Possible Role of Phosphorylation-Dephosphorylation in the Regulation of Calcium Metabolism in Cardiovascular Tissues of SHR

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1 Possible Role of Phosphorylation-Dephosphorylation in the Regulation of Calcium Metabolism in Cardiovascular Tissues of SHR RAMESH C. BHALLA, PH.D., RAM V. SHARMA, PH.D., AND S. RAMANATHAN, PH.D. SUMMARY Spontaneously hypertensive rats (SHR) and Wistar-Kyoto normotenslte rats (WKY) were compared for phosphorylatioa-depbosphorylation mechanlsm(s) in aorta, caudal artery, inferior vena cava, and right and left ventricles. Reduction of camp-induced pbospborylation of mlcrosomes and campdependent protein kinase activity was significant in the aorta and caudal artery of SHR compared with WKY. These changes were not observed in the vena cava of SHR. Pfaosphoprotein phosphatase activity was significantly increased (p < 0.05) in the soluble fraction of arterial smooth muscle. No changes were observed, however, in the myocardium or vein. Furthermore, the extent of pbospborylation, and Ca 1+ uptake ability and the protein kinase activity in the soluble and the microsomal fractions were not reduced in the myocardium of SHR compared with WKY. These data suggest that phosphorylation-depbosphorylation mechanisms are altered in the microsomal fraction of the aorta and caudal artery of SHR, which may result in reduced Ca l+ uptake by the intracellular organelle. The changes observed could have a significant effect on vasodilatation of arteries hi the hypertensive state. The lesion appears specific to the arterial smooth muscle in the cardiovascular tissues. (Hypertension 2: , 1980) KEY WORDS spontaneous hypertension membrane phosphorylation IN considering possible explanations for increased peripheral resistance, which may be regarded as a primary characteristic of essential hypertension, the physiological state of vascular smooth muscle cell appears pivotal. Vascular smooth muscle strips from hypertensive rats show reduced relaxation compared with the normotensive control rats after treatment with dibutyryl camp 1 or treatment with isoproterenol and theophylline 1 in washout experiments following contractility induced by KC1. S This defect in the relaxing ability of the vascular smooth muscle from hypertensive animals could lead to an increased vascular tonicity and peripheral resistance. An alteration in Ca s+ regulation has been postulated as a cause of increased vascular tone in the hypertensive animals and a decrease in the rate of relaxation. 1 ' * The removal of Ca 2+ from the From the Department of Anatomy and Cardiovascular Center, University of Iowa Medical School, Iowa City, Iowa. Supported in part by U.S. Public Health Service, National Institutes of Health Grants HL and HL Address for reprints: Ramesh C. Bhalla, Ph.D., Department of Anatomy and Cardiovascular Center, University of Iowa Medical School, Iowa City, Iowa Received June 14, 1979; revision accepted October 30, calcium heart and Mood vessels cytoplasm and consequent initiation of relaxation process in smooth muscle are probably accomplished by energy-dependent calcium transport in the intracellular organelles 5 "" and extrusion across plasma membrane. 9 Insight into the molecular mechanisms involved in the postulated role of camp-dependent protein kinase in Ca 2+ transport in the sarcoplasmic reticulum (SR) comes from the studies of Hicks, Shigekawa, and Katz. 10 The phosphorylation of the 22,000 dalton protein phospholamban of cardiac SR by campdependent protein kinase is shown to accelerate Ca 2+ sequestration Furthermore, the extent of phosphorylation corresponds closely with the increased rate of Ca 1+ transport. We have recently reported some aspects of regulation of Ca 1+ transport by rat aortic microsomes." We found that campdependent protein kinase augmented phosphorylation of microsomal protein, and phosphorylated microsomes exhibited enhanced Ca 1+ uptake. These data suggest a modulatory role for camp-dependent protein kinase in Ca 2+ transport in the vascular smooth muscle, and hence in the contractionrelaxation process. The microsomal fraction of the vascular smooth muscle of spontaneously hypertensive rats (SHR) 207

