Increased Expression and Activity of Phospholipase C in Renal Arterioles of Young Spontaneously Hypertensive Rats

AJH

2007; 20:38 – 43

Kidney

Increased Expression and Activity of Phospholipase C in Renal Arterioles of Young Spontaneously Hypertensive Rats Zhangping Peng, An Dang, and William J. Arendshorst Background: The aims of this study were to document the presence of phospholipase C (PLC) isozymes ␤1, ␥1, and ␦1 in freshly isolated renal glomeruli and resistance vessels, to compare their expression and activity to that in aorta, and to contrast values between 6-week-old Wistar-Kyoto (WKY) controls and 6-week-old spontaneously hypertensive rats (SHR) during the developmental phase of genetic hypertension. Methods: Aorta, preglomerular arterioles, and glomeruli were isolated from 6-week-old rats using standard techniques. PLC isozyme protein level and activity were determined with Western blot analysis and by measuring inositol 1, 4, 5-trisphosphate (IP3) production, respectively. Results: Immunoblots indicate that all three PLC isozymes examined are detectable in freshly isolated preglomerular arterioles, glomeruli, and aorta. Increased levels of PLC-␤1, and -␦1 were found in all tested vascular

tissues of SHR v WKY. No strain difference was noted for PLC-␥1. The relative abundance for both groups was glomeruli ⬎ preglomerular arterioles ⫽ aorta. The strain difference in protein expression correlated with increased PLC activity in each vascular bed of SHR. Conclusions: Protein levels of PLC-␤1 and -␦1 and PLC activity are upregulated in the systemic and renal vasculature in 6-week-old SHR, suggesting a role in exaggerated vascular reactivity during the development of genetic hypertension. A more complete understanding of the physiologic roles of PLC isozymes and their contributions to specific aspects of cellular function should advance our understanding of vascular tone/reactivity and hypertrophy/remodeling in normal and hypertensive states. Am J Hypertens 2007;20:38 – 43 © 2007 American Journal of Hypertension, Ltd. Key Words: Genetic hypertension, renal circulation, afferent arteriole, glomerulus, SHR.

hospholipase C (PLC) is an ubiquitous enzyme family that is important in signal transduction from cell surface G-protein coupled receptors (GPCRs) and growth factor receptor tyrosine kinases to many intracellular targets. Ligand interaction with various plasma membrane receptors activates PLC members through different signaling molecules that result in a variety of functions.1–3 In general terms, PLC-␤ is widely expressed and is primarily regulated by GTP-bound ␣ and ␤␥ subunits of heterotrimeric G-proteins released from heptahelical GPCRs. Of this family, PLC-␤1 is the most sensitive to Gq␣ subunits and regulators of G-protein signaling. The PLC-␥1 is principally activated by phosphoinositol 1,4,5-trisphosphate (IP3) and Src family tyrosine kinases and phosphorylated tyrosine residues secondary to stimulation and dimerization of polypeptide growth factor receptor tyrosine kinases.

P

Downstream signaling typically involves PI3 kinase, IP3, and Ca2⫹. Nonreceptor protein tyrosine kinases also activate PLC-␥. PLC-␦ is the most sensitive to cytosolic Ca2⫹ and phosphoinositol 4,5-bisphosphate. Many of the actions of constrictor agents acting through GPCRs on vascular smooth muscle cells (VSMC) are mediated by signaling involving PLC-mediated production of IP3 and diacyl glycerol (DAG), resulting in subsequent Ca2⫹ mobilization and activation of protein kinase C (PKC), respectively. This is a dominant initiating pathway in many cell types that include vascular and cardiac myocytes as well as renal mesangial and epithelial cells.2–5 Dysregulation of this signaling cascade may cause or contribute to changes in vascular reactivity and structural remodeling that occur in models of genetic hypertension and humans with essential hypertension.5– 8

Received April 13, 2006. First decision May 18, 2006. Accepted June 5, 2006. From the Department of Cell and Molecular Physiology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina. This work was supported by Research Grant HL-02334 from the

Heart Lung and Blood Institute of the National Institutes of Health, Bethesda, Maryland. Address correspondence and reprint requests to Dr. William J. Arendshorst, 6341-B, Department of Cell and Molecular Physiology, CB # 7545 School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7545; e-mail: [email protected]

0895-7061/07/$32.00 doi:10.1016/j.amjhyper.2006.06.009

© 2007 by the American Journal of Hypertension, Ltd. Published by Elsevier Inc.

