Congenital nephrotic syndrome of the Finnish type (CNF or NPHS1) is an autosomal recessive kidney disorder resulting in severe proteinurea and renal dysfunction. Although the disease occurs predominantly in the Finnish population, many cases in other populations have also been reported. The disease gene (NPHS1) encodes nephrin, a podocyte transmembrane protein that is an essential component of the podocyte slit diaphragm, the renal ultrafilter. Since the discovery of the gene, many mutations have been reported in the NPHS1 gene in patients with diverse ethnic background. A surprisingly large number of these mutations are missense mutations resulting in single amino acid substitutions. In order to study the pathomechanism of these missense mutations, we have investigated the fate of 21 such mutations hitherto identified in NPHS1 patients. Immunostaining of stable transfected cells expressing the nephrin mutants demonstrated that most of the mutants showed only endoplasmic reticulum (ER) staining and no detectable cell surface localization. Immunoelectron microscopy of cells expressing the wild-type and a mutant nephrin further confirmed that the mutant nephrin could be abundantly found in the ER but not on the plasma membrane. Subcellular fractionation of wild-type and a mutant cell line clearly showed an altered subcellular distribution and molecular mobility of the mutant nephrin. In summary, our data indicate that a defective intracellular nephrin transport, most likely due to misfolding, is the most common consequence of missense mutations in NPHS1.

Intracellular application of the sulfonylurea tolbutamide during whole-cell patch-clamp recordings stimulated exocytosis >5-fold when applied at a cytoplasmic Ca2+ concentration of 0.17 microM. This effect was not detectable in the complete absence of cytoplasmic Ca2+ and when exocytosis was elicited by guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). The stimulatory action could be antagonized by the sulfonamide diazoxide, by the Cl--channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), by intracellular application of the antibody JSB1 [originally raised against a 170-kDa multidrug resistance (mdr) protein], and by tamoxifen (an inhibitor of the mdr- and volume-regulated Cl- channels). Immunocytochemistry and Western blot analyses revealed that JSB1 recognizes a 65-kDa protein in the secretory granules. This protein exhibited no detectable binding of sulfonylureas and is distinct from the 140-kDa sulfonylurea high-affinity sulfonylurea receptors also present in the granules. We conclude that (i) tolbutamide stimulates Ca2+-dependent exocytosis secondary to its binding to a 140-kDa high-affinity sulfonylurea receptor in the secretory granules; and (ii) a granular 65-kDa mdr-like protein mediates the action. The processes thus initiated culminate in the activation of a granular Cl- conductance. We speculate that the activation of granular Cl- fluxes promotes exocytosis (possibly by providing the energy required for membrane fusion) by inducing water uptake and an increased intragranular hydrostatic pressure.

Intracellular application of the sulfonylurea tolbutamide during whole-cell patch-clamp recordings stimulated exocytosis >5-fold when applied at a cytoplasmic Ca21 concentration of 0.17 mM. This effect was not detectable in the complete absence of cytoplasmic Ca21 and when exocytosis was elicited by guanosine 5*-O-(3-thiotriphosphate) (GTPgS). The stimulatory action could be antagonized by the sulfonamide diazoxide, by the Cl2-channel blocker 4,4*-diisothiocyanatostilbene-2,2*-disulfonic acid (DIDS), by intracellular application of the antibody JSB1 [originally raised against a 170-kDa multidrug resistance (mdr) protein], and by tamoxifen (an inhibitor of the mdrand volume-regulated Cl2 channels). Immunocytochemistry and Western blot analyses revealed that JSB1 recognizes a 65-kDa protein in the secretory granules. This protein exhibited no detectable binding of sulfonylureas and is distinct from the 140-kDa sulfonylurea high-affinity sulfonylurea receptors also present in the granules. We conclude that (i) tolbutamide stimulates Ca21dependent exocytosis secondary to its binding to a 140-kDa high-affinity sulfonylurea receptor in the secretory granules; and (ii) a granular 65-kDa mdr-like protein mediates the action. The processes thus initiated culminate in the activation of a granular Cl2 conductance. We speculate that the activation of granular Cl2 f luxes promotes exocytosis (possibly by providing the energy required for membrane fusion) by inducing water uptake and an increased intragranular hydro-

Inositol hexakisphosphate (InsP6), the dominant inositol phosphate in insulin-secreting pancreatic beta cells, inhibited the serine-threonine protein phosphatases type 1, type 2A, and type 3 in a concentration-dependent manner. The activity of voltage-gated L-type calcium channels is increased in cells treated with inhibitors of serine-threonine protein phosphatases. Thus, the increased calcium channel activity obtained in the presence of InsP6 might result from the inhibition of phosphatase activity. Glucose elicited a transient increase in InsP6 concentration, which indicates that this inositol polyphosphate may modulate calcium influx over the plasma membrane and serve as a signal in the pancreatic beta cell stimulus-secretion coupling.

