Construction and expression of a modular gene encoding bacteriophage T7 RNA polymerase

Tachykinins inhibit salt appetite when applied intracranially in a number of brain regions and may function as endogenous inhibitors of sodium intake. To test the hypothesis that induced increases in salt appetite might involve disinhibition via a reduction in endogenous tachykinin expression, we used a semi-quantitative in situ hybridization analysis to investigate changes in brain areas expressing preprotachykinin-A (PPT-A) and preprotachykinin-B (PPT-B) mRNAs of rats after 1 day of sodium depletion (1d Na dep). PPT-A mRNA levels were detected in neurons of the olfactory tubercle (Tu), the nucleus of the olfactory tubercle (LOT), the dorsal and ventral caudate-putamen (d-CPu and v-CPu), the bed nucleus of the stria terminalis (BNST), the medial preoptic area (mPOA), the habenula (Hb) and the postero-dorsal part of the amygdala (MePD). PPT-B mRNA levels were measured in fundus striati (FStr), d-CPu, v-CPu, BNST, mPOA, dorsomedial hypothalamic nucleus (DMD), arcuate nucleus (Arc), central amygdaloid nucleus (CeL), basolateral amygdaloid nucleus (BLV), LOT, Hb and basal nucleus of Meynert (B). 1d Na dep reduced by 33-61% the mean number of PPT-A grains/cell in Tu, LOT, d-CPu, BNST, mPOA, Hb and MePD compared to control animals. Levels of PPT-B mRNA were not reduced as much by 1d Na dep, although statistically significant reductions of 26, 34 and 17% were found in v-CPu, BNST and B, respectively. These findings, therefore, support the hypothesis that endogenous tachykinins exert an inhibitory influence over sodium appetite.     

Heart and red blood cell antioxidant status and plasma lipid levels in the spontaneously hypertensive and normotensive Wistar-Kyoto rat

Heart and red blood cell endogenous antioxidant status and plasma lipids were investigated in hypertensive, 14-week-old spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats fed a standard commercial rat chow. Specific heart and red blood cell antioxidant enzyme activities, as well as the susceptibility of tissues to H2O2-induced glutathione (GSH) depletion and lipid peroxidation, were measured. Systolic blood pressure in SHR was greater than in WKY rats at 13 weeks of age (197 +/- 12 vs. 132 +/- 14 mmHg (1 mmHg = 133.3 Pa); p < or = 0.05), confirming the presence of hypertension in SHR. Red blood cell catalase (CAT) and superoxide dismutase (SOD) activities were greater (p < or = 0.05) in SHR than WKY rats. Red blood cell CAT activity was positively correlated (r = +0.634; p = 0.026) with SOD, which in turn was correlated (r = +0.709; p = 0.049) with systolic blood pressure. Heart SOD activity was higher (p < or = 0.05) in SHR, while glutathione reductase (GSSG-Red) activity was lower (p < or = 0.05) than in WKY rats. This reduced ability to recycle GSH in the heart coincided with greater (p < or = 0.05) levels of H2O2-induced lipid oxidation products in SHR. Plasma total cholesterol and triacylglycerol levels were lower (p < or = 0.05) in SHR than WKY rats, with no visible signs of atherosclerosis in either SHR or WKY rats. In summary, hypertension in SHR was associated with alterations in antioxidant enzyme profiles of red blood cells and heart, with the latter showing an increased susceptibility to in vitro lipid oxidation. Although hypertension is a recognized factor in the development of human atherosclerosis, spontaneously hypertensive rats did not exhibit signs of aortic plaque, reflecting the resistance of this species to the development of atherosclerosis.     

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 microM)-dependent manner in WKY rats while it failed to inhibit the enzyme activity in SHR. Dopamine (10 microM)-induced inhibition of Na+,K(+)-ATPase activity in WKY rats was significantly blocked by mepacrine (10 microM), 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 microM) manner in WKY rats while the inhibition in SHR was significantly attenuated (IC50: 7.5 and 80 microM in WKY rats and SHR, respectively). Furthermore, lower concentrations of arachidonic acid stimulated (30% at 1 microM) Na+,K(+)-ATPase activity in SHR. This suggests a defect in the metabolism of arachidonic acid in SHR. Proadifen (10 microM), 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.     

