R (p < 0.001) and 48.8 for Dahl S (p<0.0001) in A23187. (b) Lower graph shows arachidonate liberation in the VSMC of Dahl S and Dahl R rats in response to various stimuli. F values were 8.5 for Dahl R (p < 0.005) and 1.0 for Dahl S (p>0.1) in PDGF , 11.6 for Dahl R (p< 0.001) and 0.8 for Dahl S (p >0.1) in bradykinin and 10.1 for Dahl R (p< 0.002) and 59.7 for Dahl S (p< 0.0001) in A23187. Number represents the percentage change in Dahl S rats when compared with the respective values in Dahl R rats. Each value represents an average of independent determinations obtained from 6 rats.

Table 1. Characterization of Involved in Prostaglandin Synthesis in Cultured Vascular Smooth Muscle Cells Uehara et al: Phospholipase and Dahi Rats 109

Fig. 2. Enzyme properties of phospholipase A2 in cultured vascular smooth muscle cell from Dahl rats. (a) The percent of phospholipase A2 activity as a function of calcium concentration in VSMC. (b) The percent of phospholipase A2 activity as a function of pH in VSMC. Open circles represent values obtained in Dahl R rats, while closed circles represent values obtained in Dahl S rats. Each value represents an average of independent determinations obtained from 6 rats. Differences between Dahl S and Dahl R rats were analyzed by two way analysis of variance. F values were 0.0077 for calcium require- ment (p>0.1) and 0.058 for pH profile (p>0.1).

Table 2. Cytosolic Calcium Levels in Vascular Smooth Muscle Cells

level must be higher in the VSMC from Dahl S rats with the calcium ionophore, A23187, suggesting than in Dahl R rats. that the enzyme system for PGI2 biosynthesis is kept intact. Similarly, free arachidonate liberation Experiment II. in response to PDGF or bradykinin was decreased Alterations of prostaglandin synthetic enzymes in significantly in the VSMC from Dahl S rats. Howev- the vascular wall were investigated. On a low salt er, arachidonate liberation was enhanced in Dahl S (0.3% NaCI) diet, systolic blood pressure was rats when the VSMC were stimulated with the cal- slightly higher in Dahl S rats (136 ± 3 (SE) vs. 121 cium ionophore, A23187. Based on these data, the ± 2 mmHg, p < 0.05); however, the blood pressure subnormal response of PGI2 synthesis in Dahl S rats in Dahl S rats was well within a normotensive stage. may be attributable to a decrease in calcium sensi- Aortic PGI2 synthesis was significantly decreased in tivity or an abnormality in intracellular calcium Dahl S rats, compared with that in Dahl R rats mobilization. In support of this possibility, Craven (Table 4). Although PGI2 synthesis was reduced in et al. (5) and Reid et al. (4) have found that Dahl S rats, PLA2 activity in the aortic homogenate vasopressin-mediated calcium mobilization is signif- was equivalent in Dahl S and Dahl R rats. In- icantly lower in the renal papillary interstitial cells triguingly, the PIP2-PLC activity was significantly of Dahl S rats, resulting in decreased prostaglandin decreased in the arterial wall of Dahl S rats. PGI2 E2 synthesis. In fact, we demonstrated in this study synthase activity was not significantly different be- that basal cytosolic calcium levels were decreased in tween the two strains. VSMC from Dahl S rats, compared with those from Dahl R rats. Moreover, response to angiotensin II was also lowered in VSMC from Dahl S rats. Thus, Discussion it is clearly indicated that the abnormal PGI2 synthe- We demonstrated in the present study that PGI2 sis in Dahl S rats is due to a derangement of in- synthesis in response to receptor-mediated stimula- tracellular calcium mobilization rather than to an al- tion by PDGF or bradykinin is significantly blunted teration in PLA2 or other enzymes involved in PGI2 in the VSMC from Dahl S rats. This subnormal re- synthesis. sponse was not seen when the cells were stimulated To test this hypothesis, we analyzed PLA2 activ- 110 Hypertens Res Vol . 16, No. 2 (1993)

