Role of Na+-H+-antiport in restitution of isolated guinea pig gastric epithelium after superficial injury

In addition to its pH_i regulatory function Na⁺-H⁺-antiport is also involved in volume regulation of epithelial cells, particularly in neutral conditions. It is also known that the antiport is activated after ligand binding following growth factor receptor activation. The aim of the present study was to evaluate the role of the antiport in restitution of gastric mucosa and whether its activity is dependent on the type of superficial injury. Therefore the fundic epithelium of guinea pig stomach was perfused in an Ussing chamber in neutral conditions. Na⁺-H⁺- and Cl^–-HCO ₃ ^– -antiports were inhibited with 1.0 mM amiloride, 1.0 mM SITS, or with HCO ₃ ^– removal and Na⁺-K⁺-2Cl^2–-cotransporter with 0.3 M furosemide during 4 hr of restitution after superficial injury induced either by 1.25 M NaCl or by 1.0% Triton. Luminal exposure of the epithelium to amiloride had no effect on restitution but serosal application abolished the process completely. The inhibitory effect of amiloride was similar after both NaCl and Triton injury. The inhibition of Cl^–-HCO ₃ ^– -antiport with SITS interfered with the process as well, while HCO ₃ ^– removal had no significant inhibitory effect, nor did the inhibition of Na⁺-K⁺-2Cl^–-cotransporter. The morphologic findings were in accordance with the electrophysiologic measurements in each pair of tissues. It is concluded that the Na⁺-H⁺-antiport is essential for the epithelial cells during restitution even in neutral conditions, but a functional Cl^–-HCO ₃ ^– -antiport is also required. The activity of Na⁺-H⁺-antiport is sensitive to basolateral amiloride and is necessary regardless of the type of chemical injury.This study was supported by grants from Astra Co., the Orion Corporation Research Foundation, the Sigrid Juselius Foundation, the Academy of Finland, the Research Foundation of Gastrointestinal Diseases, the Clinical Research Institute of Helsinki University Central Hospital, Helsinki, Finland.     

Effects of thyroid hormone on the renal dopaminergic system

This study determined the effects of thyroid hormone on the renal dopaminergic system. Surgical thyroidectomy (Tx) and treatment with 2-thiouracil (Thio) decreased renal cortex Na⁺/K⁺ ATPase activity and urinary volume. Tx also decreased urinary Na⁺ and urinary l-DOPA without changing urinary excretion of Dopamine (DA). Thio treatment decreased slightly urinary l-DOPA and Na⁺, but increased urinary excretion of DA. In both models of thyroid hormone deficiency, the ratio urinary DA/DOPA increased. Changes after Thio treatment were reversed after one month of drug withdrawal. Treatment with T₃ via osmotic minipump increased Na⁺/K⁺ ATPase activity and urinary l-DOPA, did not change urinary DA, and increased the ratio DA/DOPA. To further analyze the effects of thyroid hormone deficiency, we administered selective DA₁ (SCH-23390), DA₂ (Sulpiride), and a non selective (Haloperidol) DA receptor antagonists to Thio treated and control animals. The DA₁ antagonist decreased diuresis, natriuresis and urinary l-DOPA in control, but had no effect in Thio treated rats. Sulpiride had no effect in either group. The combination of SCH-23390 plus Sulpiride decreased urinary l-DOPA and urinary volume only in Thio treated animals. Haloperidol decreased urinary volume in Thio treated animals, but had no effect in controls. Our findings suggest that renal DA synthesis is to some extent dependent on thyroid hormone levels, and that the response of DA receptors is altered by thyroid hormone deficiency, indicating a role of this hormone in the regulation of the renal dopaminergic system.     