Transformation of the methylotrophic actinomycete Amycolatopis methanolica with plasmid DNA: stimulatory effect of a pMEA300-encoded gene

The concentration of endothelin-1 (ET-1) in the brain regions, heart, and throacic aorta of 1-, 4-, 6- and 8-week-old spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats was determined using radioimmunoassay. ET-1-like immunoreactivity in the brain regions of 1-week-old WKY and SHR rats was lower compared to older (6 and 8 weeks) rats. ET-1 levels in the central nervous system gradually increased with age in both SHR and WKY rats. However, the concentration of ET-1 in 8-week-old rats was lower in the brain regions of SHR compared to WKY rats. The concentration of ET-1 in the thoracic aorta of SHR (224 +/- 43 pg/g tissue) rats was lower than that of WKY (452 +/- 11 pg/g tissue) rats at 1 week of age. However, ET-1 levels gradually increased with age in SHR rats. By 8 weeks of age, levels of ET-1 in SHR (623 +/- 33 pg/g tissue) rats were higher compared to WKY (439 +/- 62 pg/g tissue) rats. In the heart, ET-1 levels were similar in WKY and SHR rats at 4 weeks of age, but at 8 weeks of age ET-1 levels were higher in SHR rats (364 +/- 33 pg/g tissue) compared to WKY rats (260 +/- 31 pg/g tissue). It appears that at 8 weeks of age when hypertension is fully expressed in rats, ET-1 levels are lower in the central nervous system and are higher in the thoracic aorta and heart of SHR compared to WKY rats.     

Increase of the background human exposure to mercury through fish consumption due to gold mining at the Tapajós River region, Pará State, Amazon

Plasma and lipoprotein lipid composition and endogenous hepatic antioxidant status were investigated in hypertensive, 14-week-old spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats fed a standard commercial rat chow. Total plasma calcium and magnesium concentrations were similar between both rat strains; however, systolic blood pressure in SHR was greater than in WKY at 13 weeks of age (197 +/- 12 vs. 132 +/- 14 mmHg; p < or = 0.05), confirming hypertension in SHR. Total plasma cholesterol and triacylglycerol concentrations were lower (p < or = 0.05) in SHR compared with WKY. A lower (p < 0.05) HDL cholesterol level in SHR plasma resulted in a higher LDL to HDL cholesterol ratio compared with WKY counterparts. No significant differences in the relative proportion of HDL apolipoprotein A-I fraction were observed between SHR and WKY. Both SHR VLDL and HDL triacylglycerol fractions were lower (p < 0.05) in SHR than WKY. Analysis of liver antioxidant enzyme activities showed no differences in rat liver superoxide dismutase (SOD), but lower (p < 0.05) liver glutathione peroxidase (GSH-Px) activity in SHR. However, liver glutathione (GSH) levels were similar in SHR and WKY counterparts. A possible compensatory effect to the oxidative status of SHR was suggested by the significant (p < 0.05) increase in both liver catalase (CAT) and glutathione reductase (GSSG-Red) activities. Despite these results, in vitro oxidative challenge studies with H2O2 demonstrated a greater susceptibility of liver to GSH depletion in the SHR, although no parallel change in thiobarbituric acid reactive substances (TBARS) production was observed. The comparatively lower plasma cholesterol observed in hypertensive SHR paralleled specific differences in liver catalase and glutathione redox antioxidant enzyme activities.     

