Renal Nerve-Mediated Proximal Tubule Solute Reabsorption Contributes to Hypertension in Spontaneously Hypertensive Rats

Renal nerve activity increases (Na⁺, K⁺)-ATPase activity and contributes to the development of hypertension in young SHR. The present study was designed to examine the effect of sodium intake on blood pressure and proximal tubule solute reabsorption in sham-operated or renal denervated, 5-week old SHR and WKY. Three-week old SHR and WKY rats underwent sham surgery or renal denervation with 10% phenol and were maintained for 10 days on either a 0.6% or 2.2% NaCl diet. Blood pressure was obtained by indirect tail cuff measurements during this interval. Of the eight groups, only sham-operated SHR on a high sodium diet had hypertension, 122.0 × 4.2 mm Hg vs. 98.7 × 3.3 mm Hg (mean for remaining groups). Renal plasma flow (RPF), glomerular filtration rate (GFR), and the fractional excretion of lithium (FE_Lj) were determined in rats maintained on a 2.2% sodium diet at 5 weeks of age. FE_li was less in sham-operated SHR, 5.3 × 0.7%, compared to WKY, 9.4 × 2.8% (P>0.02). Furthermore, denervation ameliorated the reduced FE_Li in SHR, 10.2 × 1.2%, without affecting FE_Li in WKY. RPF and GFR were similar between sham-operated and renal denervated SHR and WKY. No significant difference could be detected in net sodium balance between WKY and SHR during this period. These findings demonstrate 1) from the basis of FE_Li, young SHR, of this strain, exhibit enhanced proximal tubule solute reabsorption and hypertension while on a high sodium diet and, 2) renal denervation ameliorates both the enhanced proximal tubule solute reabsorption and the early development of hypertension. These data support the concept that renal nerve activity of young SHR is augmented and contributes to the development of hypertension by enhancing salt retention.     

Renal protein phosphatase 2A activity and spontaneous hypertension in rats

The impaired renal paracrine function of dopamine in spontaneously hypertensive rats (SHR) is caused by hyperphosphorylation and desensitization of the renal D(1) dopamine receptor. Protein phosphatase 2A (PP(2A)) is critical in the regulation of G-protein-coupled receptor function. To determine whether PP(2A) expression and activity in the kidney are differentially regulated in genetic hypertension, we examined the effects of a D(1)-like agonist, fenoldopam, in renal cortical tubules and immortalized renal proximal tubule cells from normotensive Wistar-Kyoto rats (WKY) and SHR. In cortical tubules and immortalized proximal tubule cells, PP(2A) expression and activities were greater in cytosol than in membrane fractions in both WKY and SHR. Although PP(2A) expressions were similar in WKY and SHR, basal PP(2A) activity was greater in immortalized proximal tubule cells of SHR than WKY. In immortalized proximal tubule cells of WKY, fenoldopam increased membrane PP(2A) activity and expression of the regulatory subunit PP(2A)-B56alpha, effects that were blocked by the D(1)-like antagonist SCH23390. Fenoldopam had no effect on cytosolic PP(2A) activity but decreased PP(2A)-B56alpha expression. In contrast, in immortalized proximal tubule cells of SHR, fenoldopam decreased PP(2A) activity in both membranes and cytosol but predominantly in the membrane fraction, without affecting PP(2A)-B56alpha expression; this effect was blocked by the D(1)-like antagonist SCH23390. We conclude that renal PP(2A) activity and expression are differentially regulated in WKY and SHR by D(1)-like receptors. A failure of D(1)-like agonists to increase PP(2A) activity in proximal tubule membranes may be a cause of the increased phosphorylation of the D(1) receptor in the SHR.     