SALT INTAKE AND SENSITIVITY OF INTESTINAL AND RENAL NA+ -K+ ATPase TO INHIBITION BY DOPAMINE IN SPONTANEOUS HYPERTENSIVE AND WISTAR-KYOTO RATS

The present study evaluated the activity of jejunal Na⁺-K⁺-ATPase and its sensitivity to inhibition by dopamine in spontaneous hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats during low (LS), normal (NS) and high (HS) salt intake. Basal jejunal Na⁺-K⁺-ATPase activity in SHR on LS intake was higher than in WKY rats. Jejunal Na⁺-K⁺-ATPase activity in WKY rats, but not in SHR, on LS intake was significantly reduced (20% decrease) by dopamine (1 μM) and SKF 38393 (10nM), but not quinerolane (10 nM), this being antagonized the D₁ receptor antagonist (SKF 83566). Changing from LS to NS or HS intake in WKY rats increased basal jejunal Na⁺-K⁺-ATPase activity and attenuated the inhibitory effect of dopamine. In SHR, changing from LS to NS or HS intake increased basal jejunal Na⁺-K⁺-ATPase activity. Basal renal Na⁺-K⁺-ATPase activity in SHR on LS intake was similar to that in WKY rats and was insensitive to inhibition by dopamine . Changing from LS to NS or HS intake in WKY rats increased basal renal Na⁺-K⁺-ATPase activity without affecting the inhibitory effect of dopamine. In SHR, changing from LS to NS or HS intake failed to alter basal renal Na⁺-K⁺-ATPase activity. It is concluded that inhibition of jejunal Na⁺-K⁺ ATPase activity by D₁ dopamine receptor activation is dependent on salt intake in WKY rats, and SHR animals fail to respond to dopamine, irrespective of their salt intake.     

Na+ Channel Activators as Positive Inotropic Agents for the Treatment of Chronic Heart Failure

The Na⁺ channel agonists DPI 201-106, BDF 9148 and BDF 9198 are a new group of positive inotropic agents which increase cardiac contractility in a cAMP independent manner. The most likely mechanism by which positive inotropy is mediated is an enhancement of Na⁺/Ca²⁺ exchange activity in response to a Na⁺ channel agonist induced increase in the cardiac myocyte intracellular Na⁺ concentration. While the positive inotropic effect of drugs which exert their effects in a cAMP dependent manner is blunted in failing compared to nonfailing myocardium, the efficacy and potency of Na⁺ channel agonists is not only maintained, but enhanced in failing myocardium. This finding makes these substances interesting for the treatment of patients with heart failure. The positive inotropic effects of the Na⁺ channel agonists, however, are accompanied by a potential increase in the incidence of cardiac arrhythmias. These side effects might limit the clinical use of Na⁺ channel agonists and demand future development of Na⁺ channel modulators without significant arrhythmogenic effects.     

BIPHASIC EFFECTS OF DOPAMINE ON 86RUBIDIUM UPTAKE IN RAT RENAL PROXIMAL TUBULES

The mechanism(s) by which dopamine inhibits Na⁺ -K⁺ -ATPase activity in the renal proximal tubule is still controversial. We studied the short-term effects of dopamine on the sodium pump in rat renal proximal tubule suspensions with the ⁸⁶Rb uptake method. Dopamine and the D₁-like agonist, SKF81297, initially stimulated Na⁺ -K⁺ -ATPase activity at 5 min and subsequently inhibited it at 10 min and 20 min; the inhibition by 10 μM dopamine at 20 min was 21.3 ± 4.5 %. The inhibitory effect of dopamine on Na⁺ -K⁺ -ATPase activity was mimicked by thymeleatoxin (a classical protein kinase C [PKC] agonist) while Sp-8-CPT-cAMPS (a protein kinase A [PKA] agonist) had no effect. However, the combination of the PKC and PKA agonists mimicked the biphasic effects of dopamine and SKF81297. Rp-8-CPT-cAMPS (a PKA inhibitor), U-73122 (a phospholipase C inhibitor), or calphostin C (a PKC inhibitor), blocked the dopamine-mediated biphasic effects on Na⁺ -K⁺ -ATPase activity. It is suggested that the biphasic effects of dopamine on Na⁺ -K⁺ -ATPase activity (an initial stimulation and a subsequent inhibition) are transduced by activating both PKA and PKC through a D₁-like receptor.     

