Kidney Plasma Membranes Research Articles

Structural Requirements for Association of Neurofascin with Ankyrin

This paper presents the first structural analysis of the cytoplasmic domain of neurofascin, which is highly conserved among the L1CAM family of cell adhesion molecules, and describes sequence requirements for neurofascin-ankyrin interactions in living cells. The cytoplasmic domain of neurofascin dimerizes in solution, has an asymmetric shape, and exhibits a reversible temperature-dependent beta-structure. Residues Ser56-Tyr81 are necessary for ankyrin binding but do not contribute to either dimerization or formation of structure. Transfected neurofascin recruits GFP-tagged 270-kDa ankyrinG to the plasma membrane of human embryo kidney 293 cells. Deletion mutants demonstrate that the sequence Ser56-Tyr81 contains the major ankyrin-recruiting activity of neurofascin. Mutations of the FIGQY tyrosine (Y81H/A/E) greatly impair neurofascin-ankyrin interactions. Mutation of human L1 at the equivalent tyrosine (Y1229H) is responsible for certain cases of mental retardation (Van Camp, G., Fransen, E., Vits, L., Raes, G., and Willems, P. J. (1996) Hum. Mutat. 8, 391). Mutations F77A and E73Q greatly impair ankyrin binding activity, whereas mutation D74N and a triple mutation of D57N/D58N/D62N result in less loss of ankyrin binding activity. These results provide evidence for a highly specific interaction between ankyrin and neurofascin and suggest that ankyrin association with L1 is required for L1 function in humans.

Elevated 5'-nucleotidase and alkaline phosphodiesterase activities in trout gill localize to endothelial (pillar) cells

Tissue homogenates from rainbow trout gill had three- to fivefold higher specific activity for 5'-nucleotidase (5'NT) and more than twofold greater alkaline phosphodiesterase (APD) activity than liver or kidney homogenates. In isolated plasma membranes, gill 5'NT activity was 3-5 times greater than that of the kidney or liver; gill and kidney plasma membranes had similar APD specific activities, both more than five times that of liver. 5'NT and APD activities were localized by histochemistry to the endothelial (pillar) cells of trout gill secondary lamellae. Staining was consistent with the concentration of both activities at the apical plasma membranes of pillar cells (i.e. at the lamellar microvascular surfaces). This localization may reflect a capacity for processing nucleotide metabolites circulating in the blood, perhaps relating to purinergic regulation of local lamellar hemodynamics. There was no histochemical evidence of either 5'NT or APD activity in the gill epithelial (pavement) cells that interface directly with the environment. In contrast, in trout kidney, both enzyme activities localized to the apical region of tubule epithelial cells. The absence of 5'NT and APD activity in pavement cells reinforces the unique structural and functional character of the gill-environment epithelial barrier. The results indicate that 5'NT and APD activities have particular potential application as markers in efforts to isolate and characterize specific gill plasma membrane fractions.

Characterization of [formula omitted] binding to dopamine D 2-like receptors expressed in cell lines

Recently, [ 125I]S(−)5- OH-PIPAT[5- hydroxy-2-(N- n-propyl-N-3′- iodo-2′- propenyl)aminotetralin] , a derivative of S(−)5-OH-DPAT(5-hydroxy- N, N-di-n-propyl-aminotetralin), was reported to be a better radioiodinated dopamine D 2-like receptor ligand than the previously reported iodinated ligand, [ 125I]R(+)7- OH-PIPAT . Therefore, in the present study, the binding profile of [ 125I]S(−)5- OH-PIPAT to D 2-like receptors expressed in cell lines was established. High binding affinity ( K d = 0.3–0.4 nM) and NaCl sensitivity were displayed with this ligand in membranes of human embryonic kidney (HEK293) cells expressing either human D 2 or rat D 3 receptors and in Chinese hamster ovary (CHO) cells expressing human dopamine D 4 receptors. Specific binding to D 2 and D 4 receptors was significantly increased in the presence of 2 mM MgCl 2 and decreased in the presence of 100 μM 5′-guanylyl-imidodiphosphate (GMP-PNP). This finding is consistent with reports that 2-aminotetralin compounds display agonist properties. The specific binding to D 3 receptors however, was not affected by either MgCl 2 or GMP-PNP. This lack of GMP-PNP sensitivity for D 3 receptors may result from inadequate G protein-receptor coupling in this cell line. The rank order of potency for inhibition of [ 125I]S(−)5- OH-PIPAT binding with various dopamine agents was consistent with reported values for D 2, D 3 and D 4 receptors. In membranes prepared from Spodoptera frugiperda (Sf9) cells infected with baculovirus that contains DNA encoding D 3 receptors, [ 125I]S(−)5- OH-PIPAT recognized only 70% of the receptor population labeled by [ 125I]NCQ298. This new ligand offers several unique advantages, including high specific activity, high binding affinity and selectivity for D 2-like receptors, that make it an excellent probe for the investigation and the characterization of dopamine D 2-like receptors.