Modulation of endothelial cell function by antiphospholipid antibodies

AIM: To observe the effect of captopril (Cap) on intracellular pH (pHi) in aortic smooth muscle cells (ASMC). METHODS: Cultured ASMC derived from rat and rabbit aortae were loaded with the fluorescent dye BCECF and pHi was determined using digital image processing method. RESULTS: The pHi of untreated SHR and WKY rats were 7.37 +/- 0.29 and 7.19 +/- 0.31, respectively. Oral Cap decreased pHi (7.11 +/- 0.26, P < 0.05) and exaggerated pHi response to angiotensin II (Ang-II, 0.1 mumol.L-1) in ASMC of SHR rats vs WKY rats (0.14 +/- 0.05 vs 0.21 +/- 0.05 pH units, P < 0.01). Cap in vitro had no effect on Ang-II induced intracellular alkalinization in ASMC of rabbits. CONCLUSION: Oral Cap inhibits Na+(-)H+ exchange activity in ASMC of SHR rats.     

Tissue-specific regulation of angiotensinogen gene expression in spontaneously hypertensive rats

Angiotensinogen is expressed in many tissues besides the liver. Recent studies have suggested that abnormalities in the regulation of angiotensinogen gene expression may be involved in the development of hypertension. However, little information is available concerning the functional significance of tissue angiotensinogen. In this study, we measured plasma angiotensinogen concentration by radioimmunoassay and examined the expression of tissue angiotensinogen by Northern blot analysis in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). Although plasma angiotensinogen concentration in SHR was comparable to that in WKY at 6 weeks of age, it was increased significantly at 14 weeks of age in SHR and became higher than that in WKY. The levels of hepatic angiotensinogen mRNA were similar in SHR and WKY, and the levels of aortic, adrenal, and renal angiotensinogen mRNAs were lower in SHR than in WKY at both 6 and 14 weeks of age. Brain angiotensinogen expression in SHR was higher than in WKY at 6 weeks of age and was comparable to that in WKY at 14 weeks of age. On the other hand, cardiac and fat angiotensinogen mRNA levels were significantly increased at 14 weeks of age in SHR. These results demonstrate that the expression of tissue angiotensinogen is regulated differently in SHR and WKY and indicate that the development of hypertension is accompanied at least temporally with increases in plasma angiotensinogen concentration as well as cardiac and adipogenic angiotensinogen mRNA in SHR.     

Different calcium storage pools in vascular smooth muscle cells from spontaneously hypertensive and normotensive Wistar-Kyoto rats

OBJECTIVE: To evaluate whether the distribution of intracellular free calcium may be impaired in primary hypertension. DESIGN: Cytosolic free calcium and stored calcium were investigated in cultured vascular smooth muscle cells from spontaneously hypertensive rats (SHR). METHODS: The concentrations of intracellular and stored calcium were investigated in cultured vascular smooth muscle cells from spontaneously hypertensive rats aged 6 months from the Münster strain (SHR) and from age-matched normotensive Wistar-Kyoto (WKY) rats. Vascular smooth muscle cells were grown on coverslips, and fluorescence measurements of the intracellular calcium concentration were performed using fura-2. The different effects of thapsigargin, a selective Ca-ATPase inhibitor, and of angiotensin II (Ang II) on the calcium storage pools were investigated. RESULTS: In the absence of external calcium thapsigargin produced a dose-dependent transient increase in the concentration of intracellular calcium in vascular smooth muscle cells. The thapsigargin-induced maximum peak increase in the concentration of intracellular calcium was not significantly different in SHR and WKY rats. After depletion of the thapsigargin-sensitive calcium pools the addition of 100 nmol/l Ang II produced a rise in the concentration of intracellular calcium in vascular smooth muscle cells from SHR and WKY rats. Using vascular smooth muscle cells from the SHR the Ang II-induced increase in the concentration of intracellular calcium was not significantly different in the presence and absence of thapsigargin, indicating that the calcium pools depleted by thapsigargin and Ang II do not overlap significantly in vascular smooth muscle cells from SHR. In contrast, in the WKY rats the response to Ang II was significantly diminished after depletion of the thapsigargin-sensitive pool. When Ang II and thapsigargin were administered in the reverse order, i.e. Ang II before thapsigargin, the thapsigargin response was diminished in the WKY rats but not in the SHR. CONCLUSION: SHR differ from WKY rats in having vascular smooth muscle cells that contain thapsigargin-sensitive calcium storage pools that are distinct from the Ang II-sensitive calcium pools.     

