Adsorption and migration of single metal atoms on the calcite (10.4) surface.

Transition metal atoms are one of the key ingredients in the formation of functional 2D metal organic coordination networks. Additionally, the co-deposition of metal atoms can play an important role in anchoring the molecular structures to the surface at room temperature. To gain control of such processes requires the understanding of adsorption and diffusion properties of the different transition metals on the target surface. Here, we used density functional theory to investigate the adsorption of 3d (Ti, Cr, Fe, Ni, Cu), 4d (Zr, Nb, Mo, Pd, Ag) and 5d (Hf, W, Ir, Pt, Au) transition metal adatoms on the insulating calcite (10.4) surface. We identified the most stable adsorption sites and calculated binding energies and corresponding ground state structures. We find that the preferential adsorption sites are the Ca-Ca bridge sites. Apart from the Cr, Mo, Cu, Ag and Au all the studied metals bind strongly to the calcite surface. The calculated migration barriers for the representative Ag and Fe atoms indicates that the metal adatoms are mobile on the calcite surface at room temperature. Bader analysis suggests that there is no significant charge transfer between the metal adatoms and the calcite surface.

Mitogen-activated protein kinase upregulation reduces renal D1 receptor affinity and G-protein coupling in obese rats.

Reactive oxygen species play a key role in pathophysiology of cardiovascular diseases by modulating G-protein-coupled receptor signaling. We have shown that treatment of animal models of diabetes and aging with tempol decreases oxidative stress and restores renal dopamine D1 receptor (D1R) function. In present study, we determined whether oxidation of D1R and upregulation of mitogen-activated protein kinases (MAPK) were responsible for decreased D1R signaling in obese animals. Male lean and obese Zucker rats were supplemented with antioxidants tempol or lipoic acid for 2 weeks. Compared to lean, obese animals were hyperglycemic and hyperinsulinemic with increased oxidative stress, D1R oxidation and decreased glutathione levels. These animals had decreased renal D1R affinity and basal coupling to G-proteins. SKF-38393, a D1R agonist failed to stimulate G-proteins and adenylyl cyclase. Obese animals showed marked increase in renal MAPK activities. Treatment of obese rats with tempol or lipoic acid decreased blood glucose, reduced oxidative stress, and restored the basal D1R G-protein coupling. Antioxidants also normalized MAPK activities and restored D1R affinity and SKF-38393 induced D1R G-protein coupling and adenylyl cyclase stimulation. These studies show that D1R oxidation and MAPK upregulation contribute to D1R dysfunction in obese animals. Consequently, antioxidants while reducing the oxidative stress normalize the MAPK activities and restore D1R signaling.

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.

Dopamine fails to inhibit Na,H-exchanger in proximal tubules of obese Zucker rats.

Dopamine via the activation of D1-like receptors inhibits Na,K-ATPase and Na,H-exchanger and subsequently increases sodium excretion. We have previously reported that dopamine failed to inhibit Na,K-ATPase in the proximal tubules (PTs) of obese Zucker rats. The present study was designed to determine the effect of dopamine on Na,H-exchanger in PTs of lean and obese Zucker rats, and examine D1-like receptor-coupled signal transduction pathway mediating the inhibition of Na,H-exchanger. We found that dopamine inhibited Na,H-exchanger in the PTs of lean rats but this response was absent in obese rats. In brush border membranes, [3H]SCH 23390 binding revealed a approximately 45% reduction in D1-like receptor binding sites in obese compared to lean rats. Dopamine stimulated cAMP accumulation in PTs of lean but not in obese rats. Forskolin-mediated stimulation of cAMP was similar in lean and obese rats. Dopamine as well as forskolin and dibutyryl cAMP-mediated stimulation of protein kinase A (PKA) was reduced in PTs of obese compared to lean rats. The data suggest that reduction in D1-like receptor binding sites, defective coupling with signaling pathway and inability of PKA activation may be responsible for the failure of dopamine to inhibit Na,H-exchanger in PTs of obese rats. This phenomenon may contribute to an increase in sodium reabsorption and development of hypertension in obese Zucker rats.