2 208 HYPERTENSION VOL 2, No 2, MARCH-APRIL 1980 showed a reduction in Ca 2+ uptake ability compared with normotensive controls. 16 " 18 A concomitant reduction in microsomal membrane phosphorylation has been observed in the cardiovascular tissues of SHR The molecular basis for altered Ca 1+ regulation in hypertensive animals could therefore be due to a defect in phosphorylation-dephosphorylation mechanisms. Present studies were undertaken to answer the following questions: 1) Is the decreased phosphorylation of microsomal membranes due to an increased activity of phosphoprotein phosphatase or to reduced activity of camp-dependent protein kinase? 2) Are the alterations in the activities of protein kinase, phosphoprotein phosphatases, and membrane phosphorylation specific to the systemic arteries and left ventricles, which are subjected to high "stressed wall" pressure, or do they generally appear in other cardiovascular tissues? Materials and Methods Adult male Wistar-Kyoto spontaneously hypertensive rats (SHR), and Wistar-Kyoto normotensive rats (WKY) weeks old, were used. The SHR maintained at the University of Iowa are inbred descendents of the hypertensive Wistar strain developed by Okamoto and Aoki. 11 The control rats were raised under conditions identical to those used for the hypertensive animals. Preoperative systolic blood pressures were determined in the unanesthetized state by the tail plethysmographic method, with an automated cuff inflator pulse-reading system manufactured by Technilab Instruments. The "Ca (about 11 mci/mg), [T- 3 2P] ATP (2-10 Ci/mmol), cyclic [ a H] AMP (about 16 Ci/mmol), and Aquasol were obtained from New England Nuclear. The camp, camp-dependent protein kinase, ATP, bovine serum albumin, and histone type II A were purchased from Sigma Chemical Company. Filters (0.45 pm, 25 mm in diameter) were obtained from Millipore Corporation. Preparation of Subcellular Fractions Animals were sacrificed under light ether anesthesia. The aorta, caudal artery, inferior vena cava, and right and left ventricular walls were immediately removed, trimmed of adherent fat and loose connective tissue, and rinsed in cold homogenizing buffer. The tissue was homogenized three times at 4 with a Polytron (Brinkman Instruments) at a rheostat setting of 3.0 for 10 seconds with a rest interval of 30 seconds. Microsomes from vascular smooth muscle were prepared according to the procedure of Fitzpatrick et al. 7 and from myocardial homogenate according to Harigaya and Schwartz." Ca 1+ Uptake and Binding by Subcellular Fractions The following mixture was used for calcium uptake measurements: 100 mm KC1, 5 mm oxalate, 5 mm MgCl,, 5 mm ATP, 15 mm sodium azide, 0.2 "Ca, 100 nm CaCl 2, 20 mm Tris HC1 ph 7.4, and ng of protein in a total volume of 1.0 ml. After incubation at 30 C for 10 minutes, the reaction was terminated by passing the mixture through Millipore filters (0.45 fim). The filters were washed by 5 ml of buffer, and the radioactivity on the filters was determined in a liquid scintillation counter. For examination of the Ca 2+ uptake ability of the phosphorylated microsomes, the microsomes were first phosphorylated in the presence of 5 /xm camp or 5 jtm camp and 0.1 mg camp-dependent protein kinase/ml for 10 minutes, and then an aliquot of the reaction mixture (approximately ng protein) was added to the assay medium for Ca 1+ uptake. Studies of Ca 1+ binding were carried out in essentially the same way as those of calcium uptake, except that the CaClj was 10 ^M and the oxalate was omitted. Measurement of Protein Kinase Histone type II A was used as substrate for protein kinase. Activity was measured in 0.12 ml containing 40 mm acetate (ph 6.0), 18 mm NaF, 3.7 mm theophylline, 0.25 mg histone, 18 mm MgCl,, 0.1 mm [y"p] ATP (2-6 X 10 s cpm), and 5 nm camp when added. The reaction was initiated by addition of fig protein from various tissue fractions and incubated at 30 C for 10 minutes. Reaction was terminated by addition of 2.0 ml 10% ice-cold trichloroacetic acid (TCA), and filtered through Millipore filters and washed three times with 5.0 ml 10% ice-cold TCA. Phosphorylation of Microsomal Protein Microsomal vesicles were phosphorylated in 200 nl of a solution containing 0.05 M Tris HC1, ph 7.4, 18 mm NaF, 22 mm MgCl,, 0.1 mm h<-' 2 P] ATP (3-5 X 10 s cpm), and ng of microsomal protein. The reaction was initiated by the addition of microsomes and incubated at 30 C for 10 minutes. The reaction was terminated by the addition of 10% ice-cold TCA and filtered through Millipore filters. Measurement of Phosphoprotein Phosphatase For phosphoprotein phosphatase assay, "P-histone (type II A) or 3 2 P-protamine were used as substrates. The S2 P-labelled substrates were prepared according to the methods of Meisler and Langan" and Maeno and Greengard." Briefly, the method consists of incubating histone and protamine with [T-"P] ATP in the presence of camp-dependent protein kinase. One ml of the incubation mixture contained 46 ng protein kinase; 50 ^mol sodium acetate buffer, ph 6.4; 1 mg protamine or histone; 0.2 /xrnol of ATP (0.1 /xmol ATP for protamine), >- S2 P-ATP (5.10 X 10 8 cpm); 10 jtmol of magnesium acetate; 10 /xmol of sodium fluoride; 2.0 ^mol of theophylline; 0.3 fimo\ of ethylene glycol bis 08-amino ethylene) N-tetracetic acid; and 5.0 nmol of camp. The mixture was incubated at 37 for 45 minutes and the reaction was terminated by adding 0.25 ml of 100% TCA. The