Increased proximal tubule NHE-3 and H+-ATPase activities in spontaneously hypertensive rats

OBJECTIVE: To investigate renal proximal tubular sodium-hydrogen exchanger 3 (NHE-3) and H+-ATPase activities in young (5-week-old) spontaneously hypertensive rats (SHR) and normotensive Donryu (DRY) rats, in the period during which high blood pressure is developing. METHODS: Five-week-old SHR and DRY rats were weighed and systolic blood pressure recorded. Proximal tubule cells were isolated, loaded with the intracellular pH dye, 2'-7'-bis-carboxyethyl-5(6)-carboxyfluorescein-acetoxymethyl-ester and acidified with a NH4+/NH3 prepulse. Na+-independent intracellular pH recovery rate (H+-ATPase activity) and initial Na+-dependent intracellular pH recovery rate (NHE-3 activity) were assessed. NHE-3 activity was assessed during inhibition of H+-ATPase with Bafilomycin A1 and during inhibition of any possible NHE-1 activity with Hoe 694. RESULTS: Mean body weight and systolic blood pressures of 5-week-old SHR and DRY rats were not significantly different. NHE-3 activity was higher in SHR, 1.08 +/- 0.1 pH units/min compared with DRY rats, 0.73 +/- 0.1 pH units/min (P < 0.05) H+-ATPase activity was also higher in SHR, 0.119 +/- 0.02 pH units/min, compared with DRY rats, 0.051 +/- 0.02 pH units/min (P < 0.05). CONCLUSIONS: Proximal tubule cells of 5-week-old SHR have higher NHE-3 and H+-ATPase activities compared with age-matched DRY rats. Enhanced proximal tubular fluid reabsorption is likely to contribute to development of high blood pressure in young SHR.     

Renal dopamine receptor signaling mechanisms in spontaneously hypertensive and Fischer 344 old rats

Dopamine plays an important role in the regulation of renal sodium excretion. The activation of D1-like receptors located on the proximal tubules causes inhibition of tubular sodium reabsorption by inhibiting Na,H-exchanger and Na,K-ATPase activity. The D1-like receptors are linked via G proteins to the multiple cellular signaling systems namely adenylyl cyclase and phospholipase C (PLC). A defective renal dopamine receptor function exists in spontaneously hypertensive rats (SHR). In the proximal tubules of SHR, the stimulation of adenylyl cyclase and PLC caused by dopamine was significantly reduced in comparison with Wistar-Kyoto (WKY) rats. Also unlike the effects seen in WKY, D1-like receptor activation did not inhibit Na,K-ATPase and Na,H-exchanger activities in SHR. In addition, reduced quantity of Gq/11alpha proteins was detected in the basolateral membranes of SHR compared to WKY rats. Studies revealed that there may be a primary defect in D1-like receptors leading to an altered signaling system in the proximal tubules and reduced dopamine-mediated effect on renal sodium excretion in SHR. Recently, it has been shown that the disruption of D1A receptors at the gene level causes hypertension in mice. Similar to SHR, dopamine and D1-like receptor agonist failed to inhibit Na,K-ATPase activity in the proximal tubules of old Fischer 344 rats. Unlike the observations in SHR where D1-like receptors were equal to WKY rats, there is a 50% decrease in D1-like receptor number in basolateral membranes of the old rats compared to the adult rats. Dopamine was unable to stimulate G proteins in the basolateral membranes of old rats compared to the adult rats. It is suggested that a defective dopamine receptors/signaling system may contribute to the development and maintenance of hypertension. Also, the inability of dopamine to inhibit Na,K-ATPase may lead to a reduced renal sodium excretion in response to dopamine in old rats.     