Salt intake and intestinal dopaminergic activity in adult and old Fischer 344 rats.

We have earlier shown that the renal dopaminergic system failed to respond to high salt (HS) intake in old (24-month-old) Fisher 344 rats (Hypertension 1999;34:666-672). In the present study, intestinal Na+,K+-ATPase activity and intestinal dopaminergic tonus were evaluated in adult and old Fischer 344 rats during normal salt (NS) and HS intake. Basal intestinal Na+,K+-ATPase activity (nmol Pi/mg protein/min) in adult rats (142+/-6) was higher than in old Fischer 344 rats (105+/-7). HS intake reduced intestinal Na+,K+-ATPase activity by 20% (P<0.05) in adult, but not in old rats. Dopamine (1 microM) failed to inhibit intestinal Na+,K+-ATPase activity in both adult and old Fischer 344 rats (NS and HS diets). In adult animals, co-incubation of pertussis toxin with dopamine (1 microM) produced a significant inhibitory effect in the intestinal Na+,K+-ATPase activity. L-DOPA and dopamine tissue levels in the intestinal mucosa of adult rats were higher (45+/-9 and 38+/-4 pmol/g) than those in old rats (27+/-9 and 14+/-1 pmol/g). HS diet did not change L-DOPA and DA levels in both adult and old rats. DA/L-DOPA tissue ratios, an indirect measure of dopamine synthesis, were higher in old (1.1+/-0.2) than in adult rats (0.6+/-0.1). Aromatic L-amino acid decarboxylase (AADC) activity in the intestinal mucosa of old rats was higher than in adult rats. HS diet increased the AADC activity in adult rats, but not in old rats. It is concluded that intestinal dopaminergic tonus in old Fisher 344 rats is higher than in adult rats and is accompanied by lower basal intestinal Na+,K+-ATPase activity. In old rats, HS diet failed to alter the intestinal dopaminergic tonus or Na+,K+-ATPase activity, whereas in adult rats increases in AADC activity were accompanied by decreases in Na+,K+-ATPase activity. The association between salt intake, increased dopamine formation and inhibition of Na+,K+-ATPase at the intestinal level was not as straightforward as that described in renal tissues.

Activation of dopamine D(1)-like receptor causes phosphorylation of alpha(1)-subunit of Na(+),K(+)-ATPase in rat renal proximal tubules.

Dopamine causes inhibition of Na(+),K(+)-ATPase activity via activation of dopamine D(1)-like receptors. It is the phosphorylation of Serine(18) of the alpha(1)-subunit of Na(+),K(+)-ATPase which results in the inhibition of the enzyme activity; however, such a phosphorylation by dopamine D(1)-like receptor agonist has not been demonstrated in the proximal tubules. We show here by immunoprecipitation and detection with phosphoserine antibody that SKF 38393, a dopamine D(1)-like receptor agonist, causes phosphorylation of the alpha(1)-subunit of Na(+),K(+)-ATPase. The effect of (+/-)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrochloride, SKF 38393, is blocked by R(+)-7-choro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-benzazepine hydrochloride, SCH 23390, a dopamine D(1)-like receptor antagonist, and staurosporin, a protein kinase C inhibitor. The phosphorylation is also increased by phorbol 12-13 dibutyrate ester. However, Rp-cAMP triethylamine, an inhibitor of protein kinase A, does not affect the SKF 38393-mediated phosphorylation of Na(+),K(+)-ATPase. Therefore, these results provide the evidence that dopamine D(1)-like receptor activation causes phosphorylation of the alpha(1)-subunit of Na(+),K(+)-ATPase in renal proximal tubules via protein kinase C pathway.

Dopamine inhibits na,h-exchanger via D1-like receptor-mediated stimulation of protein kinase a in renal proximal tubules.