Endogenous dopamine regulates phosphate reabsorption but not NaK-ATPase in spontaneously hypertensive rat kidneys

Dopamine's modulatory actions on signal transduction in the spontaneously hypertensive rat (SHR) proximal tubule are blunted; therefore, it was predicted that dopamine does not regulate phosphate (Pi) reabsorption in SHR. To test this hypothesis, dopamine production was inhibited with carbidopa (10 mg/kg ip) 18 h before and during clearance measurements of chronically denervated SHR and Wistar-Kyoto (WKY) rat kidneys. Dopamine excretion decreased 80% from SHR and 85% from WKY rats. Pi excretion decreased 60 to 67%. Plasma Pi and calcium, inulin clearance, and Na excretion did not change. Citrate excretion, which reflects proton secretion by proximal tubules, decreased 72% from WKY rats. Citrate excretion was significantly lower from SHR (5 +/- 10 pmol/min) than from WKY rats (73 +/- 11 pmol/min) and was not altered by carbidopa. Carbidopa, injected 18 and 1 h before kidneys were collected, increased NaK-ATPase in cortical basolateral membranes from WKY rats (27%) but not in membranes from SHR. After the incubation of renal cortical minceates for 15 min with L-DOPA (10(-5) M), there was no change in brush border membrane vesicle uptake of 32Pi, (3H)glucose, or (14C)citrate. Incubation with carbidopa (10(-4) M) increased 32Pi uptake by 11% (P < 0.001) and (3H)glucose uptake by 9% (P = 0.02). (14C)citrate uptake was not increased by carbidopa but was higher in SHR (977 +/- 2 pmol/10 s.mg) than in WKY rats (823 +/- 43 pmol/10 s.mg; P = 0.04). In summary, dopamine produced in WKY rat and SHR proximal tubules decreases Pi uptake by using a signaling process distinct from those that regulate NaK-ATPase and the antiporter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Decreased flow-induced dilation and increased production of cGMP in spontaneously hypertensive rats

-We investigated flow (shear stress)- and agonist-induced cGMP release in mesenteric vascular beds of spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY). The mesenteric vascular bed was perfused in situ with Tyrode's solution. Vascular relaxation and cGMP release in the perfusate were determined on stimulation by flow or by acetylcholine (0.1 micromol/L) or sodium nitroprusside (0.1 mmol/L). Flow-induced release of cGMP was significantly greater in SHR than in WKY (P<0.01), despite a lower flow-induced dilation in SHR. In both strains, NG-nitro-L-arginine methyl ester (L-NAME) completely inhibited cGMP release in response to flow (P<0.001), although flow-induced dilation was not affected by L-NAME in SHR. Moreover, the activity of the constitutive nitric oxide synthase (NOS) was significantly greater in SHR (82+/-3.5 fmol/min) than in WKY (66+/-3.5 fmol/min; P<0.05) and was associated with increased expression of endothelial NOS mRNA in SHR. Sodium nitroprusside induced larger increases in cGMP release in SHR (3593+/-304 fmol/min) than in WKY (2467+/-302 fmol/min; P<0.05). The release of cGMP in response to acetylcholine was significantly lower in SHR (292+/-80 fmol/min) than in WKY (798+/-218 fmol/min; P<0.05) in parallel with smaller acetylcholine-induced relaxation in SHR. Despite increased cGMP production in response to flow and NOS activity, flow-induced dilation was decreased in SHR, suggesting an upregulation of the NO/cGMP pathway to compensate for the increased vascular tone in SHR.     