IGF-I receptor binding, autophosphorylation, and kinase activity in kidney and muscle of acutely uremic rats.

Following acute tubular necrosis (ATN), kidney plasma membrane insulin-like growth factor-I (IGF-I) receptor number increases markedly, although IGF-I receptor mRNA levels do not change. To determine whether this increase could represent a redistribution of intracellular receptors and whether receptor function is intact in acute uremia, rats with ATN of 2 days duration and pair-fed controls were studied. Skeletal muscle receptor binding was unchanged. In contrast, binding to receptors in solubilized cortex and isolated cortical plasma membranes increased significantly due to an increase in receptor number. However, the increase in membrane binding was threefold greater than the increase in solubilized cortex binding. This indicates that the increase in total cellular IGF-I receptors can only account for a minor portion of the increase in abundance of plasma membrane receptors number and is consistent with a redistribution of receptors from an intracellular to a membrane location as the major mechanism. Autophosphorylation and receptor kinase activity were unaffected by the uremia (blood urea nitrogen of approximately 198 mg/dl). Since these early steps of IGF-I receptor signaling are intact early in acute uremia, it is likely that at this time in the course of the disease the increase in receptor number will heighten the sensitivity to IGF-I and may thus favor its participation in renal repair.

Alpha 2C-adrenoceptors mediate contractile responses to noradrenaline in the human saphenous vein.

We have investigated the subtype of alpha 2-adrenoceptor mediating isometric contractions of human saphenous vein in comparison with alpha 2-adrenoceptor ligand binding sites. Postjunctional alpha 2-adrenoceptors in the human saphenous vein were investigated in terms of the ability of alpha 2-adrenoceptor antagonists to shift the contractile potency of noradrenaline. The following antagonists were employed (potencies, pKB, in human saphenous vein in parentheses): chlorpromazine (6.98 +/- 0.24), BDF 8933 (7.60 +/- 0.06), prazosin (6.62 +/- 0.15), ARC 239 (7.19 +/- 0.15), yohimbine (7.23 +/- 0.09), HV 723 (7.52 +/- 0.14), WB 4101 (7.90 +/- 0.06), SKF 104078 (6.55 +/- 0.08), BRL 44408 (5.72 +/- 0.21). Antagonist potency at postjunctional alpha 2-adrenoceptors was correlated with antagonist affinity at alpha 2-adrenoceptor ligand binding sites in membranes of human platelet (alpha 2A), rat kidney (alpha 2B) and Sf9 cells expressing human recombinant receptors (alpha 2C), labelled with [3H]yohimbine. The correlation with the postjunctional alpha 2-adrenoceptor mediating contraction of the human saphenous vein was best for the human recombinant alpha 2C-adrenoceptor ligand binding site (r = 0.92, n = 8, P < 0.001), as compared to correlations with the alpha 2B-adrenoceptor ligand binding site of rat kidney (r = 0.62, n = 8, n.s.) and with the alpha 2A-adrenoceptor ligand binding site of human platelet (r = 0.23, n = 8, n.s.). It is concluded that the functional postjunctional alpha 2-adrenoceptor mediating contractions of the human saphenous vein closely resembles the human recombinant alpha 2C-adrenoceptor ligand binding site.