Dopamine causes natriuresis and diuresis via activation of D1-like receptors located in the renal proximal tubules. It is reported that this response to dopamine results from the inhibition of Na,H-exchanger and Na,K-ATPase. Earlier studies have suggested a role of protein kinase A (PKA) in the inhibition of Na,H-exchanger, however, the effect of dopamine or the dopamine receptor subtype responsible for the stimulation of PKA has not been reported. Present study was designed to examine the effect of dopamine and D1-like receptor agonist, SKF 38393, on the stimulation of PKA activity in rat renal proximal tubules. Dopamine and SKF 38393 (1 nM - 1 microM) caused stimulation of PKA activity, an effect which was antagonized by a D1-like receptor antagonist, SCH 23390 (10 microM). Stimulation of PKA activity was also seen with forskolin and di-butyryl cAMP. We also observed that dopamine and SKF 38393 inhibited Na,H-exchanger activity in the proximal tubules. This response was blocked by SCH 23390 and Rp-cAMPS triethylamine, a selective inhibitor of PKA. Similarly, forskolin and di-butyryl cAMP inhibited Na,H-exchanger activity. The data provide direct evidence showing that dopamine, through the activation of D1-like receptors stimulates PKA activity which in turn inhibits Na,H-exchanger in the proximal tubules.

Diminished natriuretic response to dopamine in old rats is due to an impaired D1-like receptor-signaling pathway.

BACKGROUND: Dopamine (DA) causes natriuresis and diuresis, which results from activation of D1-like receptor (D1R) located on proximal tubules. Earlier, we reported that DA failed to inhibit Na,K-ATPase in proximal tubules of old Fischer 344 rats. The present study was designed to investigate the functional consequence of this phenomenon. METHODS: Measurements of the functional (natriuretic and diuretic) response to intravenously infused DA and SKF 38393 (D1R agonist) in adult (6 month) and old (24 month) Fischer 344 rats were taken. Biochemical measurements were carried out to determine the potential defects in D1R and its signaling pathway in proximal tubules of old rats. RESULTS: We found that intravenous infusion of DA and SKF 38393 caused natriuresis and diuresis in adult rats, but this response was blunted in old rats. In the isolated proximal tubules, DA and SKF 38393 inhibited Na,H-exchanger (NHE) in adult rats; however, this inhibition was attenuated in old rats. Radioligand binding revealed approximately 46% reduction in D1R binding sites in brush border membranes (BBMs) in old compared with adult rats. SKF 38393 stimulated [35S]GTPgammaS binding in BBM in adult rats, but not in old rats, suggesting an impaired D1R-G protein coupling. DA and SKF 38393 stimulated adenylyl cyclase (AC) activity in adult but not in the old rats. Forskolin and NaF stimulated AC activity in a comparable manner in adult and old rats, indicating no defect in AC and G proteins. DA and SKF 38393 failed to stimulate protein kinase A (PKA) activity in proximal tubules of old rats. Dibutyryl-cAMP-mediated PKA activation was also absent in old rats. CONCLUSIONS: A decrease in D1R binding sites, a coupling defect with G proteins, and a defect in PKA activation lead to diminished DA-mediated inhibition of NHE in old rats, which may contribute to the blunted natriuretic response to DA in these animals.

The role of intrarenal nitric oxide in the natriuretic response to dopamine-receptor activation.