INTESTINAL DOPAMINERGIC ACTIVITY IN OBESE AND LEAN ZUCKER RATS: RESPONSE TO HIGH SALT INTAKE

The present study examined intestinal dopaminergic activity and its response to high salt (HS, 1% NaCl over a period of 24 hours) intake in obese (OZR) and lean Zucker rats (LZR). The basal Na⁺,K⁺-ATPase activity (nmol Pi/mg protein/min) in the jejunum of OZR was higher than in LZR on normal salt (NS) (OZR-NS=111.3±6.0 vs. LZR-NS=88.0±8.3). With the increase in salt intake, the basal Na⁺,K⁺-ATPase activity significantly increased in both animals (OZR-HS=145.9±11.8; LZR-HS=108.8±6.7). SKF 38393 (10?nM), a specific D₁-like dopamine receptor agonist, inhibited the jejunal Na⁺,K⁺-ATPase activity in OZR on HS intake, but failed to inhibit enzyme activity in OZR on NS intake and LZR on NS and HS intakes. The aromatic L-amino acid decarboxylase (AADC) activity in OZR was lower than in LZR on NS intake. The HS intake increased AADC activity in OZR, but not in LZR. During the NS intake the jejunal monoamine oxidase (MAO) activity in OZR was similar to that in LZR. The HS intake significantly decreased MAO activity in both OZR and LZR. The jejunal COMT activity in OZR was higher than in LZR on NS intake. The HS intake reduced COMT activity in OZR but not LZR. It is concluded that inhibition of jejunal Na⁺,K⁺-ATPase activity through D₁ dopamine receptors is dependent on salt intake in OZR, whereas in LZR, the enzyme failed to respond to the activation of D₁ dopamine receptors irrespective of their salt intake.     

Cell Volume Regulation During Hyperosmotic Shrinkage Is Mediated by Na+/K+-ATPase and Na+–K+–2Cl− Cotransporter in Necturus Gastrics Surface Epithelial Cells

Cell volume regulation was investigated in gastric surface epithelial cells during hypertonic conditions. Isolated Necturus antral mucosa was perfused on the serosal side with Ringer's solution (pH 7.25, 95%O₂/5%CO₂) and on the mucosal side successively with 150–500 mM NaCl. Amiloride, ouabain, and bumetanide were used to experimentally inhibit Na⁺/H⁺, Na⁺/K⁺ ATPase or Na⁺–K⁺–2Cl^– ion transporters. Intracellular sodium activity and cell volume changes were measured with liquid sensor microelectrodes. The increase in intracellular sodium activity caused by luminal hyperosmolar exposure was mainly due to cell shrinkage. Inhibition of Na⁺/K⁺ ATPase or Na⁺–K⁺–2Cl^– cotransporter increased hyperosmotic cell shrinkage (−52 ± 5%, −85 ± 19%, and −77 ± 9% for control, ouabain, and bumetanide, respectively). Inhibition of Na⁺/K⁺ ATPase increased intracellular sodium activity (from 18 ± 4 to 52 ± 12 mM). Cell volume regulation in gastric epithelial surface cells during mucosal hyperosmolar exposure is maintained by the basolateral Na⁺–K⁺–2Cl^– cotransporter, while Na⁺/K⁺ ATPase maintains sodium balance, but Na⁺/H⁺ antiport seems to have a less important role. This work was supported by the Research Foundation of Helsinki University Central Hospital (EVO), Academy of Finland, Antti and Jenny Wihuri Foundation, and Biomedicum Research Foundation, Helsinki, Finland. The authors thank Mrs Paula Kokko for excellent technical assistance.     