3 PHOSPHORYLATION-DEPHOSPHORYLATION AND Ca METABOLISM/fl/iatfa et al. 209 resulting precipitate was centrifuged, washed two times by suspending it in water and reprecipitating with 20% TCA, and then dialyzed against distilled water. The amount of phosphate incorporated was calculated from the "P-phosphate incorporated. The "P-histone contained 25 nmol of "P/mg histone, and 32 P protamine contained 8 nmol of "P/mg protamine. For the measurement of phosphoprotcin phosphatase activity, the reaction mixture in 0.15 ml contained 50 mm MgCl 2, 1 mm dithiothreitol, 100 Mg 32 P-labeled substrate, and /ig tissue fraction. The incubation was carried out at 30 C for 10 minutes, and the reaction was terminated by adding 0.4 ml of 25% TCA and 0.1 ml of 0.625% bovine serum albumin. After centrifugation, 0.4 ml of supernatant was added into tubes containing 50 fi\ of 100 mm KH 2 PO 4 and 150 n\ of 5% ammonium molybdate. The phosphomolybdate complex was extracted with 1.0 ml of isobutanol, and the radioactivity in 0.5 ml of isobutanol was counted. g»c4.o co 3.0 o w oo 1 - I 4 <o is OS 500 E400 1 Right Left Ventricle Ventricle B xjr Results The average blood pressure of SHR was 170 ± 8 mm Hg as compared with 140 ± 6 mm Hg for WKY. Biochemical characterization of the microsomal fraction, as reported earlier" for the vascular smooth muscle, was carried out by the determination of cytochrome oxidase activity. The activities per milligram of protein were less than 8% of that in the mitochondrial fraction and there were no differences between SHR and WKY. Electron micrographs of the microsomal preparation from myocardium showed that this fraction consisted of smooth membrane vesicular structures and was devoid of contractile proteins. No intact mitochondrial fragments could be identified in this preparation. There was no significant difference in the yield of microsomal protein for the caudal artery, aorta, right ventricle, and left ventricle between hypertensive and normotensive control rats. Determinations of microsomal Ca 2+ uptake and binding and protein kinase activities were carried out under conditions of linearity with respect to time of incubation and protein concentration. Calcium Binding and Uptake We have shown earlier 1 " 1 ie that calcium uptake by microsomal vesicles isolated from aortae of hypertensive rats was significantly reduced (p < 0.05) compared to normotensive controls. In contrast, Ca 2+ binding (fig. 1A) and Ca J+ uptake (fig. IB) in the sarcoplasmic reticulum (SR) isolated from right or left ventricles of hypertensive rats were not changed in SHR compared with the normotensive controls. Similar results were obtained when Ca 1+ uptake was studied in the SR phosphorylated in the presence of 5 nm camp or 5 ^M CAMP and 0.1 mg/ml campdependent protein kinase. Comparison of the Ca 2+ binding and uptake ability between left and right ventricles of the same type of animal showed consistently higher values for the left ventricles than for the right ventricles. c 100 CAMP (-) Proton/\ / \ Right Ventricle ght Ventricle FIGURE 1. Calcium binding (A) and calcium uptake (B) in the sarcoplasmic reticulum (SR) isolated from right and left ventricular walls of spontaneously hypertensive rats (shaded bars) and Wistar-Kyoto normotensive rats (open bars) in the presence and absence of camp and camp plus campdependent protein kinase. Each value is the mean ± SE of five experiments. For each experiment SR were prepared from the myocardium of four to five rats. camp-dependent and -Independent Protein Kinase Activity The camp-dependent and -independent protein kinase activities were determined in the microsomes (fig. 2A) and soluble fraction (100,000 X g supernatant; fig. 2B) obtained from homogenates of vascular smooth muscle. In hypertensive rats, campdependent protein kinase activities were significantly (p < 0.05) reduced both in the aorta and caudal artery. The magnitude of differences was marked in the caudal artery, which is a muscular artery compared to aorta. Further, the extent of stimulation by camp was also reduced in the hypertensive rats. These changes were not observed in the inferior vena cava. There were no significant differences in the camp-dependent and -independent protein kinase activity in the soluble fraction of right and left ventricles of hypertensive rats as compared to normotensive rats (fig. 3). Contrary to the observations in the aorta and caudal artery, the protein kinase activity was higher in the microsomal fraction isolated from the left ventricle of the hypertensive rats compared to normotensive rats.