Transforming growth factor-beta and receptor tyrosine kinase-activating growth factors negatively regulate collagen genes in smooth muscle of hypertensive rats

Previous studies have suggested that differences in vascular smooth muscle cell (VSMC) proliferative responses between spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats can be attributed to transforming growth factor-beta (TGF-beta) actions. Because vascular collagen content is reported to be lower in SHR than in WKY rats, in this study we investigated in cell culture whether the differences in collagen content might also be attributed to differential actions of TGF-beta on VSMCs from the two strains. Exposure of VSMCs from WKY to the TGF-beta isoforms -beta1, -beta2, or -beta3 induced rapid, transient elevations in mRNAs encoding collagens alpha1(I), alpha2(I), and alpha1(III); maximum increases were apparent by 2 hours and ranged from twofold [collagen alpha1(III)] to ninefold [collagen alpha1(I)]. Thereafter they returned to near basal levels. When VSMCs from SHR were exposed to these TGF-beta isoforms, only reductions in collagen mRNA levels were observed, persisting for 24 hours. Basic fibroblast growth factor and epidermal growth factor, factors known to stimulate production of the TGF-beta1 isoform in VSMCs, also induced a pattern of gene responses similar to those induced by the TGF-beta isoforms in VSMCs from SHR and WKY rats. The simultaneous presence of TGF-beta did not affect the time course or magnitude of the changes in collagens alpha1(I), alpha2(I), or alpha1(III) mRNA levels in SHR or WKY VSMCs. Examination of the induction of c-myc mRNA and immunoreactive oncoprotein content indicated that c-myc is a likely contributor to the downregulation of the collagen gene activity in both SHR and WKY VSMCs despite the differential regulation of its mRNA by TGF-beta1 in the two VSMC lines. Together these data suggest that in VSMCs from SHR, a number of gene responses to TGF-beta, in addition to cell proliferation, appear to be abnormal compared with WKY rats, and the lower than normal collagen levels observed in the vasculature of SHR may be in part due to abnormalities in TGF-beta responsiveness.     

Gadolinium chloride removes pulmonary intravascular macrophages and curtails the degree of ovine lentivirus-induced lymphoid interstitial pneumonia

Estrogens are produced by the aromatization of androgens. These steroids exert their actions after binding to their receptors. Past studies have shown that estrogen receptors (ER) and aromatase enzyme (AROM) reside in many of the same brain regions. Few studies, however, have examined the neural co-localization of these important components involved in estrogen-activated behaviors. In the present study we examined the co-localization of ER and AROM immunoreactive (ir) neurons in musk shrew (Suncus murinus) brains. Data were collected from a representative section from three neural regions, the bed nucleus of the stria terminalis (BNST), medial preoptic area (mPOA), and ventromedial nucleus of the hypothalamus (VMN). Here we report a sex difference in the number of ER-ir neurons from the analyzed section of the mPOA and BNST. Females have more ER-ir neurons in the mPOA and males have more in the BNST. In the sections we examined, males tended to have more aromatase containing neurons than females. Although there were no significant differences in the numbers of double-labeled cells, the VMN contains the greatest percentage of these cells in both males and females; followed by the mPOA and the BNST. In addition, in the mPOA of both sexes, a distinct nucleus of aromatase containing neurons which was devoid of ER immunoreactivity was noted. Area measurements of the AROM-ir nucleus showed that it was significantly larger in males than in females. Taken together, these data suggest that there is not extensive cellular co-localization of estrogen receptors and aromatase enzyme in the musk shrew brain. However, the presence of other genomic forms of ER (membrane and/or ERbeta) in AROM containing neurons has not been ruled out by this study. Thus, we hypothesize that estrogens produced in brain affect behavior by binding to ER in neurons other than those that contain aromatase enzyme.     