AJH–January 2007–VOL. 20, NO. 1

PLC ISOZYMES IN PREGLOMERULAR RESISTANCE ARTERIOLES

Elevated PLC-␤ levels and activity may account for the previously well-documented exaggerated renal vasoconstriction and Ca2⫹ signaling in preglomerular arterioles produced by angiotensin II (Ang II), thromboxane A2 (TxA2), and vasopressin (AVP) in 6-week-old spontaneously hypertensive rats (SHR) versus normotensive animals.9 –17 Moreover, enhanced activity of PLC-␥ and downstream PI3 kinase and Akt may explain enhanced proliferation of VSMC of genetically hypertensive rats.18 –21 Available data on PLC expression in the vasculature of SHR is based almost exclusively on studies of cultured aortic VSMC. There is no comparable information for small diameter resistance arterioles of any vascular bed, either freshly isolated or cultured. It is important to know about the expression and activity of PLC isozymes in the most physiologically relevant resistance arterioles ⬍100 ␮m in diameter, because they critically regulate organ blood flow and total peripheral vascular resistance in health and disease. Little is known about the role of PLC in the initiating phase of hypertension and its contribution to enhanced vascular reactivity in the critical developmental phase, which in SHR occurs between 5 and 8 weeks of age. We tested the hypothesis that there are genetic differences in PLC expression and activity in SHR compared with agematched Wistar-Kyoto (WKY) and these differences are evident in young SHR and thus seem likely to be primary to the development of hypertension. The present study had two objectives. One was to document the presence of PLC isozymes ␤1, ␥1, and ␦1 in freshly isolated vascular preparations and to compare the relative abundance among preglomerular arterioles, glomeruli, and aorta of normotensive WKY. A second goal was to compare PLC expression and activity in freshly isolated renal resistance vessels with that of the conduit aorta of 6-week-old SHR during the developmental phase of genetic hypertension versus WKY controls.

39

Western Blot Analysis We tested for the presence of PLC-␤1, -␥1, and -␦1. These isoforms were targeted because they are commonly characterized in the vasculature and the kidney and provide comparisons with previous studies.24 –31 For Western blot analysis,23 the concentration of protein from vascular preparations was determined using a BCA Protein Assay Kit (Pierce, Rockford, IL) following the manufacturer’s protocol. Samples were boiled for 5 min. Equal amounts of whole cell lysate protein (10 ␮g/lane) were separated by 10% sodium dodecyl sulfate (SDS)– polyacrylamide gel and electrotransfered to nitrocllulose (BioRad, Hercules, CA). Membranes were blocked with 5% (w/v) nonfat milk powder in Tris-buffered saline containing 0.05% (v/v) Tween-20 (pH 7.6, TBST) overnight at 4°C, and incubated in paired fashion with affinity purified rabbit polyclonal anti-PLC-␤1 (SC-205), or PLC-␥1 (SC-81) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), or mouse monoclonal anti-PLC-␦1 (5-343, Upstate Biotechnology, Lake Placid, NY), and mouse monoclonal anti-␤-actin antibodies (A-5441, Sigma, St. Louis, MO) for 2 h at room temperature. These antibodies are specific for designated PLC isozymes and have been used often to characterize their relative abundance in the vasculature and renal tissue.27,31–33 Dilutions were 1:500 for PLC isozymes and 1:50,000 for ␤-actin. After washing in TBST, the blots, paired for PLC isozymes and ␤-actin, were incubated for 2 h at room temperature with goat antirabbitand antimouse horseradish peroxidase (HRP) IgGs (Santa Cruz and Upstate Biotechnology, respectively); dilutions were 1:1000 for PLC isozymes and 1:2000 for ␤-actin. The blots were extensively washed in TBST and band intensities were detected using Chemiluminescence Luminol reagent (Santa Cruz Biotechnology) with exposed to film for 1 to 2 min and developed. Blots were quantified using an Image Station 440 CF (Kodak Digital Science, Rochester, NY). Preliminary studies established that these conditions resulted in band intensities in the mid-range of linearity.