Interaction of angiotensin II type 1 and D5 dopamine receptors in renal proximal tubule cells

Angiotensin II type 1 (AT1) receptor and D1 and D3 dopamine receptors directly interact in renal proximal tubule (RPT) cells from normotensive Wistar-Kyoto rats (WKY). There is indirect evidence for a D5 and AT1 receptor interaction in WKY and spontaneously hypertensive rats (SHR). Therefore, we sought direct evidence of an interaction between AT1 and D5 receptors in RPT cells. D5 and AT1 receptors colocalized in WKY cells. Angiotensin II decreased D5 receptors in WKY cells in a time- and concentration-dependent manner (EC50=2.7x10(-9) M; t(1/2)=4.9 hours), effects that were blocked by an AT1 receptor antagonist (losartan). In SHR, angiotensin II (10(-8) M/24 hours) also decreased D5 receptors (0.96+/-0.08 versus 0.72+/-0.08; n=12) and to the same degree as in WKY cells (1.44+/-0.07 versus 0.92+/-0.08). However, basal D5 receptors were decreased in SHR RPT cells (SHR 0.96+/-0.08; WKY 1.44+/-0.07; n=12 per strain; P<0.05) and renal brush border membranes of SHR compared with WKY (SHR 0.54+/-0.16 versus WKY 1.46+/-0.10; n=5 per strain; P<0.05). Angiotensin II decreased AT1 receptor expression in WKY (1.00+/-0.04 versus 0.72+/-0.08; n=8; P<0.05) but increased it in SHR (0.96+/-0.04 versus 1.32+/-0.08; n=8; P<0.05). AT(1) and D5 receptors also interacted in vivo; renal D5 receptor protein was higher in mice lacking the AT1A receptor (AT1A-/-; 1.61+/-0.31; n=6) than in wild-type littermates used as controls (AT1A+/+; 0.81+/-0.08; n=6; P<0.05), and renal cortical AT1 receptor protein was higher in D5 receptor null mice than in wild-type littermates (1.18+/-0.08 versus 0.84+/-0.07; n=4; P<0.05). We conclude that D5 and AT1 receptors interact with each other. Altered interactions between AT1 and dopamine receptors may play a role in the pathogenesis of hypertension.     

Altered Arachidonic Acid Metabolism Contributes to the Failure of Dopamine to Inhibit Na+, K+-ATPase in Kidney of Spontaneously Hypertensive Rats

Dopamine decreases tubular sodium reabsorption in part by inhibition of Na⁺, K⁺-ATPase activity in renal proximal tubules. The signaling mechanism involved in dopamine-mediated inhibition of Na⁺, K⁺-ATPase is known to be defective in spontaneously hypertensive animals. The present study was designed to evaluate the role of phospholipase A2 (PLA2) and its metabolic pathway in dopamine-induced inhibition of Na⁺, K⁺-ATPase in renal proximal tubules from Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). Renal proximal tubular suspensions were prepared and Na⁺, K⁺-ATPase activity was measured as ouabain-sensitive adenosine triphosphate hydrolysis. Dopamine inhibited Na⁺,K⁺-ATPase activity in a concentration (1 nM - 10 μM)-dependent manner in WKY rats while it failed to inhibit the enzyme activity in SHR. Dopamine (10μM)-induced inhibition of Na⁺,K⁺-ATPase activity in WKY rats was significantly blocked by mepacrine (10 μM), a PLA2 inhibitor, suggesting the involvement of PLA2 in dopamine-mediated inhibition of Na⁺,K⁺-ATPase. Arachidonic acid (a product released by PLA2 action) inhibited Na⁺,K⁺-ATPase in a concentration-dependent (1-100 μM) manner in WKY rats while the inhibition in SHR was significantly attenuated (IC₅₀: 7.5 and 80 μM in WKY rats and SHR, respectively). Furthermore, lower concentrations of arachidonic acid stimulated (30% at 1 μM) Na⁺,K⁺-ATPase activity in SHR. This suggests a defect in the metabolism of arachidonic acid in SHR. Proadifen (10 μM), an inhibitor of cytochrome P-450 monoxygenase (an arachidonic acid metabolizing enzyme) significantly blocked the inhibition produced by arachidonic acid in WKY rats and abolished the difference in arachidonic acid inhibition of Na⁺,K⁺-ATPase between WKY rats and SHR. These data suggest that PLA2 is involved in dopamine-induced inhibition of Na⁺,K⁺-ATPase and altered arachidonic acid metabolism may contribute to reduced dopaminergic inhibition of Na⁺,K⁺-ATPase activity in spontaneously hypertensive rats.     

