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

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

Actions of endothelin-1 on cultured rat renomedullary interstitial cells are modulated by nitric oxide.

1. Cultured renomedullary interstitial cells (RMIC) isolated from 4-week-old Sprague-Dawley rat kidneys possess ETA receptors, as identified by reverse transcription-polymerase chain reaction (RT-PCR). 2. Treatment with endothelin (ET)-1 (10(-6) mol/L) increases the intracellular inositol 1,4,5-trisphosphate concentrations within 10 s and intracellular calcium concentrations after 7 s. 3. Endothelin-1 (10(-7) and 10(-10) mol/L) induced increases in intracellular cAMP concentrations, but only in the presence of N omega-nitro-L-arginine, a nitric oxide synthase (NOS) inhibitor. Addition of ET-1 (10(-10) mol/L) to the RMIC culture led to increases in intracellular cGMP concentrations through activation of NOS. 4. In the presence of ET-1 (10(-7) and 10(-10) mol/L) and during NOS inhibition, RMIC responded with increased cell proliferation and extracellular matrix (ECM) synthesis. These responses were abolished by BQ-123 (10(-6) mol/L), suggesting mediation via the ETA receptor subtype. The proliferative effect of ET-1 was also abolished by atrial natriuretic peptide (10(-6) mol/L). 5. The present study provides evidence that binding of ET-1 to ETA receptors on RMIC activates several intracellular second messenger systems that mediate cell proliferation and ECM synthesis. 6. These results also highlight an important interaction between ET-1 and nitric oxide in the control of RMIC function.

Inhibition of proximal tubular fluid absorption by nitric oxide and atrial natriuretic peptide in rat kidney.

Atrial natriuretic factor (ANF) and nitric oxide (NO) stimulate production of guanosine 3',5'-cyclic monophosphate (cGMP) and are natriuretic. Split-drop micropuncture was performed on anesthetized rats to determine the effects of ANF and the NO donor sodium nitroprusside (SNP) on proximal tubular fluid absorption rate (Jva). Compared with control solutions, SNP (10(-4) M) decreased Jva by 23% when administered luminally and by 35% when added to the peritubular perfusate. Stimulation of fluid uptake by luminal angiotensin II (ANG II; 10(-9) M) was abolished by SNP (10(-4) and 10(-6) M). In proximal tubule suspensions, ANF (10(-6) M) increased cGMP concentration to 143%, whereas SNP (10(-6), 10(-5), 10(-4), 10(-3) M) raised cGMP to 231, 594, 687, and 880%, respectively. S-nitroso-N-acetylpenicillamine (SNAP) also raised cGMP concentrations with similar dose-response relations. These studies demonstrate inhibition by luminal and peritubular NO of basal and ANG II-stimulated proximal fluid absorption in vivo. The ability of SNP to inhibit basal fluid uptake whereas ANF only affected ANG II-stimulated transport may be because of production of higher concentrations of cGMP by SNP.

Effects of angiotensin II on cultured rat renomedullary interstitial cells are mediated by AT1A receptors.

Renomedullary interstitial cells (RMICs) are prominent in the inner medullary interstitium and have binding sites for several vasoactive agents, including angiotensin II (ANG II). Although the functional role of RMICs remains largely unknown, it is likely that the interaction between RMICs and vasoactive peptides is important in the regulation of renal function. The current investigation characterizes the cellular responses following treatment of RMICs with ANG II. Studies were performed on RMICs isolated from Sprague-Dawley rat kidneys. 125I-labeled [Sar1,Ile8]ANG II specifically bound to RMICs at sites determined by reverse transcription-polymerase chain reaction to be of the AT1A subtype. ANG II (10(-6) and 10(-10) M) had no effect on either basal or forskolin-stimulated adenosine 3',5'-cyclic monophosphate accumulation in RMICs but increased intracellular inositol 1,4,5-trisphosphate concentration after 10 s and intracellular calcium concentration after 18 s. For RMICs plated at low densities, ANG II (10(-6) M) induced an increase in [3H]thymidine incorporation, mediated through the AT1-receptor subtype. For RMICs plated at high densities, ANG II (10(-6) M) induced an increase in extracellular matrix synthesis as detected by trans-35S incorporation, an effect also mediated by AT1 receptors. We conclude that ANG II AT1A receptors on cultured RMICs are coupled to intracellular second messenger pathways leading to hyperplasia and synthesis of extracellular matrix.