Methods

PLC Activity Assay

Experiments were performed with 6-week-old WKY and SHR from our Chapel Hill breeding colony in compliance with the guidelines and practices of University of North Carolina at Chapel Hill Institutional Animal Care and Use committees. For each experiment, two to three rats were anesthetized by intraperitoneal injection of pentobarbital sodium. Mean arterial pressure (MAP) was measured in some animals through a femoral arterial catheter connected to a Statham pressure transducer.9,10 Glomeruli and preglomerular arterioles (interlobular artery and afferent arterioles) were isolated using techniques standard for our laboratory.9,11,22 The media of aorta was separated from endothelial lining and perivascular adventia.23

The PLC-specific activity in whole cell lysates was measured by IP3 production after hydrolysis of [3H]phosphatidylinositol 4,5-bisphosphate (PIP2).32,33 Briefly, 80 ␮g of protein (50 ␮L) was mixed with 50 ␮L of reaction buffer containing 100 mmol/L NaCl, 0.6% sodium deoxycholate, 2 mmol/L CaCl2, 4 mmol/L EGTA, 20 mmol/L Tris-HCl, pH 7.0 and [3H]-PIP2 (specific activity: 6.5 Ci/ mmol, 100,000 disintegrations per minute (dpm)/reaction, PerkinElmer Life Sciences, Inc., Boston, MA). Samples were incubated at 37°C for 30 min. The reaction stopped by addition of 100 ␮L of ice-cold chloroform:methanol:12 N HCl (100:100:6, v/v/v). Of the upper aqueous layer, 100 ␮L were transferred to 6 mL of scintillation cocktail (Economical Safety LSC-cocktail, Fisher Scientific, Fair Lawn, NJ) and radioactivity was determined using a liquid

40

PLC ISOZYMES IN PREGLOMERULAR RESISTANCE ARTERIOLES

AJH–January 2007–VOL. 20, NO. 1

FIG. 1. (Top) Representative Western blots of PLC-␤1 (150 kDa), -␥1 (145 kDa), and -␦1 (85 kDa) and ␤-actin (42 kDa) protein expression in freshly isolated glomeruli, preglomerular arterioles, and aorta of 6-week-old WKY and SHR. (Bottom) Group means ⫾ SEM for the relative densities of PLC isozymes (percentage of WKY; n ⫽ 4 to 8 preparations per group). WKY values are given in Table 1. Strain difference: *P ⬍ .05, **P ⬍ .01, ***P ⬍ .001 v WKY.

scintillation counter (Wallace 1414, Shelton, CT). Data are presented as disintegrations per minute per milligram of protein per minute of incubation. Data Analysis Statistical analyses of results among groups were performed by ANOVA, with the post-hoc test of Holm-Sidak (Sigma-Stat). The PLC differences within rat strain were analyzed by Student t test as samples of both SHR and WKY were paired in each determination. A P value ⬍ .05 was considered statistically significant.

Results The MAP tended to be higher in anesthetized 6-week-old SHR, but was not statistically different from that in WKY (113 ⫾ 5 v 106 ⫾ 2 mm Hg, respectively; n ⫽ 5 each; P ⬎ .05). These values agree with values obtained from larger groups in previous studies.9,10 Expression of PLC Isozyme Protein Immunoblots indicate that all three PLC isozymes examined are detectable in freshly isolated glomeruli, preglomerular arterioles, and aorta of these rat strains at 6 weeks

of age. Representative blots are shown in Fig. 1 (top). Table 1 summarizes data for group averages of the density of each PLC isozyme examined normalized to ␤-actin in each sample, and ratios of values in SHR to those for WKY. Group means for SHR values normalized to those in WKY are shown graphically in Fig. 1 (bottom). Our results indicate that protein levels for PLC-␤1 and PLC-␦1 are significantly higher in the vasculature of SHR versus WKY. No strain difference was noted for PLC-␥1. The PLC isozyme levels in WKY were roughly two- to fourfold greater in glomeruli than preglomerular arterioles and aorta, with the latter two being similar to each other (WKY values; Table 1). Absolute values for ␤-actin did not differ markedly between vessel preparations or rat strain, except for the fact that ␤-actin levels were 15% higher in glomeruli than in preglomerular arterioles of WKY (Table 1). Within SHR, the density of ␤-actin was similar among vessels. Relative to WKY, SHR tended to have 13% more ␤-actin on average, a nonstatistically significant difference. PLC Activity The PLC specific activity in whole cell lysates was measured by IP3 production. Our results indicate that overall