ABSTRACT: This study examines the ontogeny of the regulation of Na+,K+-ATPase activity in the proximal tubule (PT) by a first messenger, dopamine (DA), and by direct stimulation of a third messenger, protein kinase C (PKC). PT segments dissected from 10− (PT10), 15-(PT15), 20− (PT20), and 40− (PT40) d-old rats were preincubated with DA 10−5 M, diacylglycerol (DAG) 10−5 M (an endogenous activator of PKC), or phorbol 12,13-dibutyrate (PDBu) 10−6 M (an exogenous activator of PKC). DA inhibited Na+,K+-ATPase activity in PT40. In PT20, DA also inhibited Na+,K+-ATPase activity, but the inhibitory effect in PT20 was less pronounced than in PT40. In PT15, DA had no effect on Na+,K+-ATPase activity. DAG significantly inhibited Na+,K+-ATPase activity in PT40. DAG also inhibited Na+,K+-ATPase activity in PT20, but the inhibition was slightly less pronounced than in PT40. DAG had no effect on Na+,K+-ATPase activity in PT15. Na+,K+-ATPase activity in PT40 and PT20 preincubated with PDBu was significantly lower than with vehicle. The inhibitory effect in PT20 was less pronounced than in PT40. When PT40 and PT20 were preincubated with both PDBu and 5 × 10−5 M sphingosine, an inhibitor of PKC activation, the inhibitory effect of PDBu was abolished. In both PT40 and PT20 incubated with 4-α-12,13 phorbol didecanoate 10−7 M, a phorbol ester that will not activate PKC, Na+,K+-ATPase activity was not different from the control. In PT10, Na+,K+-ATPase activity was the same after PDBu incubation and after vehicle incubation. Na+,K+-ATPase activity in PT10 incubated with both PDBu and sphingosine was not different from the control. PDBu inhibited Na+,K+-ATPase activity in a dose-dependent way in PT40 and PT20 but not in PT10. We conclude that the short-term regulation of renal tubular Na+,K+-ATPase activity undergoes postnatal changes.

Dopamine was shown to affect Na+,K(+)-ATPase activity in basolateral membranes of the rat kidney via a pertussis toxin dependent mechanism. In order to examine if some form of pertussis toxin sensitive G-protein is present exclusively in the basolateral membrane of the rat renal cortex we examined the G-protein composition of both apical and basolateral membrane vesicles. Western blots showed an essentially uniform distribution of G alpha total, G alpha S and G beta over the two membranes. Go could not be detected with western blot technique in the vesicle preparations. By contrast, the distribution of ADP-ribosylation with the bacterial toxins pertussis toxin and cholera toxin depended on the amount of detergent in the assay and perhaps other factors, and thus could not be used to evaluate the relative amounts of G-protein subunits. Thus, in contrast to the situation in cultured renal cells, unequal distribution of receptor and G-protein substrates is apparently not paralleled by an unequal distribution of the detected forms of G-proteins under physiological conditions.

This study examines the role of endogenous dopamine (DA) for the regulation of renal tubular sodium (Na) transport. The enzyme L-amino acid decarboxylase (L-AADC) that converts L-dopa to DA has been localized to the proximal tubule cells with immunocytochemistry. Locally formed DA will inhibit the activity of Na-K-ATPase, the enzyme that yields energy to active Na transport. The effect is of physiological importance during high salt diet. The phosphoprotein DARPP-32, a DA1 receptor associated third messenger is abundant in the medullary thick ascending limb of Henle (mTAL). DARPP-32 is phosphorylated after activation of DA1 receptors. DARPP-32 is in its phosphorylated form a potent phosphatase inhibitor. Activation of the DA1 receptor in mTAL with the DA1 agonist SKF 82526 causes dose-dependent inhibition of Na-K-ATPase activity. The effect involves activation of cAMP protein kinase. It is likely that this effect is potentiated by DARPP-32.

The short term regulation of the activity of the Na,K-pump (Na+,K(+)-ATPase) is just beginning to be understood. By using single microdissected proximal tubule segments (PCT) (permeabilized in order to clamp Na entry), it was possible to study regulation of Na+,K(+)-ATPase activity in its own environment and in a well defined cell population. The Na+,K(+)-ATPase activity can be regulated over a short term via guanidine triphosphate (GTP) dependent regulatory proteins. However the guanidine proteins are not directly coupled to the Na,K-pump and the mechanism involves the activation of complex intracellular signalling system. Locally produced dopamine induces a dose dependent inhibition of Na+,K+ ATPase activity. This inhibition is mediated by a complex mechanism that requires the activation of both membrane dopamine receptors, DA-1 and DA-2. It involves the activation of a pertussis toxin sensitive GTP-binding protein and activation of protein kinase C. A DA-2 agonist only inhibits Na+,K(+)-ATPase activity when it is incubated together with dibutyryl cAMP or Forskolin. We have therefore concluded that an increase in cellular cAMP levels plays a permissive role for DA-2 inhibition of Na+,K(+)-ATPase activity. A fully differentiated cell is required for dopamine inhibition of Na+,K(+)-ATPase activity. An abnormal regulation of proximal tubule Na+,K(+)-ATPase activity might be of importance in the pathogenesis of certain types of hypertension.