Regulation of renal tubular sodium transport by angiotensin II and atrial natriuretic factor.

1. The effects of angiotensin II (AngII) on water and electrolyte transport are biphasic and dose-dependent, such that low concentrations (10(-12) to 10(-9) mol/L) stimulate reabsorption and high concentrations (10(-7) to 10(-6) mol/L) inhibit reabsorption. Similar dose-response relationships have been obtained for luminal and peritubular addition of AngII. 2. The cellular responses to AngII are mediated via AT1 receptors coupled via G-regulatory proteins to several possible signal transduction pathways. These include the inhibition of adenylyl cyclase, activation of phospholipases A2, C or D and Ca2+ release in response to inositol-1,4,5,-triphosphate or following Ca2+ channel opening induced by the arachidonic acid metabolite 5,6,-epoxy-eicosatrienoic acid. In the brush border membrane, transduction of the AngII signal involves phospholipase A2, but does not require second messengers. 3. Angiotensin II affects transepithelial sodium transport by modulation of Na+/H+ exchange at the luminal membrane and Na+/HCO3 cotransport, Na+/K(+)-ATPase activity and K+ conductance at the basolateral membrane. 4. Atrial natriuretic factor (ANF) does not appear to affect proximal tubular sodium transport directly, but acts via specific receptors on the basolateral and brush border membranes to raise intracellular cGMP levels and inhibit AngII-stimulated transport. 5. It is concluded that there is a receptor-mediated action of ANF on proximal tubule reabsorption acting via elevation of cGMP to inhibit AngII-stimulated sodium transport. This effect is exerted by peptides delivered at both luminal and peritubular sides of the epithelium and provides a basis for the modulation by ANF of proximal glomerulotubular balance. The evidence reviewed supports the concept that in the proximal tubule, AngII and ANF act antagonistically in their roles as regulators of extracellular fluid volume.

Regulation of renal tubular sodium transport by angiotensin II and atrial natriuretic factor.

1. The effects of angiotensin II (AngII) on water and electrolyte transport are biphasic and dose-dependent, such that low concentrations (10(-12) to 10(-9) mol/L) stimulate reabsorption and high concentrations (10(-7) to 10(-6) mol/L) inhibit reabsorption. Similar dose-response relationships have been obtained for luminal and peritubular addition of AngII. 2. The cellular responses to AngII are mediated via AT(1) receptors coupled via G-regulatory proteins to several possible signal transduction pathways. These include the inhibition of adenylyl cyclase, activation of phospholipases A(2), C or D and Ca(2+) release in response to inositol-1,4,5,-triphosphate or following Ca(2+) channel opening induced by the arachidonic acid metabolite 5,6,-epoxy-eicosatrienoic acid. In the brush border membrane, transduction of the AngII signal involves phospholipase A(2), but does not require second messengers. 3. Angiotensin II affects transepithelial sodium transport by modulation of Na(+) /H(+) exchange at the luminal membrane and Na(+)/HCO(3) cotransport, Na(+)/K(+)-ATPase activity and K(+) conductance at the basolateral membrane. 4. Atrial natriuretic factor (ANF) does not appear to affect proximal tubular sodium transport directly, but acts via specific receptors on the basolateral and brush border membranes to raise intracellular cGMP levels and inhibit AngII-stimulated transport. 5. It is concluded that there is a receptor-mediated action of ANF on proximal tubule reabsorption acting via elevation of cGMP to inhibit AngII-stimulated sodium transport. This effect is exerted by peptides delivered at both luminal and peritubular sides of the epithelium and provides a basis for the modulation by ANF of proximal glomerulotubular balance. The evidence reviewed supports the concept that in the proximal tubule, AngII and ANF act antagonistically in their roles as regulators of extracellular fluid volume.