Relation between gastric emptying of glucose and plasma concentrations of glucagon-like peptide-1

-Dopamine, via D1-like receptors, stimulates the activity of both protein kinase A (PKA) and protein kinase C (PKC), which results in inhibition of renal sodium transport. Since D1-like receptors differentially regulate sodium transport in normotensive and hypertensive rats, they may also differentially regulate PKC expression in these rat strains. Thus, 2 different D1-like agonists (fenoldopam or SKF 38393) were infused into the renal artery of anesthetized normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) (n=5 to 6/drug/strain). Ten or 60 minutes after starting the D1-like agonist infusion, both the infused kidney and the noninfused kidney that served as control were prepared for analysis. The D1-like agonists produced a greater diuresis and natriuresis and inhibited Na+,K+-ATPase activity in proximal tubule (PT) and medullary thick ascending limb (mTAL) to a greater extent in WKY (Delta20+/-1%) than in SHR (Delta7+/-1%, P<0.001). D1-like agonists had no effect on PKC-alpha or PKC-lambda expression in either membrane or cytosol but increased PKC-theta expression in PT in both WKY and SHR at 10 minutes but not at 60 minutes. However, membranous PKC-delta expression in PT and mTAL decreased in WKY but increased in SHR with either 10 or 60 minutes of D1-like agonist infusion. D1-like agonists also decreased membranous PKC-zeta expression in PT and mTAL in WKY but increased it in PT but not in mTAL in SHR. We conclude that there is differential regulation of PKC isoform expression by D1-like agonists that inhibits membranous PKC-delta and PKC-zeta in WKY but stimulates them in SHR; this effect in SHR is similar to the stimulatory effect of norepinephrine and angiotensin II and may be a mechanism for their differential effects on sodium transport.     

Parathyroid hormone-related protein expression in vascular smooth muscle of spontaneously hypertensive rats: evidence for lack of response to angiotensin II

OBJECTIVE: We studied the expression of parathyroid hormone (PTH)-related protein in vascular smooth muscle cells of spontaneously hypertensive rats (SHR) using Wistar-Kyoto (WKY) and Sprague-Dawley rats as normotensive controls. METHODS: Aortae from 4- and 18-week-old SHR versus age-matched WKY and Sprague-Dawley rats were excised to obtain total RNA or smooth muscle cells. The cells were subcultured in Dulbecco's Modified Eagle's Medium containing 10% fetal calf serum, then serum-deprived for 72 h and stimulated with 0.1 micromol/I angiotensin II. PTH-related protein, c-myc and angiotensin II type qa receptor (AT1aR) messenger (m)RNA levels were measured by Northern blot, using total RNA extracted by phenol/chloroform. The effects of PTH-related protein(1-34)NH2 intravenous injections on arterial blood pressure and the heart rate were studied in anesthetized SHR and WKY rats. RESULTS: The Northern blots showed a significantly higher abundance of PTH-related protein mRNA in aortae of SHR versus WKY rats in the prehypertensive state but no significant difference in adult animals. In cultured aortic smooth muscle cells, angiotensin II induced a four- to sixfold increase in PTH-related protein mRNA levels in smooth muscle cells from normotensive animals, but failed to elicit a significant response in smooth muscle cells derived from SHR in either the prehypertensive or the hypertensive state. This lack of response to angiotensin II in SHR smooth muscle cells was not due to decreased expression or responsiveness of the AT1aR, since SHR smooth muscle cells had more AT1aR mRNA than Sprague-Dawley smooth muscle cells, and angiotensin II-induced activation of c-myc was faster and greater in smooth muscle cells derived from 4- or 18-week-old SHR than in Sprague-Dawley smooth muscle cells. In contrast, PTH-related protein(1-34)NH2 induced a long-lasting dose-dependent hypotensive and tachycardic response in both SHR and WKY rats, indicating that SHR retained responsiveness to the vasodilator. CONCLUSIONS: PTH-related protein gene expression in response to angiotensin II is impaired in SHR arteries. A deficiency in this potent local vasodilator may contribute to the development and/or maintenance of arterial hypertension in this model.     