Altered AT1 receptor regulation of ETB receptors in renal proximal tubule cells of spontaneously hypertensive rats

The renin-angiotensin and endothelin systems regulate blood pressure, in part, by affecting renal tubular sodium transport. In rodents, ETB receptors decrease proximal tubular reabsorption, whereas AT1 receptors produce the opposite effect. We hypothesize that ETB and AT1 receptors interact at the receptor level, and that the interaction is altered in spontaneously hypertensive rats (SHRs). In immortalized renal proximal tubule (RPT) cells from Wistar-Kyoto (WKY) rats, angiotensin II, via AT1 receptors, increased ETB receptor protein in a time- and concentration-dependent manner. In contrast, in SHR RPT cells, angiotensin II (10(-8) M/24 hours) had no effect on ETB receptor protein. AT1/ETB receptors colocalized and co-immunoprecipitated in both rat strains but long-term angiotensin II (10(-8) M/24 hours) treatment increased AT1/ETB co-immunoprecipitation in WKY but not in SHR cells. Short-term angiotensin II (10(-8) M/15 minutes) treatment decreased ETB receptor phosphorylation in both WKY and SHR cells, and increased ETB receptors in RPT cell surface membranes of RPT cells in WKY but not SHRs. Basal cell surface membrane ETB receptor expression was also higher in WKY than in SHRs. We conclude that AT1 receptors regulate ETB receptors by receptor interaction and modulation of receptor expression. The altered AT1 receptor regulation of ETB receptors in SHRs may play a role in the pathogenesis of hypertension.     

Activation of MAPKs in proximal tubule cells from spontaneously hypertensive and control Wistar-Kyoto rats

The aim of this study was to test the hypothesis that differences exist in the activity and/or expression of mitogen-activated protein kinases (MAPKs) between spontaneously hypertensive rats (SHR) and control Wistar-Kyoto rats (WKY) and that these differences may account for the enhanced activity of the Na(+)/H(+) exchanger (NHE) previously observed in the renal proximal tubule of SHR. Therefore, the activities of c-jun N-terminal kinase(1) (JNK(1)), extracellular signal-regulated kinase(1/2) (ERK(1/2)), and p38 were investigated. A reduced amount of ERK(1) and JNK(1) protein was found in renal cortex specimens of SHR as compared with WKY; however, their activities were the same. To study the cellular basis of this difference, immortalized proximal tubule cell lines were grown on Millicell-CM filter inserts where the cell lines organize as polarized monolayers with separate access to apical and basolateral compartments. Although basal JNK(1) and ERK(1/2) activities were not significantly different between WKY and SHR cells, anisomycin stimulated JNK(1) activity in WKY cells more than in SHR cells (eg, at 15 minutes 300% versus 30%, respectively). Similarly, angiotensin II increased JNK(1) and ERK(1/2) activity in a time- and concentration-dependent manner in WKY cells but not in SHR cells. Western blot analyses showed a deficit in JNK(1) and ERK(1) protein in SHR (0.25 and 0.5, respectively, of the levels in WKY cells), although ERK(2) and p38 protein levels were the same. These observations suggest that, although angiotensin II activates MAPKs and MAPKs have been shown to regulate NHE, this regulatory pathway is unlikely to account for the increased activity of NHE in the proximal tubular epithelium of SHR.     