Effects of atrial natriuretic factor on cyclic nucleotides in rabbit proximal tubule.

Atrial natriuretic factor induces renal sodium excretion by several mechanisms, including inhibition of angiotensin II-stimulated sodium reabsorption in the proximal tubule. In most tissues, the action of atrial natriuretic factor involves generation of the intracellular second messenger, cyclic GMP, but in the proximal tubule the presence of this signal transduction pathway has remained controversial. We used intrarenal arterial infusion of iron oxide followed by enzymatic dispersion and magnetic separation to obtain suspensions of rabbit kidney cortex enriched with either glomeruli or proximal tubules. When suspensions enriched with proximal tubules or preparations of microdissected proximal tubules were incubated with atrial natriuretic factor (1 mumol/L), cyclic GMP concentrations increased significantly. Addition of angiotensin II (1 mumol/L) together with atrial natriuretic factor had no significant effect on the stimulation of cyclic GMP accumulation observed with atrial natriuretic factor alone. Neither atrial natriuretic factor nor angiotensin II altered intracellular concentrations of cyclic AMP in tubule-enriched suspensions or microdissected tubules. We conclude that cyclic GMP acts as a second messenger for atrial natriuretic factor in rabbit proximal tubule. However, we found no evidence to support the view that alterations in intracellular cyclic AMP levels are involved in the proximal tubular actions of angiotensin II and have not been able to demonstrate that interactions between cyclic AMP and cyclic GMP underlie the antagonistic effect of atrial natriuretic factor on angiotensin II-stimulated proximal sodium transport.

A monoclonal antibody that interferes with the post-aggregation adhesion of Dictyostelium discoideum cells.

A monoclonal antibody that interferes with the EDTA-resistant adhesion of Dictyostelium discoideum slug cells recognised a carbohydrate epitope on four major antigens (95, 90, 35 and 30 kDa) in slug cells. The 35 and 30 kDa antigens were specific for stalks and spores, respectively. The 30 kDa antigen was identified as the cell surface glycoprotein, PsA. Cyclic AMP, acting via cell surface receptors, induced only the 90 kDa slug cell antigen. Slug cell adhesion proteins may be involved in cell-sorting and the glycosylation of the 95 and 90 kDa antigens appeared to be abnormal in a mutant defective in cell-sorting. Previously, a 150 kDa glycoprotein has been strongly implicated in slug cell adhesion and the present work suggests that additional glycoprotein(s) are involved.

Polysaccharides influence the aggregation of Dictyostelium discoideum cells and bind to developmentally regulated cell surface proteins.

Six of ten anionic polysaccharides studied were found to significantly reduce the adhesion of growth-phase Dictyostelium discoideum cells. However, only hyaluronic acid, chondroitin-4-sulfate and chondroitin-6-sulfate interfered with the adhesion of aggregation-competent cells. Neither EDTA-stable nor EDTA-sensitive adhesion of postaggregation cells were affected by the polyanions. The two chondroitin sulfates influenced the aggregation of cells in submerged cultures, long and broad aggregation streams being formed and the broad sheets of cells eventually building multilayered aggregates. Radioiodination of cell surface proteins followed by cellulose fiber affinity chromatography identified the same nine proteins bound by hyaluronic acid and the chondroitin sulfates, six of which were regulated during development. Protease-resistant anionic material isolated from cells bound the same surface proteins as the three glycosaminoglycans. Discoidin I bound to the uncoupled cellulose fibers, suggesting a structural role for the lectin in the extracellular slime sheath. Anionic polysaccharides and cell surface lectins that bind them may be involved in the cell recognition, cell aggregation, and the cell sorting that occurs during pattern formation.

Implication of developmentally regulated Concanavalin A binding proteins of Dictyostelium in cell adhesion and cyclic AMP regulation.

Contact sites A and cyclic AMP phosphodiesterase are Concanavalin A binding membrane proteins. Both are characteristic for the aggregation phase of Dictyostelium discoideum. Extracellular cyclic AMP phosphodiesterase and an inhibitor of this enzyme can be recovered from the extracellular medium by binding to Concanavalin A-Sepharose.