Possible mechanism of the potent vasoconstrictor actions of ryanodine on femoral arteries from spontaneously hypertensive rats

1. The Ca2+ buffering function of sarcoplasmic reticulum (SR) in the resting state of arteries from spontaneously hypertensive rats (SHR) was examined. Differences in the effects of ryanodine that removes the function of SR, on tension and cellular Ca2+ level were assessed in endothelium-denuded strips of femoral arteries from 13-week-old SHR and normotensive Wistar-Kyoto rats (WKY). 2. The addition of ryanodine to the resting strips caused a concentration-dependent contraction in SHR. This contraction was extremely small in WKY. In the presence of 10(-5) M ryanodine, caffeine (20 mM) failed to cause a further contraction in SHR, but it caused a small contraction in WKY. After washout of the strips with a Krebs solution, the resting tone was greatly elevated in SHR when compared with WKY. 3. The elevated resting tone in SHR strips was abolished by 10(-7) M nifedipine. The ryanodine-induced contraction was also abolished by 10(-7) M nifedipine. Nifedipine itself caused a relaxation from the resting tone of SHR strips, suggesting the maintenance of myogenic tone. 4. In strips preloaded with fura-PE3, the addition of 10(-5) M ryanodine caused a large and moderate elevation of cytosolic Ca2+ level ([Ca2+]i) in SHR and WKY, respectively. After washout, the resting [Ca2+]i was greatly elevated in SHR. The ryanodine-induced elevation of [Ca2+]i was decreased by 5 x 10(-6) M verapamil in SHR. Verapamil itself caused a decrease in resting [Ca2+]i which was significantly greater in SHR than in WKY, and caused a relaxation only in SHR. 5. The resting Ca2+ influx in arteries measured by a 5 min incubation with 45Ca was significantly increased in SHR when compared with WKY. The resting Ca2+ influx was not increased by 10(-5) M ryanodine in both SHR and WKY. The net cellular Ca2+ uptake in arteries measured by a 30 min incubation with 45Ca was decreased by 10(-5) M ryanodine in both strains. 6. The resting Ca2+ influx was decreased by 10(-7) M nifedipine in the SHR artery, but it was unchanged in the WKY artery. 7. These results suggest that (1) the Ca2+ influx via L-type voltage-dependent Ca2+ channels was increased in the resting state of the SHR femoral artery, (2) the greater part of the increased Ca2+ influx was buffered by Ca2+ uptake into the SR and some Ca2+ reached the myofilaments resulting in the maintenance of the myogenic tone, and (3) therefore the functional removal of SR by ryanodine caused a potent contraction in this artery.     

Dopamine fails to stimulate protein kinase C activity in renal proximal tubules of spontaneously hypertensive rats

We have previously reported that dopamine-1 receptor-mediated activation of phospholipase C is diminished in renal cortical slices of spontaneously hypertensive rats. The present study was carried out to examine the effect of dopamine on protein kinase C (PKC), which is one of the enzymes involved in the signal-transduction pathway leading to dopamine-induced inhibition of Na+/K(+)-ATPase in the renal proximal tubule. Renal proximal tubule suspensions were obtained from spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats of 10-12 weeks old. The tubules were incubated with dopamine in the presence or absence of DA-1 receptor antagonist SCH 23390. The PKC activity was measured by using a specific fluorescent peptide substrate (sequence, PKSRTLSVAAK). We found that dopamine produced a concentration-dependent increase in protein kinase C activity in the WKY rats, however, it failed to stimulate PKC activity in the SHR. Peak stimulation of 3.828 +/- 0.35 (ng/micrograms) protein in the WKY rats was observed at dopamine concentration of 1 microM, which was blocked in a concentration-dependent manner by SCH 23390 (0.25 microM). These results provide evidence that dopamine directly stimulates PKC activity via activation of DA-1 receptors in WKY rats. Furthermore, we discovered that dopamine fails to stimulate PKC activity in the SHR. This phenomenon may be responsible for the failure of dopamine to inhibit Na+/K(+)-ATPase activity in the hypertensive animals.