Some Properties of Erythrocyte Na+ -K+ -ATPase in Essenial Hypertension

The activity and some allosteric properties of Na⁺ -K⁺ -ATPase in erythrocytes and their membrane preparations (ghosts) from 57 patients with essential hypertension and 36 normotensive controls were studied. To reveal enzyme activity in whole erythrocytes the cells were pretreated with detergent Tween-20. It was found that in the patient erythrocytes the Na⁺ -K⁺ -ATPase activity was 33% less as compared to the control group. Moreover, in the patient erythmcytes the sensitivity of the enzyme to high concentrations of MgCI₂ was decreased. In contrast, no analogous changes of the enzyme were revealed in the patient ghosts. It is suggested that the erythrocytes of patients with essential hypertension contain an inhibitor of Na⁺ -K⁺ -ATPase.     

Kinetics of Red Cell Na+ and K+ Transport in Prague Hypertensive Rats

Kinetics of ouabain-sensitive, furosemide-sensitive (FS), bumetanide-sensitive (BS) and -resistant Na⁺ and K⁺ transport were studied in erythrocytes of Prague hypertensive rats (PHR) and Prague normotensive rats (PNR). Maximal transport rates (V_max) and apparent affinities for either intracellular Na⁺ or extracellular K⁺ (replaced by Rb⁺) were determined in red cells in which Na⁺ content varied around the physiological range and that were incubated in Na⁺ media. No major differences between PHR and PNR were disclosed in the kinetics of ion transport mediated by the Na⁺-K⁺ pump or BS inward Na⁺-K⁺ cotransport. FS Rb⁺ uptake was higher (due to a greater V_max) in red cells of PHR as compared to PNR. In cells with a lowered Na⁺ content this elevation of FS Rb⁺ uptake was largely due to an augmented K⁺-Cl^? cotransport which exhibits a low affinity for Rb⁺_o and is blocked by 1 mM furosemide but not by 10 μM bumetanide. Red cells of PHR and PNR strains did not differ in either Na⁺ or Rb⁺ leaks. A slight increase of red cell Na⁺ content in PHR was evaluated in terms of the pump-leak concept. The present study did not reveal any obvious kinetic abnormalities of red cell cation transport the presence of which in tissues involved in blood pressure regulation would favor the development or the maintenance of genetic hypertension in PHR.     

Evidence for apical Na+/H+ exchanger in bovine main pancreatic duct

The finding of a high Pco ₂ in basally secreted pancreatic juice of man and dog raises the hypothesis of proton secretion from ductal epithelial cells presumably through a Na⁺/H⁺ exchanger. To test this possibility, H⁺ luminal secretion and Na⁺ movements were measuredin vitro on samples of bovine pancreatic ducts mounted in Ussing-type chambers. The rate of luminal acidification measured by the pH stat method, using bicarbonate-free media gassed with 100% O₂, reached 2.75 Eq/cm²/hr. Proton secretion was blocked in the presence of 1 mM amiloride or in the absence of Na⁺ (replaced by choline) in the mucosal solution. Study of transepithelial²²Na fluxes in short-circuited tissue, bathed on both sides by control Ringer solution, gassed by 95% O₂-5% CO₂ demonstrated a net sodium transport from the mucosal to the interstitial side of the duct (net²²Na flux=3.23±0.8 Eq/cm²/hr). This net sodium transport was electroneutral and blocked by mucosal amiloride (0.5–1 mM/liter) or by interstitial ouabain (1 mM/liter). These results are consistent with the existence of a Na⁺/H⁺ exchanger on the luminal side of the bovine main pancreatic duct.     

INCREASED BLOOD PRESSURE AND LOSS OF ANP-INDUCED NATRIURESIS IN MICE LACKING DARPP-32 GENE

Atrial natriuretic peptide (ANP) is an important regulator of sodium metabolism and indirectly of blood pressure. Evidence has accumulated that ANP regulates sodium metabolism through a cascade of steps involving an increase in the level of cGMP, activation of cGMP-dependent protein kinase (PKG), and inhibition of renal tubular Na⁺,K⁺ -ATPase activity.