Dopamine and dopamine-1 receptor agonists produce diuresis and natriuresis by causing changes in renal hemodynamics and by the activation of dopamine-1 receptors located within the various regions of the nephron. Nitric oxide plays an important role in the maintenance of systemic and regional hemodynamics. The present study was undertaken to investigate the effect of locally generated nitric oxide on renal function and its potential influence on the renal responses to dopamine-1 receptor agonists. The intrarenal infusion of a nitric oxide synthase inhibitor, L-NAME, (50 microg/kg min for 90 min) in anesthetized rats produced significant decreases in urine volume, urinary sodium excretion, glomerular filtration rate and fractional sodium excretion. These changes in renal function were associated with a concomitant decrease in urinary nitrate excretion, an indicator of nitric oxide release. However, L-NAME at this dose did not produce any significant changes in mean arterial pressure or heart rate. Intravenous infusion of fenoldopam (1 microg/kg min for 30 min), a selective dopamine-1 receptor agonist, produced diuresis and natriuresis without causing any changes in mean arterial pressure and heart rate. These renal effects of fenoldopam were significantly attenuated in animals that received the simultaneous infusion of L-NAME (intrarenal). Similar results were obtained with dopamine in that the natriuretic and diuretic response to dopamine was also attenuated during simultaneous infusion of dopamine with L-NAME. In addition, the diuresis and natriuresis produced by fenoldopam and dopamine was associated with increases in urinary nitrate excretion. Interestingly, these increases in the nitrate levels seen with fenoldopam and dopamine were also significantly reduced in the presence of L-NAME. These results indicate that intrarenal nitric oxide plays an important role in regulating renal sodium excretion and that an intact renal nitric oxide system is required for the full expression of diuretic and natriuretic response seen during dopamine-1 receptor activation.

Defective renal dopamine D1-like receptor signal transduction in obese hypertensive rats.

It is reported that dopamine promotes renal sodium excretion via activation of D1-like dopamine receptors located on the proximal tubules. In spontaneously hypertensive rats the natriuretic and diuretic response to exogenously administered and endogenously produced dopamine is reduced, which results from a diminished dopamine-induced inhibition of the enzyme, Na+,K+-ATPase. The present study was designed to examine dopamine-receptor mediated inhibition of Na+,K+-ATPase and its associated signal transduction pathway in the proximal tubules of Zucker obese and lean control rats. The obese animals were hypertensive, hyperinsulinaemic and hyperglycaemic compared with the lean rats. While dopamine caused inhibition of Na+,K+-ATPase activity in lean rats, this effect was significantly attenuated in the obese animals. There was significant reduction in D1-like receptor numbers in the basolateral membranes of obese rats compared with lean rats with no change in the affinity to the ligand [3H]SCH 23390 between the two groups of rats. Dopamine failed to stimulate G proteins as measured by [35S]GTPgammaS binding in the obese rats. Also, dopamine was unable to cause phospholipase-C activation in obese rats, but it did activate phospholipase-C in lean rats. These results show that reduction in D1-like receptor numbers and a defect in receptor-G protein coupling may account for the inability of dopamine to activate the D1-like receptor-coupled signal transduction pathway and cause inhibition of Na+,K+-ATPase in the obese hypertensive rats.

Defective dopamine receptor function in proximal tubules of obese zucker rats.

Some of the pathophysiological consequences of obesity include insulin resistance, increased renal sodium reabsorption, and the development of hypertension. Dopamine promotes renal sodium excretion via activation of D(1)-like receptors present on the proximal tubules. Reduced dopamine-induced natriuresis and a defect in D(1)-like receptor function have been reported in the proximal tubules of hypertensive animals. The present study investigated D(1)-like dopamine receptors and associated G proteins as the initial signaling components in the proximal tubular basolateral membranes of obese Zucker and control lean Zucker rats. We found that the obese rats were hyperinsulinemic, hyperglycemic, and hypertensive compared with the lean rats. Dopamine produced concentration-dependent inhibition of Na,K-ATPase activity in the proximal tubules of lean rats, whereas the inhibitory effect of dopamine was reduced in obese rats. The D(1)-like receptors measured by [(3)H]SCH 23390 binding revealed an approximately 45% decrease in B(max) without a change in K(d) in the basolateral membranes of obese rats compared with lean rats. Although we found an increase in G(q)/11alpha and no change in G(s)alpha in the basolateral membranes of obese rats, dopamine and SKF 38393 failed to stimulate G proteins as measured by [(35)S]GTPgammaS binding in obese rats, suggesting a receptor-G protein coupling defect. We conclude that decrease in D(1)-like dopamine receptor binding sites and diminished activation of G proteins, resulting perhaps from defective coupling, led to the reduced inhibition by dopamine of Na,K-ATPase activity in the proximal tubules of obese Zucker rats. Such a defect in renal dopamine receptor function may contribute to sodium retention and development of hypertension in obese rats.