This study examines the receptor mechanisms by which dopamine (DA) inhibits Na(+)-K(+)-adenosinetriphosphatase (ATPase) activity in single permeabilized proximal tubule (PCT). Na(+)-K(+)-ATPase activity was inhibited in the presence of both DA1- and DA2-specific agonists but not by either agonist alone. The inhibition induced by DA (10(-6) M) was attenuated in the presence of either of the two DA2-specific antagonists S-sulpiride and YM 09151 at 10(-5) M and in the presence of the DA1 antagonist SCH 23390 (10(-5) M). PCT adenosine 3',5'-cyclic monophosphate (cAMP) levels were significantly increased in the presence of DA and DA1 agonist, but DA2 agonist had no effect on cell cAMP levels. Na(+)-K(+)-ATPase activity was significantly inhibited in PCT incubated with DA2 agonist (10(-5) M) and dibutyryl (DB)-cAMP (10(-6) M) but not with DA2 agonist (10(-5) M) only. PCT Na(+)-K(+)-ATPase activity was also significantly inhibited in the presence of both DA2 agonist (10(-5) M) and forskolin (10(-6) M). Neither DBcAMP (10(-6) M) nor forskolin (10(-6) M) alone inhibited Na(+)-K(+)-ATPase activity. In tubules incubated with DA (10(-8) to 10(-9) M), the presence of DBcAMP (10(-6) M) enhanced the sensitivity by which DA inhibited Na(+)-K(+)-ATPase activity. We conclude that PCT Na(+)-K(+)-ATPase activity is inhibited by a synergistic action of the DA1 and DA2 receptors, with the DA1 receptor acting to increase cell cAMP levels.

Several abnormalities of cation transport have been described in the Milan hypertensive rats (MHS). In this study we examined Na,K-ATPase activity in proximal convoluted tubules (PCT) cells and medullary thick ascending limb of Henle cells (TAL) from MHS and from the Milan normotensive rats (MNS). Na,K-ATPase activity was determined as 32P-ATP hydrolysis in single tubule segments. Na,K-ATPase activity (pmol Pi/mm t/h) was significantly higher in MHS than MNS both in PCT (903 +/- 227 n = 8 v 506 +/- 285 n = 12) and TAL (4324 +/- 800 n = 5 v 3063 +/- 625 n = 5). Na,K-ATPase dependent respiration was determined in PCT cell from MNS and MHS. Under basal condition Na,K-ATPase dependent respiration (mumol O2/mg protein/h) was higher in MHS than in MNS (24.2 +/- 1.8 n = 5 v 16.1 +/- 0.4 n = 5). When the cells were Na loaded by amphotericin Na,K-ATPase dependent respiration increased significantly more in MHS than MNS (38.4 +/- 1.6 v 26.8 +/- 2.2 n = 4). Thus, Na,K-ATPase activity is higher in renal tubule cells both at normal intracellular Na and after the cells have been Na loaded. The results indicate that regulation of Na homeostasis in renal tubule cell is different in MHS and MNS.

Activators of protein kinase C (PKC) inhibit sodium transport in proximal tubules (PT) (M. Baum and S. R. Hays. Am. J. Physiol. 254 (Renal Fluid Electrolyte Physiol. 23): F9-F14, 1988. In this study we have evaluated the effect of PKC activators on the enzyme responsible for active sodium transport, Na+-K+-ATPase. Both endogenous (diacylglycerol, DAG) and exogenous (phorbol esters, PE) activators were used. Enzyme activity was determined in permeabilized single PT segments. In vehicle-incubated PT, Na+-K+-ATPase activity (pmol Pi.mm tubule-1.-1 h) was 1,403 +/- 128. The synthetic DAG, L-alpha-l-oleoyl-2-acetoyl-sn-3-glycerol (10(-4) M) significantly inhibited Na+-K+-ATPase activity to 673 +/- 51, P less than 0.05. The PE-phorbol 12,13-dibutyrate (PDBu), induced a time- and dose-dependent inhibition of Na+-K+-ATPase activity. Inhibition was significant at 15 and maximal at 20 min. Na+-K+-ATPase activity in PT incubated with PDBu was 796 +/- 171 (10(-8) M), 570 +/- 198 (10(-7) M), and 484 +/- 130 (10(-6) M). A PE that does not activate PKC, 4-alpha-phorbol didecanoate, did not inhibit Na+-K+-ATPase activity. PDBu 10(-7) M had no effect on purified Na+-K+-ATPase. Sphingosine (SP), a PKC inhibitor, abolished the inhibitory effect of PDBu (10(-7) M) on Na+-K+-ATPase activity. Dopamine (DA) is a physiological inhibitor of Na+-K+-ATPase activity in PT [A. Bertorello, T. Hökfelt, M. Goldstein, and A. Aperia Am. J. Physiol. 254(Renal Fluid Electrolyte Physiol. 23): F795-F801, 1988].(ABSTRACT TRUNCATED AT 250 WORDS)