Perturbation of D1 dopamine and AT1 receptor interaction in spontaneously hypertensive rats

The dopaminergic and renin-angiotensin systems interact to regulate blood pressure. Because this interaction may be perturbed in genetic hypertension, we studied D1 dopamine and AT1 angiotensin receptors in immortalized renal proximal tubule (RPT) and A10 aortic vascular smooth muscle cells. In normotensive Wistar-Kyoto (WKY) rats, the D1-like agonist fenoldopam increased D1 receptors but decreased AT1 receptors. These effects were blocked by the D1-like antagonist SCH 23390 (10(-7) mol/L per 24 hours). In spontaneously hypertensive rat (SHR) RPT cells, fenoldopam also decreased AT1 receptors but no longer stimulated D1 receptor expression. Basal levels of AT1/D1 receptor coimmunoprecipitation were greater in WKY RPT cells (29+/-2 density units, DU) than in SHR RPT cells (21+/-2 DU, n=7 per group, P<0.05). The coimmunoprecipitation of D1 and AT1 receptors was increased by fenoldopam (10(-7) mol/L per 24 hours) in WKY RPT cells but decreased in SHR RPT cells. The effects of fenoldopam in RPT cells from WKY rats were similar in aortic vascular smooth muscle cells from normotensive BD IX rats, that is, fenoldopam decreased AT1 receptors and increased D1 receptors. Our studies show differential regulation of the expression of D1 and AT1 receptors in RPT cells from WKY and SHR. This regulation and D1/AT1 receptor interaction are different in RPT cells of WKY and SHR. An altered interaction of D1 and AT1 receptors may play a role in the impaired sodium excretion and enhanced vasoconstriction in hypertension.     

Free cytosolic calcium in renal proximal tubules from the spontaneously hypertensive rat

Free intracellular calcium was measured in renal proximal tubules obtained from spontaneously hypertensive rats (SHR) and from age-matched Wistar-Kyoto rats (WKY) ingesting a normal diet. Experiments were performed on renal proximal tubule suspensions using fura-2 to monitor cytosolic calcium. In 4-week-old rats, when systolic blood pressure was not significantly different between the two groups, renal proximal tubule cytosolic calcium was similar (143 +/- 28 and 144 +/- 15 nM, respectively). By the age of 5 weeks, cytosolic calcium increased significantly in both SHR and WKY (214 +/- 24 and 262 +/- 34 nM, respectively, p less than 0.05). Calcium, however, was not significantly different between the two groups, even though at this age blood pressure was higher in SHR than in WKY. As compared with values in 4-week-old rats, cytosolic calcium was also found increased in tubules from both SHR and WKY aged 10 to 12 weeks (261 +/- 42 and 279 +/- 30 nM, respectively) and 20 to 24 weeks (263 +/- 42 and 308 +/- 28 nM, respectively). However, no significant differences in cytosolic calcium were found between SHR and WKY even though at these ages systolic blood pressure increased markedly in the SHR. Moreover, regression analysis failed to reveal a correlation between cytosolic calcium and blood pressure when data from either group of rats of all ages studied were pooled. Exposure to ouabain (10(-3) M) to inhibit Na+,K+-adenosine triphosphatase and increase intracellular sodium had no significant effect on cytosolic calcium in tubules from either SHR or WKY (260 +/- 69 and 250 +/- 45 nM, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Altered responsiveness of proximal tubule fluid reabsorption of peritubular angiotensin II in spontaneously hypertensive rats

Stimulation of proximal tubular fluid reabsorption by peritubular angiotensin II (Ang II) was examined by split-drop micropuncture in 5- and 12-week-old spontaneously hypertensive rats (SHR) and age-matched normotensive Wistar-Kyoto rats (WKY). In WKY, the maximum stimulation occurred at 10(-11) mol/l and the response did not vary with age. In 5-week-old SHR, the dose-response relationship was similar in shape and in the extent of the maximum response but was shifted one half-logarithmic step to the right, indicating decreased sensitivity to Ang II. In contrast, the dose-response relationship was shifted one half-logarithmic step to the left in 12-week-old SHR compared with WKY. Alterations in the responsiveness of the proximal tubule to Ang II in young SHR could contribute to sodium retention observed during development of hypertension in these rats.     

Low-sodium diet and alpha-adrenoceptors of renal basolateral membrane in spontaneously hypertensive rats.