Aging, high salt intake, and renal dopaminergic activity in Fischer 344 rats.

The present study examined renal dopaminergic activity and its response to high salt (HS) intake in adult (6-month-old) and old (24-month-old) Fischer 344 rats. Daily urinary excretion of L-3, 4-dihydroxyphenylalanine (L-DOPA), dopamine, and its metabolites 3, 4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid was similar in adult and old rats; by contrast, daily urinary excretion of norepinephrine in old rats was almost twice that in adult animals. HS intake (1% NaCl) over a period of 24 hours resulted in a 2-fold increase in the urinary excretion of dopamine, DOPAC, and norepinephrine in adult animals but not in old animals. Norepinephrine and L-DOPA plasma levels did not change during HS intake and were similar in both groups of rats. The natriuretic response to an HS intake in old rats (from 4.7+/-0.4 to 10.7+/-2.0 nmol. kg(-1). d(-1); Delta=6.0+/-0.9 nmol. kg(-1). d(-1)) was less than in adult rats (from 5.2+/-0.4 to 13.5+/-2.5 nmol. kg(-1). d(-1); Delta=8.3+/-0.8 nmol. kg(-1). d(-1)). A diuretic response to HS intake was observed in adult rats (from 20.9+/-2.3 to 37.6+/-2.8 mL. kg(-1). d(-1)) but not in old rats (from 37.7+/-5.7 to 42.3+/-6. 0 mL. kg(-1). d(-1)). Dopamine levels and dopamine/L-DOPA ratios in the renal cortex of old rats were greater than in adult rats. HS intake increased both dopamine levels and dopamine/L-DOPA ratios in the renal cortex of adult rats but not in old rats. Aromatic L-amino acid decarboxylase activity was higher in old rats than in adult rats; HS intake increased L-amino acid decarboxylase activity (nmol. mg protein(-1). l5 min(-1)) in adult rats (from 67+/-1 to 93+/-1) but not in old rats (from 86+/-2 to 87+/-2). Dopamine inhibited Na(+),K(+)-ATPase activity in proximal tubules obtained from adult rats, but it failed to exert such an inhibitory effect in old rats. It is concluded that renal dopaminergic tonus in old rats is higher than in adult rats but fails to respond to HS intake as observed in adult rats. This may be due in part to the inability of dopamine to inhibit Na(+),K(+)-ATPase activity in old rats.

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.

Bromocriptine regulates angiotensin II response on sodium pump in proximal tubules.

-Dopamine and angiotensin II (Ang II) receptors have been reported to exhibit an interaction in renal proximal tubules. The present study was designed to investigate the regulation by a D2-like dopamine receptor of Ang II-mediated stimulation of Na,K-ATPase activity in the renal proximal tubules. Ang II (10(-13) to 10(-9) mol/L) stimulated Na,K-ATPase activity in the proximal tubules that was completely abolished when the tubules were pretreated with the D2-like receptor agonist bromocriptine (1 micromol/L) for 30 minutes. The effect of bromocriptine on Ang II response was prevented by domperidone (1 micromol/L), a D2-like dopamine receptor antagonist. Similarly, the inhibition of forskolin (1 micromol/L)-induced cAMP accumulation caused by Ang II (10 pmol/L) was also abolished in bromocriptine-pretreated tubules. Basal and forskolin-stimulated cAMP was not significantly different in bromocriptine-treated tubules compared with the control. [3H]-Ang II binding sites (angiotensin type 1 [AT1] receptors) were reduced by approximately 65% in bromocriptine-treated proximal tubules, a result that was further substantiated by Western blot analysis revealing a 50% decrease in AT1 receptors in bromocriptine-treated tubules compared with the control. Western blot analysis of G proteins revealed a 2-fold increase in Gsalpha and a 20% decrease in Gialpha1 and Gialpha2 in the bromocriptine-treated proximal tubules. Bromocriptine (1 micromol/L) alone stimulated Na,K-ATPase activity during the first 30 minutes of incubation, and thereafter the stimulation fell to the basal level. Similarly, bromocriptine-mediated inhibition of cAMP lasted only up to 20 minutes. The data suggest that preactivation of D2-like dopamine receptors abolishes Ang II-mediated stimulation of Na,K-ATPase activity and inhibition of cAMP accumulation. This phenomenon may be a consequence of a decrease in AT1 receptors and alterations in G protein levels in the proximal tubules. We propose that such a regulation of Ang II response by bromocriptine is the result of heterologous desensitization of the D2-like receptor system.