4 210 HYPERTENSION VOL 2, No 2, MARCH-APRIL 1980 o> 0.6 o c c o c CAMP (-) Aorta to S2 3.0 g-ffl 2.0? o c I 1 Aorta Caudal Artery 1 ) (+) Caudal Artery Vena Cava FIGURE 2. camp-dependent and -independent protein kinase activity in the microsomal fraction (A) and X g supernatant (B) of the aorta and caudal artery of spontaneously hypertensive (shaded bars) and control (open bars) Wistar-Kyoto normotensive rats. Each value is the mean ± SE for five experiments. For each experiment, the subcellular fractions were prepared from tissues collected from rats. 'Significantly different (p < 0.05) from normotensive control. o 6.0 CD Q. 5.0.C ^ 3.0 o 8- o c a a "5 c CAMP (-) (+) Soluble Right Ventricle Microsomes camp-stimulated Pbospboryiatioa of Microsomes The camp-dependent and -independent phosphorylation of SR isolated from right and left ventricles is shown in fig. 4. In the SR isolated from the right ventricle, there were no differences between hypertensive and normotensive rats either in the extent of stimulation achieved over the basal value by 1 nm camp or in the levels of phosphorylation. In the SR of the left ventricle, however, U P incorporation in the absence and presence of 5 nm camp was significantly higher in the hypertensive rat as compared to the normotensive rat. The net stimulation by camp was 15%-20% over the basal value. Phospfaoprotein Phosphatase Actirity We compared phosphoprotein phosphatase activity of microsomes and soluble fraction isolated from vascular smooth muscle and also in the right and left ventricles from both the groups. The enzyme activity was consistently increased in the soluble fraction of the aorta and caudal artery of SHR compared with WKY, with significant differences (p < 0.05) observed in the caudal artery (fig. 5A). No changes were observed, however, in the inferior vena cavae (fig. 5A), or in the right and left ventricles (fig. 5B). Addition of 1 mg/ml bovine heart protein kinase to the phosphoprotein assay mixture did not alter the observed differences (data not given). Phosphoprotein phosphatase activity in the soluble fraction of caudal artery was found to be linear in the range of 2-20 minutes and ng protein. At all incubation intervals tested, and at protein concentrations from 10 ^g and higher, the phosphatase activity was consistently i Soluble Left Ventricle,X* Microsomes FIGURE 3. camp-dependent and -independent protein kinase activity in the sarcoplasmic reticulum and 100,000 X g supernatant of right and left ventricles of spontaneously hypertensive rats (shaded bars) and normotensive (open bars) Wistar-Kyoto control rats. Each value is the mean ± SB for five experiments. 'Significantly different (p < 0.05) from respective control.