To clarify the effect of dietary sodium restriction on the mechanism regulating sodium and water in the development of hypertension, we determined the number of the alpha-adrenoceptors in renal basolateral membrane in spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats. The rats had been fed a low-sodium (0.5%) or normal-sodium (0.4%) diet from 3 weeks of age. The experiments were performed at 6, 8 and 20 weeks of age in both rat groups. Renal basolateral membranes were prepared using Percoll and radioligand binding studies were performed using 3H-prazosin and 3H-rauwolscine. Systolic blood pressure in SHR was already elevated at 6 weeks of age compared with that in WKY rats and rose to hypertensive levels at 8 weeks of age. The sodium balance in WKY rats on both diets decreased at 8 weeks of age, but that of SHR decreased at 20 weeks of age. The maximum number (Bmax) for the alpha 1-adrenoceptor did not differ in any groups of the WKY rats or SHR. Bmax for the alpha 2-adrenoceptors increased at 8 weeks of age in the low-sodium SHR compared with normal-sodium SHR, but did not increase in WKY rats. The data show that the increases in blood pressure in the SHR occur prior to significant increases in the alpha 2-adrenoceptor density of renal basolateral membrane, and that the modulation of alpha 2-adrenoceptor density in SHR differs from that in WKY rats under sodium restriction. The results suggest that renal alpha 2-adrenoceptors in SHR could relate the regulatory mechanism to sodium reabsorption under sodium restriction rather than to the primary cause of the development of hypertension in SHR. There may be the possibility of an abnormality in renal alpha 2-adrenoceptor mechanism in SHR.     

Effect of luminal angiotensin II receptor antagonists on proximal tubule transport.

The proximal tubule can endogenously synthesize and secrete luminal angiotensin II at a concentration approximately 100- to 1000-fold higher than that in the systemic circulation. We have recently shown that this endogenously produced and luminally secreted angiotensin II regulates proximal tubule volume reabsorption, which is a reflection of sodium transport within this segment. In this study, we use in vivo microperfusion of angiotensin II receptor antagonists into the lumen of the proximal tubule to examine the role of the luminal AT1 and AT2 receptor in the regulation of volume reabsorption. Systemically administered (intravenous) AT1 and AT2 receptor antagonists, acting through basolateral angiotensin II receptors, have previously been shown to inhibit proximal tubule transport. Luminal perfusion of 10(-6) mol/L Dup 753 (AT1 antagonist) and 10(-6) mol/L PD 123319 (AT2 antagonist) decreased proximal tubule volume reabsorption from 2.94 +/- 0.18 to 1.65 +/- 0.18 and 1.64 +/- 0.19 nL/mm x min, respectively, P < .01. Luminal perfusion of 10(-4) mol/L CGP 42112A, another AT2 antagonist, similarly decreased volume reabsorption to 1.32 +/- 0.36 nL/nm x min, P < .01. The inhibition of transport with AT1 and AT2 antagonist was additive, as luminal perfusion of 10(-6) mol/L Dup 753 plus 10(-6) mol/L 123319 resulted in a decrease in volume reabsorption to 0.41 +/- 0.31 nL/mm x min, P < .001 v control, P < .05 v Dup 753, and P < .01 v PD 123319. These results show that endogenously produced angiotensin II regulates proximal tubule volume transport via both luminal AT1 and AT2 receptors.     

Shear stress-induced changes of membrane transporter localization and expression in mouse proximal tubule cells