AJH–January 2007–VOL. 20, NO. 1

PLC ISOZYMES IN PREGLOMERULAR RESISTANCE ARTERIOLES

41

Table 1. Comparisons of PLC isozyme protein levels and PLC activity in freshly isolated glomeruli, preglomerular arterioles, and aorta of 6-week-old WKY and SHR Protein levels PLC-␤1 WKY (density PLC-␤1/␤-actin) WKY (v preglomerular arterioles) SHR (% of WKY) SHR v WKY PLC-␥1 WKY (density PLC-␥1/␤-actin) WKY (v preglomerular arterioles) SHR (% of WKY) SHR v WKY PLC-␦1 WKY (density PLC-␦1/␤-actin) WKY (v preglomerular arterioles) SHR (% of WKY) SHR v WKY ␤-actin WKY (density) WKY (v preglomerular arterioles) SHR (density) SHR v WKY PLC activity WKY (dpm/mg/min) WKY (v preglomerular arterioles) SHR (% of WKY) SHR v WKY

Glomeruli

Preglomerular arterioles

Aorta

0.55 ⫾ 0.05 (8) ⬍0.001 114 ⫾ 3 (6) ⬍0.01

0.16 ⫾ 0.01 (6)

0.13 ⫾ 0.04 (4) NS 219 ⫾ 28 (4) ⬍0.05

0.50 ⫾ 0.09 (5) ⬍0.001 99 ⫾ 5 (5) NS

0.23 ⫾ 0.03 (4)

0.32 ⫾ 0.06 (8) ⬍0.001 172 ⫾ 26 (8) ⬍0.05

0.08 ⫾ 0.01 (5)

125 ⫾ 5 (6) ⬍0.01

90 ⫾ 12 (4) NS

186 ⫾ 29 (5) ⬍0.05

643 ⫾ 81 (5) ⬍0.001 707 ⫾ 88 (5) NS

546 ⫾ 63 (5)

4222 ⫾ 532 (3) NS 194 ⫾ 29 (3) ⬍0.01

2355 ⫾ 235 (4)

643 ⫾ 54 (5) NS

179 ⫾ 20 (4) ⬍0.01

0.26 ⫾ 0.08 (4) NS 109 ⫾ 19 (4) NS 0.19 ⫾ 0.07 (6) NS 390 ⫾ 57 (6) ⬍0.001 627 ⫾ 99 (6) NS 703 ⫾ 71 (6) NS 3892 ⫾ 397 (5) NS 157 ⫾ 11 (5) ⬍0.01

NS ⫽ not significant.

enzymatic activity of PLC was 50% to 100% higher in the vasculature of SHR. This strain difference was observed for glomeruli, preglomerular arterioles, and aorta (Fig. 2). Absolute values for WKY vessels are presented in Table 1. Basal activity was greatest and similar in glomeruli and aorta.

Discussion A goal of our study was to document the presence and compare the relative abundance of PLC-␤1, -␥1, and -␦1 between freshly isolated renal preglomerular resistance

FIG. 2. Group means ⫾ SEM for the total activity of PLC (percentage of WKY; n ⫽ 3 to 5 preparations per group). WKY values are given in Table 1. Strain difference: **P ⬍ .01 v WKY.

arterioles, glomeruli, and aorta. In this regard, our studies provide a comprehensive characterization of selected PLC isozymes in small diameter resistance arterioles versus a large diameter systemic conduit vessel and also the glomerular capillary bed. Our new information indicates a similarity of protein levels of PLC-␤1 and -␥1 in fresh renal arterioles (interlobular arteries and afferent arterioles) and the aorta in normotensive control rats, with roughly two- and fourfold more abundance in glomeruli. The PLC-␦1 tended to be greater in aorta than in preglomerular arterioles of WKY. A related aim was to compare PLC expression and activity in fresh conduit and resistance vessels between 6-week-old SHR during the developmental phase of genetic hypertension versus normotensive WKY. An important finding in the present study was that these vessels of SHR have greater amounts of PLC-␤1 and PLC-␦1 than those of WKY, with larger strain differences noted for aorta. Paralleling these protein levels was overall PLC activity, which was greater in SHR developing hypertension at 6 weeks of age. This was the case for IP3 production by preglomerular arterioles and glomeruli as well as aorta. These data are the first on PLC isozyme expression in fresh preglomerular resistance vessels of normotensive animals and of animals prone to genetic hypertension. Preglomerular arterioles are a major site of intrarenal vascular resistance that contributes significantly to the regulation of glomerular filtration rate, renal blood flow,