One of the major substrates for PKG is DARPP-32. In the present study we observed that ANP does not induce natriuresis in mice that lack DARPP- 32. In contrast, there was a 4-fold increase in urinary sodium excretion following ANP administration to wild type mice. ANP as well as Zaprinast, a selective inhibitor of cGMP phosophodiesterase, inhibited renal Na⁺,K⁺-ATPase activity in wild type mice but had no such effect in mice lacking DARPP-32. Mean arterial blood pressure, measured in conscious animals, was significantly increased in DARPP-32 deficient mice as compared to wild type mice.

The results confirm that DARPP-32 acts as a third messenger in the ANP signaling pathway in renal tissue and suggest an important role of DARPP-32 in the maintenance of normal blood pressure.     

Angiotensin II AT1 Receptor/Signaling Mechanisms in The Biphasic Effect of The Peptide on Proximal Tubular Na+,K+-ATPase

The present study was designed to determine the cellular signaling mechanisms responsible for mediating the effects of angiotensin II on proximal tubular Na⁺,K⁺-ATPase activity. Angiotensin II produced a biphasic effect on Na⁺,K⁺-ATPase activity: stimulation at 10^-13-10^-10M followed by inhibition at 10^-7-10^-5M of angiotensin II. The stimulatory and inhibitory effects of angiotensin II were antagonized by losartan (1nM) suggesting the involvement of AT, receptor. Angiotensin IT produced inhibition of forskolin-stimulated CAMP accumulation at 10^-13-10^-10M followed by a stimulation in basal CAMP levels at 10^-7-10^-5M. Pretreatment of proximal tubules with losartan (1nM) antagonized both the stimulatory and inhibitory effects of angiotensin II on CAMP accumulation. Pretreatment of the proximal tubules with pertussis toxin (PTx) abolished the stimulation of Na⁺,K⁺-ATPase activity but did not affect the inhibition of Na⁺,K⁺-ATPase activity produced by angiotensin II. Pretreatment of the tubules with cholera toxin did not alter the biphasic effect of angiotensin II on Na⁺,K⁺-ATPase activity. Mepacrine (l0μM), a phospholipase A2 (PLA2) inhibitor, reduced only the inhibitory effect of angiotensin II on Na⁺,K⁺-ATPase activity. These results suggest that the activation of AT₁angiotensin II receptors stimulates Na⁺,K⁺-ATPase activity via a PTx-sensitive G protein-linked inhibition of adenylyl cyclase pathway, whereas the inhibition of Na⁺,K⁺-ATPase activity following AT₁receptor activation involves multiple signaling pathways which may include stimulation of adenylyl cyclase and PLA2     

Cardiovascular remodelling and red blood cell Li+/Na+ countertransport in patients with type 1 diabetes and essential hypertension

Patients with type 1 (insulin-dependent) diabetes may develop a specific cardiac disease characterized by functional and structural abnormalities. The pathogenesis of the cardiac disease is poorly understood but cardiac and renal complications may coexist. Patients with overt diabetic nephropathy have increased red cell Li⁺/Na⁺ countertransport (CT), which reflects abnormal kinetic properties of the red cell membrane Na⁺/H⁺ exchange. Since the activation of Na⁺/H⁺ exchange has a key role in cell proliferation and cell growth, as well as in the regulation of cell sodium and cell pH and in the renal reabsorption of Na⁺ and bicarbonate, we have looked for relationships between red cell Li⁺/Na⁺ CT, Na⁺/H⁺ exchange and cardiovascular remodeling in patients with type 1 diabetes, essential hypertension and idiopathic familiar cardiomyopathy. In type 1 diabetes the maximal velocity of Li⁺/Na⁺ CT is positively correlated with interventricular septum thickness and the left ventricular wall to lumen ratio. Similar results were obtained in patients with essential hypertension. In these patients an increased Li⁺/Na⁺ CT is also associated with severe and drug-resistant hypertension and with significant vascular remodelling, estimated by the minimal post-ischaemic vascular resistance in the calf. Finally, Li⁺/Na⁺ CT is negatively correlated with diastolic relaxation of the left ventricle in familiar hypertrophic cardiomyopathy. From these data it appears that widespread abnormal kinetic properties of Na⁺/H⁺ exchange, estimated by increased red cell Li⁺/Na⁺ CT, may have epistatic effects on the pathogenesis of cardiac complications of type 1 diabetes and essential hypertension.     