Comparative Activity of ADP-ribosylation Factor Family Members in the Early Steps of Coated Vesicle Formation on Rat Liver Golgi Membranes

We have compared the abilities of mammalian ADP-ribosylation factors (ARFs) 1, 5, and 6 and Saccharomyces cerevisiae ARF2 to serve as substrates for the rat liver Golgi membrane guanine nucleotide exchange factor and to initiate the formation of clathrin- and coatomer protein (COP) I-coated vesicles on these membranes. While Golgi membranes stimulated the exchange of GTPgammaS for GDP on all of the ARFs tested, mammalian ARF1 was the best substrate, with an apparent Km of 5 microM. In all cases myristoylation of ARF was required for stimulation. Agents that inhibit the Golgi membrane guanine nucleotide exchange factor (the fungal metabolite brefeldin A and trypsin treatment) selectively inhibited the guanine nucleotide exchange on mammalian ARF1. Taken together, these data indicate that of the ARFs tested, only mammalian ARF1 is activated efficiently by the Golgi guanine nucleotide exchange factor. The other ARFs are activated mainly by another mechanism, possibly phospholipid-mediated. Once activated, all of the membrane-associated, myristoylated ARFs promoted the recruitment of coatomer to about the same extent. Mammalian ARFs 1 and 5 were the most effective in promoting the recruitment of the AP-1 adaptor complex, whereas yeast ARF2 was the least active. These data indicate that the specificity for ARF action on the Golgi membranes is primarily determined by the Golgi guanine nucleotide exchange factor, which has a strong preference for myristoylated mammalian ARF1.

Stimulation of inorganic phosphate transport by insulin-like growth factor I and vanadate in opossum kidney cells is mediated by distinct protein tyrosine phosphorylation processes.

Insulin-like growth factor I (IGF-I) stimulates sodium-dependent inorganic phosphate (Pi) transport across the apical plasma membrane of confluent opossum kidney (OK) cells. Previous studies indicated that vanadate, at doses known to inhibit protein tyrosine phosphatases, mimicked the effect of IGF-I and suggested the involvement of tyrosine phosphorylation processes in Pi transport regulation. In this study, protein tyrosine phosphorylation and activation of several cellular signaling pathways were investigated in confluent OK cells in response to IGF-I and vanadate. We report that IGF-I and vanadate induced tyrosine phosphorylation of distinct proteins. Tyrosine phosphorylation of p95 (IGF-I receptor beta-subunit) was rapidly and dose dependently increased in response to IGF-I. Associated with phosphorylation of the receptor, the increase in tyrosine phosphorylation of a protein of 50 kDa was observed. Vanadate did not mimic the effect of IGF-I, but increased phosphorylation of seven major proteins of 170, 140, 100, 83, 70-82, 60, and 35 kDa. Among the different tyrosine kinase inhibitors tested, only staurosporine affected Pi transport up-regulation by IGF-I and vanadate, attenuating the effect of IGF-I and completely blocking the response to vanadate. Staurosporine decreased tyrosine phosphorylation of several constitutively phosphorylated proteins and interfered with the increase in tyrosine phosphorylation induced by vanadate. Phosphorylation of p95 in response to IGF-I was not affected. Staurosporine also markedly decreased constitutive association of the adapter protein Nck with tyrosine-phosphorylated proteins and attenuated increases in phosphotyrosine-associated Nck induced by IGF-I and vanadate. In contrast, signaling to other downstream effectors common to IGF-I and vanadate, such as mitogen-activated protein kinase and phosphatidylinositol-3-kinase, was not affected by staurosporine. In conclusion, our results suggest that although IGF-I and vanadate induce distinct protein tyrosine phosphorylation in OK cells, they activate an overlapping set of signaling molecules, among which Nck appears as an interesting candidate to link activation of tyrosine kinases to the stimulation of Pi transport.