42

PLC ISOZYMES IN PREGLOMERULAR RESISTANCE ARTERIOLES

and excretion of salt and water. Well-documented exaggerated renal vascular reactivity to vasoconstrictor agents such as Ang II, AVP, and TxA2 characterizes renal function in 6-week-old SHR during the development of genetic hypertension.9,10,12,16,17 Postreceptor signaling may be involved in the pathogenesis and maintenance of hypertension.5,11,12,14,15 Principal controllers of vasomotor tone include PLC and its products IP3 and DAG, which trigger increases in cytosolic Ca2⫹ concentration and PKC activation. The Ca2⫹ signaling in preglomerular arterioles of young SHR is exaggerated in response to Ang II, AVP, and norepinephrine.11–15 In view of this background, we postulated that PLC activity is enhanced in the renal vasculature of young SHR. We targeted the PLC-␤1, -␥1, and -␦1 isoforms as being representative and they are commonly characterized in the vasculature and the kidney, thus allowing comparisons with the literature.24 –28 Previous studies have identified PLC isozymes in the general renal regions of the cortex and medulla, with some localization to glomeruli and nephron segments of normotensive rats. Very little is known about subtypes along the renal vasculature in the cortex in the functionally relevant resistance vessels, specifically the interlobular arteries and the afferent and efferent arterioles. Immunohistochemistry of kidney slices reveals the presence of PLC-␥1 and -␦1 in specific nephron segments but not in glomeruli, with no mention of any staining along arteries or arterioles.24 Another immunohistochemical study reports that PLC-␥1 is the major PLC in the main renal artery, where it is limited to the internal elastic region rather than VSMC in the media.25 The PLC-␤1 and -␦1 appear to predominate in vasa recta in the renal medulla. However, no immunohistochemical staining for PLC-␤1 or -␦1 was seen in renal cortical arteries and arterioles. Western blots reveal the presence of PLC␤1, -␥1, and -␦1 in isolated glomeruli and microdissected proximal tubules and cortical collecting ducts.25 Our data extend these studies by clearly demonstrating that isolated preglomerular arterioles express PLC-␤1, -␥1, and -␦1, with levels similar to the aorta and less than in the glomeruli. Such differences in PLC expression and activity observed between 6-week-old SHR and WKY implicate the higher levels in renal cortical arterioles during the development of genetic hypertension in SHR. High levels of PLC may explain, at least in part, increased production of IP3, enhanced cytosolic Ca2⫹ signaling, and exaggerated renal vasoconstriction in 6-week-old SHR.9 –17 The reason for the increased PLC levels in young SHR is not known. Likely candidates include increased receptor density and defective buffering of second messengers associated with vasoconstrictor GPCRs.10 –17 The possible roles of Ang II, AVP, TxA2, and catecholamines await further investigation. In this regard, norepinephrine has been shown to increase expression of PLC-␤1, decrease PLC-␥1, and have no effect on PLC-␦1 in renal cortical but not medullary membranes.26 On the other hand, based on the liter-