Na+/H+ exchange and its inhibition in cardiac ischemia and reperfusion

Summary The characterization of various ion transport systems has led to a better understanding of the effects, which seem to take part in the impairment of ischemic and reperfused cardiac tissue. This review discusses the role of the Na⁺/H⁺ exchange system in the pathophysiology of ischemia and reperfusion and the beneficial effects of its inhibition.At the onset of ischemia intracellular pH (pH_i) decreases due to anaerobic metabolism and ATP hydrolysis, leading to an activation of Na⁺/H⁺ exchange. This in turn increases intracellular Na⁺ (Na⁺ _i) and activates Na⁺/K⁺ ATPase, with a consecutive increase of energy consumption. Since cellular Na⁺ and Ca⁺⁺ transport are coupled by the Na⁺/Ca⁺⁺ exchange system, which depends on the Na⁺ gradient, the high Na⁺ _i leads to increased intracellular Ca⁺⁺ (Ca⁺⁺ _i). After a certain period, Na⁺/H⁺ exchange is inactivated by a decrease of extracellular pH.In case of reperfusion the acid extracellular fluid is washed out, which reactivates Na⁺/H⁺ exchange, leading to an unfavourably fast restoration of pH_i and a second time to Na⁺ and Ca⁺⁺ _i overflow.High Ca⁺⁺ _i is assumed to be one of the main reasons for ischemic and reperfusion injury, like arrhythmias, myocardial contracture, stunning and necrosis.It seems that the inhibition of Na⁺/H⁺ exchange can interrupt this process at an early phase and prevent or delay the consequences of ischemia and reperfusion as demonstrated by numerous investigators.     

The Effect of Melittin on Na+ and Rb+ Transport in Cultured Skin Fibroblasts of the Spontaneously Hypertensive Rat

Melittin effect on transport of Na⁺ and Rb⁺(K⁺ analog) was examined in cultured skin fibroblasts originating from the Spontaneously Hypertensive, Wistar Kyoto and Wistar rats. Melittin increased both Na⁺ (²²Na⁺₊) uptake and 86Rb⁺ efflux as well as the activity of the Na⁺-pump (ouabain sensitive ⁸⁶Rb⁺ uptake) in all three preparations. The effect of the toxin was maximal at a dose of 160–240ng/10⁵ cells/ml. At this does, fibroblasts of the Spontaneously Hypertensive rat demonstrated the greatest response to melittin with respect to the increase in Na⁺ and Rb⁺ fluxes and increase in the intracellular Na⁺ concentrations. It is concluded that melittin can be utilized as a probe to delineate subtle differences in the cellular regulation of Na⁺ and K⁺ in the Spontaneously Hypertensive rat as compared with its normotensive controls.     