Our previous studies of microperfused single proximal tubule showed that flow-dependent Na(+) and HCO(3)(-) reabsorption is due to a modulation of both NHE3 and vacuolar H(+)-ATPase (V-ATPase) activity. An intact actin cytoskeleton was indicated to provide a structural framework for proximal tubule cells to transmit mechanical forces and subsequently modulate cellular functions. In this study, we have used mouse proximal tubule (MPT) cells as a model to study the role of fluid shear stress (FSS) on apical NHE3 and V-ATPase and basolateral Na/K-ATPase trafficking and expression. Our hypothesis is that FSS stimulates both apical and basolateral transporter expression and trafficking, which subsequently mediates salt and volume reabsorption. We exposed MPT cells to 0.2 dynes/cm(2) FSS for 3 h and performed confocal microscopy and Western blot analysis to compare the localization and expression of both apical and basolateral transporters in control cells and cells subjected to FSS. Our findings show that FSS leads to an increment in the amount of protein expression, and a translocation of apical NHE3 and V-ATPase from the intracellular compartment to the apical plasma membrane and Na/K-ATPase to the basolateral membrane. Disrupting actin by cytochalasin D blocks the FSS-induced changes in NHE3 and Na/K-ATPase, but not V-ATPase. In contrast, FSS-induced V-ATPase redistribution and expression are largely inhibited by colchicine, an agent that blocks microtubule polymerization. Our findings suggest that the actin cytoskeleton plays an important role in FSS-induced NHE3 and Na/K-ATPase trafficking, and an intact microtubule network is critical in FSS-induced modulation of V-ATPase in proximal tubule cells.     

Protein phosphatase 2A B56alpha during development in the spontaneously hypertensive rat

Mistargeting of the regulatory subunit of protein phosphatase 2A (PP2A), B56alpha is involved in the hyperphosphorylation and desensitization of the D1 dopamine receptor in renal proximal tubules of spontaneously hypertensive rats (SHRs). However, the renal expression of B56alpha before hypertension develops is not known. Therefore, we studied the expression of B56alpha and PP2A activity in the kidney during development in the SHR and its normotensive control, the Wistar-Kyoto (WKY) rat. PP2A B56alpha was expressed in proximal and distal tubules with no differences in the pattern of expression in WKY and SHRs at any age. In brush border membranes of renal proximal tubules, PP2A B56alpha protein was greatest in the immature rats and decreased with development. However, PP2A activity did not change with age. PP2A B56alpha protein and PP2A activity were similar in WKY and SHRs except at 2 weeks when both PP2A B56alpha protein and PP2A activity were higher in SHRs than in WKY rats. The PP2A catalytic subunit co-immunoprecipitated with the D1 receptor in renal proximal tubule cells. It is possible that the increased expression of PP2A B56alpha and increased basal PP2A activity in the young, especially in the SHRs, may serve as a compensatory mechanism in the increased phosphorylation and decreased renal D1 receptor function, including D1-receptor mediated stimulation in renal proximal tubules of SHRs.     

Some enzyme characteristics of spontaneously hypertensive rats myocardium

In order to ascertain the pathogenesis of myocardial cell vulnerability in spontaneously hypertensive rats (SHR), several enzyme activities were examined by using subcellular fractions of myocardium and compared to those in Wistar-Kyoto rats (WKY). In the normotensive WKY heart, both 5'-nucleotidase and Na+/K(+)-ATPase, which are plasma membrane associated enzymes, increased with age. But in the SHR heart, both enzymes were lower at 16 weeks than they were at 10 weeks of age. Moreover, at 16 weeks of age they were lower in SHR than in WKY. On the other hand, NADP(+)-isocitrate dehydrogenase activity, a mitochondria associated enzyme, was higher in SHR than in WKY at 6 weeks, but lower at 10 and again at 16 weeks of age. The activities of both acid phosphatase and N-acetyl-beta-glucosaminidase, which are lysosomal enzymes, decreased with age in SHR but not in WKY. These results suggest that an enzymatic alteration in the plasma membrane and mitochondria may be one of important factors behind myocardial vulnerability in the SHR heart.     