Renal dopamine receptor function in hypertension.

Dopamine plays an important role in the regulation of renal sodium excretion. The synthesis of dopamine and the presence of dopamine receptor subtypes (D1A, D1B, as D1-like and D2, and D3 as D2-like) have been shown within the kidney. 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 to the multiple cellular signaling systems (namely, adenylyl cyclase, phospholipase C, and phospholipase A2) in the different regions of the nephron. Defective renal dopamine production and/or dopamine receptor function have been reported in human primary hypertension as well as in genetic models of animal hypertension. There may be a primary defect in D1-like receptors and an altered signaling system in the proximal tubules that lead to reduced dopamine-mediated effects on renal sodium excretion in hypertension. Recently, it has been shown in animal models that the disruption of either D1A or D3 receptors at the gene level causes hypertension in mice. Dopamine and dopamine receptor agonists also provide therapeutic potential in treatment of various cardiovascular pathological conditions, including hypertension. However, because of the poor bioavailability of the currently available compounds, the use of D1-like agonists is limited to the management of patients with severe hypertension when a rapid reduction of blood pressure is clinically indicated and in acute management of patients with heart failure. In conclusion, there is convincing evidence that dopamine and dopamine receptors play an important role in regulation of renal function, suggesting that a defective dopamine receptor/signaling system may contribute to the development and maintenance of hypertension. Further studies need to be directed toward establishing a direct correlation between defective dopamine receptor gene in the kidney and development of hypertension. Subsequently, it may be possible to use a therapeutic approach to correct the defect in dopamine receptor gene causing the hypertension.

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(-10) M followed by inhibition at 10(-7) - 10(-5) M of angiotensin II. The stimulatory and inhibitory effects of angiotensin II were antagonized by losartan (1nM) suggesting the involvement of AT1 receptor. Angiotensin II produced inhibition of forskolin-stimulated cAMP accumulation at 10(-13) - 10(-10) M followed by a stimulation in basal cAMP levels at 10(-7) - 10(-5) M. 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 (10microM), 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 AT1 angiotensin 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 AT1 receptor activation involves multiple signaling pathways which may include stimulation of adenylyl cyclase and PLA2.

Stimulation of protein kinase C rapidly reduces intracellular Na+ concentration via activation of the Na+ pump in OK cells.

Na+ reabsorption is regulated in proximal tubules by hormones that stimulate protein kinase C (PKC). To determine whether stimulation of PKC causes a reduction in intracellular Na+ concentration ([Na+]i) that might link Na+ pump activation to increased Na+ reabsorption, [Na+]i was measured in kidney cells loaded with the Na+-sensitive fluorescent indicator SBFI. Rapid digital imaging fluorescence microscopy determinations were performed in epithelial kidney cells transfected with the rodent Na+ pump alpha1 cDNA. In 42 determinations, the basal [Na+]i was 19.7 +/- 2.4 mM. Stimulation of PKC reduced the [Na+]i to 5.6 +/- 0.6 mM in approximately 10 sec. This drastic change in [Na+]i requires a transient 74-120-fold increase in Na+ pump activity. After the new steady state [Na+]i is reached, the Na+ pump is 58% activated. The entry of Na+ into the cells is not affected by stimulation of PKC; therefore, the reduction in [Na+]i is exclusively dependent on activation of the Na+ pump. Accordingly, PKC stimulation does not affect the [Na+]i of cells expressing a mutant Na+ pump that is not stimulated by PKC. The decrease in [Na+]i observed in cells transfected with the rodent Na+ pump alpha1 cDNA is large and sufficiently fast that it is expected to stimulate rapidly passive Na+-influx into the cells, thereby accounting for the observed PKC-induced stimulation of Na+ reabsorption.