Isoform-specific alterations in cardiac and erythrocyte Na ,K-ATPase activity induced by norepinephrine

Background: Myocardial Na⁺,K⁺-ATPase activities are decreased in congestive heart failure because of an increase in plasma norepinephrine levels, but it is difficult to monitor the activities in the clinical setting.Methods and Results: This study investigated whether erythrocyte Na⁺,K⁺-ATPase activity can reflect myocardial enzyme activity and whether isoform-specific alterations occur in the presence of catecholamine. Na⁺,K⁺-ATPase activity was measured by the colorimetric method by using the left ventricular myocardium and erythrocytes prepared from eight rabbits given norepinephrine for 7 days and from eight control rabbits that received saline. The protein levels of total catalytic subunit and α₁ - or α₃-isoform of Na⁺,K⁺-ATPase were determined by Western blot analysis. Na⁺,K⁺-ATPase activity was lower in both myocardium and erythrocytes from norepinephrine-treated rabbits than control rabbits (P < .01 and P < .01, respectively). There was a close correlation in Na⁺,K⁺-ATPase activity between myocardium and erythrocytes (r = .963). Total catalytic subunit protein level was lower in myocardium from norepinephrine-treated rabbits than control rabbits, but the α₁-isoform level was similar between the two groups. The α₃-isoform level was lower in norepinephrinetreated rabbits than control rabbits. In erythrocytes, α₁-isoform was lower in norepinephrinetreated rabbits than control rabbits.Conclusions: Na⁺,K⁺-ATPase activity in myocardium could be reflected in erythrocyte membrane, although there was a difference in isoform-specific regulation between the two.     

Inhibition of Rat Na+/K+-ATPase Isoforms by Endogenous Digitalis Extracts from Neonatal Human Plasma

An unique endogeneous digitalis-like factor (EDLF) has been previously purified from human newborn cord plasma and its differential effects tested on the three well defined functional isoforms (α₁, α₂and α₃) of the alpha subunits of Na⁺/K⁺-ATPase in rat

EDLF specifically inhibits the enzymatic activity. It differs from ouabain by three criteria: a preincubation with the membranes is required for full activity, no effect on the rat cerebral α₃isoform and a steep dose-response curve with the same apparent potency for rat α₂and α₁isoforms of high (10^-7M) and low affinity (3 × 10^-5M) for ouabain. These results indicate that the Na⁺/K⁺-ATPase inhibitor involved in the regulation of sodium and body fluid volume and present in neonate and adult human plasmas is distinct from ouabain     

Decreased expression of Na+-H+ exchanger isoforms 1 and 3 in denervated spontaneously hypertensive rat kidney

To determine whether the sympathetic nerve plays a role in the regulation of Na⁺-H⁺ exchange (NHE) in the kidney of spontaneously hypertensive rats (SHR), we investigated the expression of NHE and NHE regulatory protein family (NHERF) in the denervated kidneys compared with intact kidneys. Twelve-week-old male SHR and age-matched Wistar Kyoto (WKY) rats were used. SHR were randomly assigned to the renal denervated (RDNX, n = 8) or Sham (n = 8) groups. The protein and mRNA expression of NHE1, NHE3, NHERF1 and NHERF2 were assessed in the kidney of the groups. Following the renal denervation, immunohistochemistry and western blot analysis showed that NHE1 and NHE3 protein were signi?cantly decreased in the kidney compared with Sham group. NHERF1 protein expression was signi?cantly increased in RDNX compared with Sham group, whereas NHERF2 protein expression was signi?cantly decreased after renal denervation. Similar results were observed at the mRNA level of NHE1, NHE3, NHERF1 and NHERF2 expression. The present ?ndings suggest that the renal sympathetic nervous system plays a role in the regulation of NHE1 and NHE3 in the kidney of SHR, and NHERF1 may be involved in the expression of NHE3 in the kidney of SHR.     

ATP-dependent Ca2+ Transport and Na+/Ca2+ Exchange System in Renal Basolateral Plasmamembranes of Spontaneously Hypertensive Rats