Table 3. Messenger RNA Content in VSMC under Basal Conditions

Table 4. Analyses of Enzymes Involved in Prostaglandin Synthesis in the Arterial Walls

ity in VSMC homogenates and showed that the bas- metabolism in prostacyclin generation in Dahl S rats al PLA2 activity was equivalent in Dahl S and Dahl will be the subject of future investigations. R rats. Moreover, there was no difference in the Few studies have investigated PI turnover in the PLA2 calcium requirement or pH profile between cardiovascular system of Dahl S rats. Craven and the two strains. Determination of the PLA2 Km and DeRubertis have observed that the cytosolic cal- Vmax values revealed no significant difference be- cium level is decreased in Dahl S rats (5,6). In addi- tween Dahl S and Dahl R rats. These data strongly tion, abnormal calcium metabolism and decreased suggest that the enzymatic properties of PLA2 in prostaglandin synthesis is found in the papillary col- VSMC homogenates are quite similar in Dahl S rats lecting tubules of Dahl S rats (4). Other resear- and Dahl R rats. chers, however, have reported that PI turnover is The Vmax value reflects the activity of PLA2. enhanced and cytosolic calcium level may be in- Since the Vmax value was equivalent in the two creased in various tissues in spontaneously strains, it follows that PLA2 activity must also be hypertensive rats (SHR) (11,19-22) . SHR have similar. However, we demonstrated that the amount also been found to have enhanced prostaglandin of PLA2 mRNA was increased in the VSMC from synthesis in their vascular walls and kidneys (23- Dahl S rats. The discrepancy between the unaltered 25). There is, therefore, an apparent discrepancy in PLA2 activity and the increased PLA2 mRNA level cytosolic calcium metabolism between the two gene- may indicate an abnormality in mRNA translation. tic models of hypertensive rats. This discrepancy In fact, translation at the ribosomal level is a needs to be considered in a proposed mechanism in calcium-dependent event (17) . An abnormality in PI which a calcium level induced by decreased PI turn- metabolism with a probable subsequent decrease in over results in hypertension in Dahl S rats. Two the cytosolic free calcium concentration could con- points are worthy of mention in this regard. Firstly, ceivably account for the observed decrease in PLA2 a decrease in PI turnover lowers protein kinase C translation. activity. This kinase is known to have dual effects We demonstrated that the PIP2-PLC concentra- on smooth muscle contraction (26). It phosphoryl- tion was significantly lower in both the VSMC and ates myosin light chain to facilitate smooth muscle the arterial walls from Dahl S rats. This observation contraction. On the other hand, it phosphorylates is confirmed as well by the fact that the IP3 content myosin light chain kinase, which lowers its affinity is lower in the VSMC from Dahl S rats. The reduc- to calcium calmodulin complex and is inhibitory to tion of cytosolic IP3 content and a subsequent de- the contraction mechanism. The decreased PI turn- crease in intracellular calcium mobilization would over and lowered protein kinase C activity disturbs be expected to lower PLA2 activity. Moreover, a this inhibitory mechanism, thereby leading to the decrease in the amounts of diacylglycerol, a bypro- elevation of blood pressure in Dahl S rats. In addi- duct of PI breakdown, leads to decreased free tion, it is possible that the calcium messenger sys- arachidonate via diacylglycerol (18). This tem following elevated cytosolic calcium is somehow mechanism may underlie the subnormal prostacyclin upregulated in Dahl S rats. The decreased PI turn- synthesis in Dahl S rats. The role of diacylglycerol over may be enough to signal smooth muscle con- Uehara et al: Phospholipase and Dahl Rats 111 traction. To further define these possibilities, it is eration in DOCA-salt hypertensive rats. Am J necessary to analyze alterations of the receptor- Hypertens 1991; 4: 667-673. 13. Chomczynski P, Sacchi N: Single-step method of GTP-binding protein-phospholipase C complex and the function of calcium-dependent contraction RNA isolation by acid guanidium thiocyanate- mechanisms. phenol-chloroform extraction. Anal Biochem 1987; 162: 156-159. 