Increased systolic performance with diastolic dysfunction in adult spontaneously hypertensive rats

Hypertensive heart disease is characterized by early development of hypertrophy and fibrosis that leads to heart failure (HF). HF develops in spontaneously hypertensive rats (SHR) after 18 months; however, it is not clear whether hypertrophy leads to altered cardiac performance at an earlier age in these rats. We studied cardiac performance in 10- to 11-month-old SHR and age-matched Wistar-Kyoto rats (WKY), using presssure-volume (PV) conductance catheter system to evaluate systolic and diastolic function in vivo at different preloads, including preload recruitable stroke work (PRSW), +dP/dt, and its relation to end-diastolic volume (+dP/dt-EDV) and preload-adjusted maximal power (PWR(max)-EDV(2)) as well as the time constant of left ventricular pressure decay, tau (tau), as an index of relaxation. The slope of the end-diastolic pressure-volume relation (EDPVR) and the ex vivo PV relation, both indexes of stiffness, were also calculated for each heart, and the Doppler E/A ratio was determined. In addition, plasma samples were obtained to assess B-type natriuretic peptide levels (BNP). We found that PRSW was higher in SHR than in WKY (174.5+/-15.6 versus 92.6+/-18.9 mm Hg; P<0.01). +dP/dt and +dP/dt-EDV were also enhanced in SHR versus WKY (9125+/-662 versus 6633+/-392 mm Hg/sec, P<0.01, and 28.14+/-4.35 versus 12.7+/-2.8 mm Hg/s per micro L, P<0.02). In addition, PWR-EDV(2) was elevated in SHR (7.3+/-1.5 versus 3.1+/-0.6 mW/ micro L(2)). Tau was prolonged in SHR (14.5+/-1 ms versus 10.8+/-0.8 for WKY, P<0.02) and EDPVR was significantly greater in SHR than in WKY (0.01+/-0.005 versus 0.004+/-0.001, P<0.05). The ex vivo pressure-volume relation was also steeper for SHR and the E/A ratio was 2.53+/-0.15 for SHR versus 1.67+/-0.08 for WKY (P<0.02). BNP was 45+/-2.5 pg/mL for SHR and 33.3+/-1.8 pg/mL for WKY (P<0.02). Taken together, these data suggest that at 10 to 11 months of age, before HF develops, SHR have increased systolic performance accompanied by delayed relaxation and increased diastolic stiffness.     

Enhanced protein phosphorylation in hypertensive hypertrophy

OBJECTIVE: Chronic pressure overload in spontaneously hypertensive rats (SHR) is accompanied by heart hypertrophy and signs of heart failure. Since there is growing evidence for a possible pathophysiological role of altered protein phosphorylation in heart hypertrophy and failure, we studied here cardiac regulatory phosphoproteins and the kinases and phosphatases which regulate their phosphorylation state. METHODS: The experiments were performed in ventricles of SHR (12-13 weeks old) and age-matched normotensive Wistar-Kyoto rats (WKY). RESULTS: Basal as well as isoproterenol (Iso)-stimulated force of contraction (FOC) was markedly decreased in isolated electrically driven papillary muscles of SHR. Iso (3 micromol/l, 10 min) increased FOC by 0.91+/-0.20 mN in SHR and by 3.88+/-0.52 mN in WKY, respectively. Ca(2+)-uptake by sarcoplasmic reticulum (SR) at low ionized Ca(2+)-concentration was increased in homogenates from SHR. This was not due to altered expression of phospholamban (PLB), SR-Ca(2+)-ATPase and calsequestrin. However, PLB-phosphorylation at threonine-17 (PLB-PT-17) and the activity of Ca(2+)/calmodulin dependent protein kinase (Ca(2+)/Cam-PK) was increased in SHR. In addition, we found an enhanced protein kinase A (PKA)-dependent phosphorylation of the inhibitory subunit of troponin (TnI). In contrast, there was no difference in the activity or expression (protein- and mRNA-level) of protein phosphatases type 1 or type 2A between SHR and WKY. CONCLUSIONS: It is suggested that increased Ca(2+)/Cam-PK-activity with resulting increase of PLB-PT-17 enhanced SR-Ca(2+)-uptake in SHR and might contribute to the pathophysiological changes in cardiac hypertrophy of SHR.