A common abnormality of cellular Ca²⁺ handling in most tissues of spontaneously hypertensive rats (SHR) has been suggested. Therefore we investigated the ATP-dependent Ca²⁺ transport and Na⁺/Ca²⁺ exchange system in basolateral membrane vesicles (BLMV) of renal cortices from SHR and normotensive Wistar-Kyoto rats (WKY) at 12 and 20 weeks of age. In WKY the maximal transport rate of the ATP-dependent Ca²⁺ transport was 5.7 nmol/min/mg prot with an affinity for Ca²⁺ of 0.14 µM. These values were not significantly different in SHR at both ages studied. High concentrations of Na⁺ inhibited ATP-dependent Ca²⁺ uptake by 40% in BLMV of SHR and WKY. Low concentrations of Na⁺ stimulated ATP-dependent Ca²⁺ transport 20% in both rats. These findings suggest equal Na⁺/Ca²⁺ exchange activity in WKY and SHR. The present study failed to show a significant change in ATP-dependent Ca²⁺ transport and Na⁺/Ca²⁺ exchange activity in renal BLMV in SHR, suggesting that the Ca²⁺ homeostasis of the cortical cells is normal in SHR as far as the plasmamembrane is concerned.     

Dopamine-1 Receptor G-Protein Coupling and the Involvement of Phospholipase A2 in Dopamine-1 Receptor Mediated Cellular Signaling Mechanisms in the Proximal Tubules of SHR

Dopamine-induced natriuretic response which results from the activation of tubular dopamine₁ (DA₁) receptors is diminished in spontaneously hypertensive rats (SHR). This may be a result of alterations occurring at the receptor level and within the cellular signaling pathway which ultimately causes inhibition of Na⁺,K⁺-ATPase. There have been reports showing that DA, receptor induced inhibition of Na⁺,K⁺-ATPase is abolished in SHR which is due to a decreased activation of PLC and PKC by dopamine. Of the mechanisms, adenylyl cyclase and phospholipase C are two known enzymes linked to DA₁ receptors via G proteins. Furthermore, the involvement of phospholipase A2 (PLA2) has also been reported in this process. However, the site of defect in DA₁ receptor signaling pathway in SHR is still not well understood. This report will (i) review the coupling of DA₁ receptor with G proteins and their levels in Wistar Kyoto (WKY) rats and SHR and (ii) discuss studies dealing with the role of PLA2 in dopamine-induced inhibition of Na⁺,K⁺-ATPase in WKY rat and SHR kidneys. Fenoldopam, DA₁ receptor selective agonist stimulated [³⁵S]GTP_γ S binding in a concentration (10^?9-10^?4 M)-dependent manner in WKY rats which was attenuated in SHR. Fenoldopam (10 μM)-induced stimulation of [³⁵S]GTP_γ S binding was significantly reduced by a DA₁ receptor selective antagonist, SCH 23390 suggesting the involvement of DA, receptor. Furthermore, the specific antipeptides Gsa, and Gq/l la significantly blocked fenoldopam-stimulation of [35S]GTPrS binding suggesting the coupling of DA, receptor with both the G proteins. Western analysis revealed a significant decrease in Gq/lla but no changes in Gsa in SHR compared to WKY rats. Dopamine inhibited Na′,K+-ATPase activity in a concentration (lo5 M)-dependent manner in WKY rats while it failed to inhibit the enzyme activity in SHR. Dopamine (10 &I)-induced inhibition in Na′,K'′ATPase activity was significantly blocked by mepacrine (a PLA2 inhibitor) suggesting the involvement of PLA2 in dopamine-mediated inhibition of Na′,K′-ATPase. Arachidonic acid (AA), a PLA2 product, inhibited Na′,K′-ATPase in a concentration (1-100 &I)-dependent manner in WKY rats while the inhibition in SHR was significantly attenuated (IC5,: 7.5, A4 in WKY and 80 &I in SHR). Furthermore, lower concentration (1 pM) of AA stimulated the enzyme activity in SHR. This suggests a defect in the metabolism of AA in SHR. Proadifen (10 pM), 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 (i) the reduced activation of G proteins following DA, receptor stimulation, (ii) reduced amount of Gq/l l a and (iii) a defect in the AA metabolism may be responsible for the reduced dopaminergic inhibition of sodium pump activity and a diminished natriuretic response to dopamine in SHR.