14. Merrick WC: Translation of exogenous mRNAs in Acknowledgments reticulocyte lysate. Methods Enzymol 1983; 101: 606- 615. The authors acknowledge Ono Pharmaceutical Co. Ltd., 15. Grynkiewicz G, Poenie M, Tsien RY: A new genera- Osaka, Japan for a gift of and related corn- tion of Ca indicators with greatly improved pounds. fluorescence properties. J Biol Chem 1985; 260: 3440-3450. 16. Karaki H, Sato K, Ozaki H, Murakami K: Effects of References sodium nitroprusside on cytosolic calcium level in vascular smooth muscle. Eur J Pharmacol 1988; 156: 1 Tobian L, Ganguli M, Johnson MA, Iwai J: In- 259-266. fluence of renal prostaglandins and dietary linoloate 17. Brostrom CO, Brostrom MA: Calcium-dependent on hypertension in Dahl S rats. Hypertension 1982; 4: regulation of protein synthesis in intact mammalian II-149-II-153. cell. Annu Rev Physio! 1990; 52: 577-590. 2 Uehara Y, Tobian L, Iwai J, Ishii M, Sugimoto T: 18. Williamson JR: Role of inositol lipid breakdown in Alterations of vascular prostacyclin and thromboxane the generation of intracellular signals. Hypertension A2 in Dahl genetical strain susceptible to salt-induced 1986; 8(suppl II): II-140-II-156. hypertension. Prostaglandins 1987; 33: 727-738. 19. Heagerty AM, Ollerenshaw JD, Swales JD: Abnor- 3 Falardeau P, Martineau A: In vivo production of mal vascular phosphoinositide hydrolysis in the spon- prostaglandin 12 in Dahl salt-sensitive and salt-resis- taneously hypertensive rat. Br J Pharmacol 1986; 89: tent rats. Hypertension 1983; 5: 701-705. 803-807. 4 Reid GM, Appel RG, Dunn MJ: Papillary collecting 20. Koutouzov S, Marche P, Girad A, Meyer P: Altered tubule synthesis of prostaglandin E2 in Dahl rats. turnover of polyphosphoinositides in the erythrocyte Hypertension 1988; 11: 179-184. membrane of the spontaneously hypertensive rats. 5 Craven PA, Studer RK, DeRubertis FR: Decreased Hypertension 1983; 5: 409-414. cytosolic calcium and prostaglandin synthesis in pre- 21. Bruschi G, Bruschi ME, Caroppo M, Orlandini G, hypertensive rats. Hypertension 1990; 15: 388-396. Spaggiari M, Cavatorta A: Cytoplasmic free (Ca2+ ) 6 Craven PA, DeRubertis FR: Calcium and prostaglan- is increased in platelets of spontaneously hyperten- din E2 in renomedullary interstitial cells. Hyperten- sive rats and essential hypertensive patients. Clin Sci sion 1991;17: 303-307. 1985; 68: 179-184. 7. Ross R, Glomset J, Kariya B, Harker L: A platelet- 22. Osanai T, Dunn MJ: Phospholipase C responses in dependent serum factor that stimulates the prolifera- cells from spontaneously hypertensive rats. Hyperten- tion of arterial smooth muscle cells in vitro. Proc sion 1992;19: 446-455. Nat! Acad Sci USA 1974; 71: 1207-1210. 23. Pace-Asciak CR, Carrara MC, Rangaraj G, Nicolaou 8 Uehara Y, Ishimitsu T, Kimura K, Ishii M, Ikeda T, KC: Enhanced formation of PGI2, a potent hypoten- Sugimoto T: Regulatory effects of eicosanoids on thy- sive substance, by aortic rings and homogenates of midine uptake by vascular smooth muscle cells of the spontaneously hypertensive rat. Prostaglandins rats. Prostaglandins 1988; 36: 847-857. 1978;15: 1005-1012. 9 Uehara Y, Numabe A, Kawabata Y, et a!: Rapid 24. Ishimitsu T, Uehara Y, Ishii M, Ikeda T, Matsuoka smooth muscle cell growth and endogeneous prosta- H, Sugimoto T: Alterations of the cardiovascular and glandin system in spontaneously hypertensive rats. renal prostaglandins and thromboxanes system in Am J Hypertens 1991; 4: 806-814. prehypertensive spontaneously hypertensive rats. Jpn 10. Numabe A, Uehara Y, Hirawa N, Takada S, Yagi S: Cir J 1989; 53: 307-312. Effects of thiazide diuretic on vascular eicosanoid sys- 25. Ishimitsu T, Uehara Y, Iwai J, et al: Vascular eicosa- tem of spontaneously hypertensive rats. J Hypertens noid production in experimental hypertensive rats 1989; 7: 493-499. with different mechanisms. Prostaglandins Leukotri 11 Uehara Y, Ishii M, Ishimitsu T, Sugimoto T: En- Essen 1991; 43: 179-184. hanced phospholipase C activity in the vascular wall 26. Kam KE, Stull JT: The function of myosin and of spontaneously hypertensive rats. Hypertension myosin light chain kinase phosphorylation in smooth 1988;11: 28-33. muscle. Annu Rev Pharmacol Toxicol 1985; 25: 593. 12. Uehara Y, Numabe A, Takada S, et al: Possible role of in altered prostacyclin gen-