Identification of a 56 kDa putative bovine herpesvirus 1 cellular receptor by anti-idiotype antibodies

Polyclonal anti-idiotype antibodies (anti-ids) to anti-bovine herpesvirus 1 (BHV-1) MAbs blocked virus infection in cell cultures, indicating that they contain internal images of a viral attachment protein(s). In the present study anti-id (anti-83) of BHV-1 gB was used as a probe to detect the cellular receptor. Anti-id specifically identified a 56 kDa protein in radioimmunoprecipitation analysis (RIPA) of Madin-Darby bovine kidney (MDBK) cell membranes suggesting the involvement of this cell surface component in BHV-1 binding. Anti-83 pretreated with MAb 83 failed to identify the 56 kDa cellular component proving its specificity for the reacting epitope of MAb 83. The recognition of 56 kDa protein by anti-id was inhibited by prior incubation of radiolabelled membrane proteins with BHV-1 suggesting that the ligands competed for the same binding sites on the cells. 35S-Radiolabelled BHV-1 virions also bound a 56 kDa protein from purified MDBK cell membrane proteins in a virus overlay protein blot assay. RIPA using anti-id as probe detected the 56 kDa protein in permissive MDBK cells but not in non-permissive bat lung cells. The protein nature of the 56 kDa component was confirmed by protease treatment of membranes which resulted in abolition of the 56 kDa signal in RIPA. In addition, purified 56 kDa protein inhibited biotinylated BHV-1 attachment in flow cytometry. These findings indicate that the 56 kDa protein identified by anti-id is a putative receptor for BHV-1.

Mutational analysis and molecular modeling of the nonapeptide hormone binding domains of the [Arg8]vasotocin receptor.

To identify determinants that form nonapeptide hormone binding domains of the white sucker Catostomus commersoni [Arg8]vasotocin receptor, chimeric constructs encoding parts of the vasotocin receptor and parts of the isotocin receptor have been analyzed by [(3,5-3H)Tyr2, Arg8]vasotocin binding to membranes of human embryonic kidney cells previously transfected with the different cDNA constructs and by functional expression studies in Xenopus laevis oocytes injected with mutant cRNAs. The results indicate that the N terminus and a region spanning the second extracellular loop and its flanking transmembrane segments, which contains a number of amino acid residues that are conserved throughout the nonapeptide receptor family, contribute to the affinity of the receptor for its ligand. Nonapeptide selectivity, however, is mainly defined by transmembrane region VI and the third extracellular loop. These results are complemented by a molecular model of the vasotocin receptor obtained by aligning its sequence with those of other G-protein coupled receptors as well as that of bacteriorhodopsin. The model indicates that amino acid residues of transmembrane regions II-VII that are located close to the extracellular surface also contribute to the binding of vasotocin.

Mesotocin Binding to Receptors in Hen Kidney Plasma Membranes

Radioligand assays were performed to demonstrate the presence of a receptor for mesotocin (MT) in the membrane fractions of the kidney of the hen. Specific [125I]MT bindings were decreased by the presence of Mg2+ and Ca2+, increased by the presence of EDTA, increased during the first 4 h of incubation and then reached a plateau, and increased with the increase in the protein concentration from 2.5 to 20 μg. The membrane fraction showed binding specificity to [125I]MT. The Scatchard plot revealed a curvilinear profile that indicated the presence of two classes of binding sites: a high affinity site and a low affinity site. The equilibrium dissociation constant was 0.08 ± 0.01 nM (χ̄ ± SEM; n = 5) in the high affinity site and 0.87 ± 0.08 nM (n = 5) in the low affinity site. The maximum binding capacity of the high and low affinity sites was 42 ± 4 and 129 ± 6 fmol/mg protein, respectively. The results suggest the presence of two distinct MT receptors in the kidney of the hen.

Evidence suggesting that the minimal functional unit of a renal cystine transporter is a heterodimer and its implications in cystinuria