AJH–January 2007–VOL. 20, NO. 1

ature, tubular levels in the renal medulla appear to be reduced in SHR. Protein levels of PLC-␦1 in the inner medulla are reduced in 4-week-old SHR versus WKY, with strain-related reductions in inner medullary PLC-␤1, -␥1, and -␦1 in 12-week-old SHR.27 Our findings of early PLC increases in the renal vasculature as well as aorta of SHR at 6 weeks of age suggest that they may be causative. Our results are consistent with the earlier finding that, compared to age-matched WKY, PLC activity is increased in the aortic wall of 4-week-old prehypertensive SHR as well as 14-week-old SHR with established hypertension.28 However, the reported similarity of aortic PLC activity at 7 weeks and increased activity at 12 weeks in SHR34 could be interpreted as age-dependent changes that are secondary to hypertension rather than being primary. The PLC enzyme activity, measured as production of IP3 and DAG, is reported to be greater in aorta of 12-week-old SHR versus WKY, a difference that may be explained by exaggerated activation of PLC-␦1 by [Ca2⫹]i greater than 1 ␮mol/L.34,35 Nevertheless, immunoblots of aortic homogenates in this study suggest agedependent decreases in PLC-␥1 and an increase in PLC-␦1 of both SHR and WKY, with no apparent strain difference.34 This is consistent with the observation of increased phosphoinositide metabolism of aorta and femoral artery of hypertensive adult SHR and SHR stroke prone rats.6 Basal PLC activity is elevated in the renal cortex of 10- to 12-week-old SHR.28 It is noteworthy that PLC levels and activity are normal in the low pressure venous system of adult SHR.36 In summary, we provide new information using Western blot analysis about the presence of PLC-␤1, -␥1, and -␦1 and their protein levels in freshly isolated preglomerular resistance, which are similar to values of the aorta, a conduit vessel, and less than in glomeruli. We demonstrate for the first time that the protein levels of PLC-␤1, and PLC-␦1 are elevated in both resistance and conduit vessel types of 6-week-old SHR versus WKY. No strain difference was noted for PLC-␥1. Overall enzymatic activity of PLC was also much higher in the renal vasculature and the aorta of SHR in a developmental phase of genetic hypertension. A more complete understanding of the physiologic roles of PLC isozymes and their contributions to specific aspects of cellular responses should advance our understanding of vascular tone/reactivity and hypertrophy/ remodeling in normal and hypertensive states.

References 1. 2.

3.

Exton JH: Phopsphoinostide phosphlipases and G protein in hormone action. Annu Rev Physiol 1994;56:349 –369. Rebecchi MJ, Pentyala SN: Structure, function, and control of phosphoinositide-specific phospholipase C. Physiol Rev 2000;80: 1291–1335. Rhee SG: Regulation of phosphoinositide-specific phospholipase C. Annu Rev Biochem 2001;70:281–312.

AJH–January 2007–VOL. 20, NO. 1

4.

5.

6.

7. 8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18. 19.

20.

21.

PLC ISOZYMES IN PREGLOMERULAR RESISTANCE ARTERIOLES

Litosch I: Phosphatidic acid modulates G protein regulation of phospholipase C-beta 1 activity in membranes. Cell Signal 2002; 14:259 –263. Schiffrin EL: Intracellular signal transduction for vasoactive peptides in hypertension [Review]. Canadian J Physiol & Pharmacol 1994;72:954 –962. Turla MB, Webb RC: Augmented phosphoinositide metabolism in aortas from genetically hypertensive rats. Am J Physiol 1990;258: H173–H178. Osanai T, Dunn MJ: Phosphoplipase C responses in cells from spontaneously hypertensive rats. Hypertension 1992;19:446 – 455. Michel JB, de Roux N, Plissonnier D, Anidjar S, Salzmann JL, Levy B: Pathophysiological role of the vascular smooth muscle cell. J Cardiovas Pharmacol 1990;16:S4 –S11. Chatziantoniou C, Arendshorst WJ: Angiotensin and thromboxane in genetically hypertensive rats: renal blood flow and receptor studies. Am J Physiol 1991;261:F238 –F247. Chatziantoniou C, Ruan X, Arendshorst WJ: Interactions of cAMPmediated vasodilators with angiotensin II in rat kidney during hypertension. Am J Physiol 1993;265:F845–F852. Fellner SK, Arendshorst WJ: Store-operated Ca2⫹ entry is exaggerated in fresh preglomerular vascular smooth muscle cells of SHR. Kidney Int 2002;61:2132–2141. Feng JJ, Arendshorst WJ: Calcium signaling mechanisms in renal vascular responses to vasopressin in genetic hypertension. Hypertension 1997;30:1223–1231. Iversen BM, Arendshorst WJ: Exaggerated Ca2⫹ signaling in preglomerular arteriolar smooth muscle cells of genetically hypertensive rats. Am J Physiol 1999;276:F260 –F270. Salomonsson M, Arendshorst WJ: Norepinephrine-induced calcium signaling pathways in afferent arterioles of genetically hypertensive rats. Am J Physiol Renal Physiol 2001;281:F264 –F272. Vagnes OB, Hansen FH, Feng JJ, Iversen BM, Arendshorst WJ: Enhanced Ca2⫹ response to AVP in preglomerular vessels from rats with genetic hypertension during different hydration states. J Physiol Renal Physiol 2005;288:F1249 –F1256. Jackson EK, Herzer WA: Angiotensin II/prostaglandin I2 interactions in spontaneously hypertensive rats. Hypertension 1993;22: 688 – 698. Jackson EK, Herzer WA: Defective modulation of angiotensin II-induced renal vasoconstriction in hypertensive rats. Hypertension 1994;23:329 –336. Berk BC: Vascular smooth muscle growth: autocrine growth mechanisms. Physiol Rev 2001;81:999 –1030. Bukoski RD, DeWan P, Bo J: Mechanism of the enhanced epidermal growth factor-induced growth response of genetically hypertensive vascular myocytes. Circ Res 1991;69:757–764. Oba T: Niflumic acid differentially modulates two types of skeletal ryanodine-sensitive Ca2⫹-release channels. Am J Physiol 1997;273: C1588 –C1595. Scott-Burden T, Resink TJ, Hahn AW, Buhler FR: Angiotensininduced growth related metabolism is activated in cultured smooth