5 PHOSPHORYLATION-DEPHOSPHORYLATION AND Ca METABOLISM/flAa//a et al. 211 ll » 5 2i CAMP (-) (+) Right Ventricle X (-) (+) Left Ventricle FIGURE 4. camp-dependent and -independent phosphorylation of sarcoplasmic reticulum isolated from right and left ventricular walls of spontaneously hypertensive (shaded bars) and normotensive (open bars) Wislar-Kyolo rats. Each value is the mean ± SE for five experiments. In each experiment, tissues from five to six rats were pooled. 'Significantly different (p < 0.05) from normotensive controls. 8 5-^2.0 O).0 i.o- 5^ 0> CD. ttq-2. 0 X I A ffl Soluble Mlcrosomes Soluble Microsomes Soluble B Aorta Soluble Microsomes Right Ventricle Caudal Artery m Soluble Mlcrosomes Left Ventricle Vena Cava FIGURE 5. Phosphoprotein-phosphatase activity of the aorta, caudal artery, inferior vena cava (A), and right and left ventricular walls (B) of spontaneously hypertensive (shaded bars) and control Wistar-Kyoto normotensive (open bars) rats. Values are mean ± SE of five separate experiments carried out in duplicate. *Significantly different (p < 0.05) from respective controls. In each experiment, left and right ventricles were pooled from five to six rats and the aorta, caudal artery and vena cava from rats. increased in hypertensive rats compared with normotensive controls (fig. 6). The increase in phosphoprotein phosphatase activity in the caudal artery of SHR was due to an increase in V^, rather than K m. The V mm values for phophohistone and phosphoprotamine were doubled in SHR compared with WKY (table 1) with no change in apparent K m. The enzyme activity was determined in the supernatant and the sarcoplasmic reticulum preparations in the presence of several divalent cations. Divalent cations Mn 2+, Mg 3+, and Ca 2+ stimulated phosphatase activity in the vascular smooth muscle and in cardiac muscle (data not given). In all the preparations tested, Mn l+ was the most potent stimulator of the divalent cations. The enzyme activity was significantly higher in the supernatant fraction of the caudal artery of SHR as compared with WKY with all the divalent cations tested; however, no differences were observed in the vena cava and myocardium between the two groups. Discussion A defect has been reported 1 ' in the relaxing ability of the vascular smooth muscle from hypertensive animals after treatment with dibutyryl camp and isoproterenol. Physiological and pharmacological regulation of contraction or relaxation of vascular smooth muscle is determined by the concentration of activator Ca a+ in the sarcoplasm. The removal of Ca 2+ from the cytoplasm and consequent initiation of relaxation in smooth muscle involves two important systems: 1) sequestration into intracellular structures, including both mitochondria and sarcoplasmic reticulum; and 2) efflux across the plasma membrane. Abnormalities in each of these mechanisms have been implicated in the pathogenesis of hypertension '"-" From studies on microsomal fraction of smooth muscle, Ford and Hess 28 concluded that the microsomes sequestered sufficient Ca 2+ and that this fraction has the capability to be both sink and source for the activator Ca 2+ in excitation-contraction coupling. Observations made in this laboratory" and by others 8 "' demonstrate that incubation of smooth muscle microsomal fraction with camp-dependent protein kinase enhances energy-dependent Ca l+ sequestration, which suggests a modulatory role for camp-dependent protein kinase in Ca 2+ transport in vascular smooth muscle. Previous observations of reduced Ca 1+ uptake by aortic microsomes of SHR compared with WKY 1* "" led us to investigate further the biochemical basis for this defect. Our results indicate that camp-dependent protein kinase activity in the microsomal and soluble fractions of the caudal artery and aorta of SHR was significantly reduced compared with those of WKY. The stimulation of protein kinase activity by camp was also reduced in SHR. This could result in a decrease in the expression of camp message due to an inherent or induced defect in camp-dependent protein kinase mediated membrane phosphorylation.

6 212 HYPERTENSION VOL 2, No 2, MARCH-APRIL 1980.C 5 B s2.0 CO CD D1.0 CO co CD Time (min.) Protein (LKJ) FIGURE 6. Phosphohistone phosphatase activity of soluble fraction from caudal artery of spontaneously hypertensive (closed circles) and control Wistar-Kyolo normotensive (open rectangles) rats. Reactions were carried out as described in Methods for indicated times (A) or with indicated amounts of protein (B)for 10 minutes. Results are mean values of two independent determinations in duplicate. In each experiment, the caudal artery from rats were pooled. 100 Another mechanism that could influence the net membrane phosphorylation and Ca 1+ metabolism is a change in the dephosphorylation process, probably due to the altered activity of phosphoprotein phosphatase. Dephosphorylation of phosphorylated cardiac sarcoplasmic reticulum has been shown to be catalyzed by both membrane associated 10 "" and soluble phosphoprotein phosphatase. It was, further, demonstrated that dephosphorylation of the 22,000 dalton phosphoprotein of cardiac sarcoplasmic reticulum catalyzed by an intrinsic phosphoprotein phosphatase was associated with a decrease in the rate of Ca 2+ transport by these membranes. 