Alterations in dopamine DA1 receptor and G proteins in renal proximal tubules of old rats.

The present study examines the effect of dopamine DA1-receptor agonists on the renal proximal tubular Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) activity and quantitates DA1 receptors and the coupled G proteins in Fischer 344 model of adult (6 mo old) and old (23 mo old) rats. Dopamine and the preferential DA1-receptor agonist, SKF-38393, produced a concentration-dependent inhibition of Na(+)-K(+)-ATPase activity in proximal tubules from adult rats, whereas the enzyme activity was unaffected by these agonists in the old rats. The binding of DA1-receptor antagonist [3H]Sch-23390 in the proximal tubular basolateral membranes showed a marked decrease (approximately 47%) in the receptor numbers in old compared with adult rats, whereas dissociation constant (Kd) values in old compared with adult rats were not significantly different. Dopamine and SKF-38393 stimulated 35S-labeled guanosine 5'-O-(3-thiotriphosphate) binding in adult rats, but there was no significant effect on the binding in the old rats. Quantification of G2 alpha and Gq/11 alpha using Western analysis revealed a significant increase in quantities of both the G proteins in old rats. The data suggest that a reduction in DA1 receptor number and subsequently reduced G protein activation may be the causative factors for the impairment in DA1 receptor-mediated inhibition of Na(+)-K(+)-ATPase activity in the proximal tubules of old rats.

Renal dopamine DA1 receptor coupling with G(S) and G(q/11) proteins in spontaneously hypertensive rats.

The dopamine DA1 receptor transduces its signal via adenylyl cyclase and phospholipase C in the renal proximal tubule, which has been suggested to be defective at the level of receptor-G protein coupling in spontaneously hypertensive rats (SHR). We prepared basolateral membranes from Wistar-Kyoto (WKY) rats and SHR to determine the coupling of DA1 receptor with G proteins, especially G(q/11). Fenoldopam, a DA1-receptor agonist, produced a time- and concentration-dependent stimulation in 35S-labeled guanosine 5'-O-(3-thiotriphosphate) ([35S]GTPgammaS) binding in WKY rats. Fenoldopam-induced (10 microM) stimulation was significantly inhibited by a DA1-receptor antagonist, Sch-23390. Specific antibodies against COOH terminals of G(S)alpha and G(q/11)alpha produced 50-60% and 40-50% inhibition, respectively, in fenoldopam stimulation of [35S]GTPgammaS binding. Western analysis of basolateral membranes with these antibodies revealed the presence of G(S)alpha (45 kDa) and G(q/11)alpha (42 kDa). Fenoldopam stimulation of [35S]GTPgammaS binding was significantly attenuated in SHR compared with WKY rats. Parathyroid hormone stimulation of [35S]GTPgammaS binding was similar in SHR and WKY rats, whereas stimulation by phenylephrine was significantly reduced in SHR. Densitometric quantification of 42-kDa band showed a reduced amount in SHR, whereas the density of 45-kDa band was not significantly different compared with WKY rats. We provide the direct evidence showing the coupling of DA1 receptor with G(q/11)alpha and G(S)alpha and propose that, in addition to a defect in the receptor-G protein coupling, a reduced amount of G(q/11)alpha observed in the hypertensive animals may also contribute to the diminished dopamine-induced inhibition of Na+-K+-adenosinetriphosphatase in SHR.