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

43

muscle cells from spontaneously hypertensive rats and WistarKyoto rats. Am J Hypertens 1991;4:183–188. Ruan X, Chatziantoniou C, Arendshorst WJ: Impaired prostaglandin E(2)/prostaglandin I (2) receptor-G(s) protein interactions in isolated renal resistance arterioles of spontaneously hypertensive rats. Hypertension 1999;34:1134 –1140. Zhu Z, Zhang SH, Wagner C, Kurtz A, Maeda N, Coffman TM, Arendshorst WJ: Angiotensin AT1B receptor mediates calcium signaling in vascular smooth muscle cells of AT1A receptor deficient mice. Hypertension 1998;31:1171–1177. Cha SH, Cha JH, Cho YJ, Noh DY, Lee KH, Endou H: Distributional patterns of phospholipase C isozymes in rat kidney. Nephron 1998;80:314 –323. Lea JP, Ertoy D, Hollis JL, Marrero MB, Sands JM: Immunolocalization of phospholipase C isoforms in rat kidney. Kidney Int 1998;54:1484 –1490. Yu PY, Asico LD, Eisner GM, Jose PA: Differential regulation of renal phospholipase C isoforms by catecholamines. J Clin Invest 1995;95:304 –308. Lee KH, Cho YJ, Cha SH, Endou H: Attenuation of renomedullary phospholipase C isozyme, PLC-delta 1, in spontaneously hypertensive rats. Biochem Mol Biol Int 1997;43:741–747. Chen CJ, Vyas SJ, Eichberg J, Lokhandwala MF: Diminished phospholipase C activation by dopamine in spontaneously hypertensive rats. Hypertension 1992;19:102–108. Blayney LM, Gapper P, Rix C: Identification of phospholipase C beta isoforms and their location in cultured vascular smooth muscle cells of pig, human and rat. Cardiovasc Res 1998;40:564 –572. LaBelle EF, Wilson K, Polyak E: Subcellular localization of phospholipase C isoforms in vascular smooth muscle. Biochimica et Biophysica Acta 2002;1583:273–278. Schelling JR, Nkemere N, Konieczkowski M, Martin KA, Dubyak GR: Angiotensin II activates the beta 1 isoform of phospholipase C in vascular smooth muscle cells. Am J Physiol 1997;272:C1558 – C1566. Schwartz Z, Shaked D, Hardin RR, Gruwell S, Dean DD, Sylvia VL, Boyan BD: 1alpha, 25 (OH)2D3 causes a rapid increase in phosphatidylinositol-specific PLC-beta activity via phospholipase A2-dependent production of lysophospholipid. Steroids 2003;68: 423– 437. Zhou CJ, Akhtar RA, Abdel-latif AA: Purification and characterization of phosphoinositide-specific phospholipase C from bovine iris sphincter smooth muscle. Biochem J 1993;289:401– 409. Kato H, Fukami K, Shibasaki F, Homma Y, Takenawa T: Enhancement of phospholipase C delta 1 activity in the aortas of spontaneously hypertensive rats. J Biol Chem 1992;267:6483– 6487. Uehara Y, Ishii M, Ishimitsu T, Sugimoto T: Enhanced phospholipase C activity in the vascular wall of spontaneously hypertensive rats. Hypertension 1988;11:28 –33. Vila E, Macrae IM, Reid JL: Differences in inositol phosphate production in blood vessels of normotensive and spontaneously hypertensive rats. Br J Pharmacol 1991;104:296 –300.