30 It has been suggested that similar mechanisms may be regulating Ca 2+ metabolism in vascular smooth muscle. 18 Our results indicated an increase in the phosphoprotein phosphatase activity in the soluble fraction of arterial smooth muscle of SHR compared with that of WKY (figs. 5 and 6). The increase in activity was associated with a change in V ma,. The combined effect of these changes, i.e., a decrease in protein kinase activity and an increase in phosphoprotein phosphatase activity, could result in a reduced membrane phosphorylation and Ca 2+ uptake by membrane vesicles in the arterial smooth muscle cell. Recently, Kuo et al. si reported the role of "wall stress" of blood vessels on the levels of cgmp- and camp-dependent protein kinase(s). To test the effect of intravascular pressure on camp-dependent and -independent protein kinases, comparisons were also made between inferior vena cava of SHR and WKY. Since we did not find differences in the campdependent and -independent protein kinases and phosphoprotein phosphatase activity in the inferior vena cava, it is suggested that the lesion is specific to the arterial smooth muscle. Furthermore, in the left and right ventricles we observed no differences between SHR and WKY for Ca J+ uptake by SR, camp-dependent and -independent protein kinase, and phosphoprotein phosphatase activity in soluble and microsomal fractions. Limas and Cohn, 10 however, have reported reduced Ca 2+ uptake and a TABLE 1. Kinetic Constants for Phosphoprotein Phosphatase in Soluble Fraction of Caudal Artery* WKY SHR WKY SHR Substrate K m y mg/ml p,m["p] mg/ml AlM["P] His tone Protamine *Each value is the mean of two determinations. ;JM "p] refers to molarity of bound "p. V^ is expressed as nm "P released per mg protein/min at 30. WKY Wistar-Kyoto normotensive rats; SHR = spontaneously hypertensive rat

7 PHOSPHORYLATION-DEPHOSPHORYLATION AND Ca METABOLISM/BAa//a et al. 213 decrease in membrane phosphorylation and protein kinase activity in the SR isolated from the myocardium of SHR compared with WKY. The reason for this discrepancy is hard to explain. There appear to be at least two possible reasons: 1) source of hypertensive and control animals; and 2) ventricular wall employed in the present study compared with the whole heart. Biochemical observations made in the present investigation correlate well with the hemodynamic responses and myocardial function in SHR. Pfeffer and Frohlich 82 ' " have shown that cardiac output in 9- to 12-week SHR was about 22% above that of agematched normotensive controls. Similar observations have been made for other experimental models of hypertension. 34 Our data of increased SR membrane phosphorylation and protein kinase activity in the left ventricle of SHR compared with WKY would provide a basis for observed increased cardiac output and myocardial function in the hypertensive state. These data suggest that alteration in phosphorylation-dephosphorylation mechanisms of SR of arterial smooth muscle has occurred in SHR. Further, these changes are specific for arterial smooth muscle among the cardiovascular tissues. In smooth muscle, increased intracellular levels of camp inhibit tension development. This effect has been attributed to enhanced Ca 1+ binding by membrane fractions, thereby lowering intracellular free Ca 1+." The data presented here are suggestive of changes that could influence calcium metabolism in the vascular smooth muscle in such a manner that it could result in an increase in free Ca I+ levels in vascular smooth muscle in the hypertensive state. These results, however, should be taken with caution because they do not unequivocally demonstrate a reduced Ca 1+ sequestering ability, with the implication of higher cytoplasmic [Ca 1+ ] and increased tone in the hypertensive animals. Another possible mechanism that could imply a direct role of the changes observed in protein kinase activity in regulating smooth muscle tone comes from the observations of Adelstein et al.* 6 They have demonstrated that a camp-dependent protein kinase in smooth muscle can phosphorylate the myosin light chain kinase." This decreases the activity of the myosin light chain kinase, and therefore the degree of myosin phosphorylation that would decrease Ca 1+ stimulated interaction between actin and myosin."" 39 Thus it is possible that the decreased campdependent protein kinase activity in the vascular smooth muscle of hypertensive animals might be associated with a more active myosin light chain kinase, more phosphorylated myosin, and a higher level of tone. It will be of interest to learn whether these enzymes are altered in the hypertensive state. References 1. Cohen ML, Berkowitz BA: Decreased vascular relaxation in hypertension. J Pharmacol Exp 196: 396, Triner L, Vulliemoz Y, Verosky M, Manger WM: Cyclic adenosine monophosphate and vascular reactivity in spontaneously hypertensive rats. Biochem Pharmacol 24: 743, Bhalla RC, Sharma RV, Webb RC: Possible role of camp and calcium in the pathogenesis of hypertension. 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