Cystinuria, one of the most common genetic disorders, is characterized by excessive excretion of cystine and basic amino acids in urine. The low solubility of cystine results in formation of kidney stones which can eventually lead to renal failure. Three types of cystinurias have been described. All involve defects in a high-affinity transport system for cystine in the brush border membranes of kidney and intestinal epithelial cells. The molecular properties of proteins involved in epithelial cystine transport are incompletely understood. A protein (NBAT, neutral and basic amino acid transporter), initially cloned by us from rat kidney and shown to be localized in the renal and intestinal brush border membranes, has been implicated in this transport, and mutations in human NBAT gene have been found in several cystinurics, making it a prime candidate for a cystinuria gene. However, mutations in NBAT were found only in Type I cystinurics and not in Types II and III suggesting that defects in other, as yet uncharacterized, genes may also be involved. NBAT has an unusual (for an amino acid transporter) membrane topology. We proposed that the protein contains four membrane-spanning domains, a model disputed by other investigators. We subsequently obtained experimental data consistent with a four membrane-spanning domain model. Furthermore, recently we showed that kidney and intestinal NBAT (85kDa) is associated with another brush border membrane protein (about 50kDa) and have proposed that the heterodimer represents the minimal functional unit of the high-affinity cystine transporter in these membranes. These findings raise the tantalizing possibilities that defects in the NBAT-associated protein might account for cystinurias in individuals with normal NBAT gene (such as the Types II and III cystinurics).

Canine kidney glucagon receptor: evidence for a structurally-different, tissue-specific variant of the glucagon receptor

125I-Glucagon was directly cross-linked to its receptor sites on the MDCK plasma membranes using a UV irradiation procedure. Analysis of the affinity labeled membranes by SDS-PAGE and autoradiography, demonstrated the presence of a single band at 74 kDa. The incorporation of radiolabeled glucagon into this band was abolished by the presence of excess unlabeled hormones, thus indicating a specificity of labeling. Also this band was observed in affinity labeled dog kidney plasma membranes. The size of the MDCK and the dog kidney glucagon receptors were consistently larger than that of the dog liver receptor as judged by electrophoretic mobility. Treatments with neuraminidase, endoglycosidase F, or N-glycanase failed to convert the renal form into the hepatic form of the receptor. Proteolytic mapping of the MDCK and the dog liver glucagon receptors revealed that major domains of both proteins are remarkably similar, yet transient variations in the size of the fragments could be detected after short duration digestions. Overall the data presents evidence that the dog renal receptor represents a structurally unique isoform of the glucagon receptor.

Phosphorylation of the Goodpasture Antigen by Type A Protein Kinases

Collagen IV is the major component of basement membranes. The human alpha 3 chain of collagen IV contains an antigenic domain called the Goodpasture antigen that is the target for the circulating immunopathogenic antibodies present in patients with Goodpasture syndrome. Characteristically, the gene region encoding the Goodpasture antigen generates multiple alternative products that retain the antigen amino-terminal region with a five-residue motif (KRGDS). The serine therein appears to be the major in vitro cAMP-dependent protein kinase phosphorylation site in the isolated antigen and can be phosphorylated in vitro by two protein kinases of approximately 50 and 41 kDa associated with human kidney plasma membrane, suggesting that it can also be phosphorylated in vivo. Consistent with this, the Goodpasture antigen is isolated from human kidney in phosphorylated and non-phosphorylated forms and only the non-phosphorylated form is susceptible to phosphorylation in vitro. Since this motif is exclusive to the human alpha 3(IV) chain and includes the RGD cell adhesion motif, its phosphorylation might play a role in pathogenesis and influence cell attachment to basement membrane.

Ba(2+)-sensitive K+ channels in basal membrane of confluent Madin-Darby canine kidney cells.

A method is described for gaining access to the basolateral membranes of confluent Madin-Darby canine kidney (MDCK) cells by surgical reflection of the cell layer overlying fluid-filled domes. Single-channel recordings from cell-attached inside-out and outside-out configurations revealed two K+ channels located in the basal membranes of the highly differentiated monolayers. With 140 mmol/l KCl in pipette, the intermediate-conductance K+ channel displayed outward rectification in cell-attached configuration with channel conductances of 65 pS for outward part and 17 pS for inward part. In excised-patch recording, this channel had a conductance of 92 pS with 140 mmol/l KCl on the extracellular side of the patch and 5 mmol/l KCl on the cytosolic side. The maximum conductance obtained in symmetrical KCl (140 mmol/l) solution was 140 pS. Ba2+ (1 mmol/l) and tetraethylammonium (5 mmol/l) blocked this channel reversibly. Channel open probability (Po) was reduced from 0.41 at cytosolic pH 7.4 to 0.14 at pH 6.8 and increased to 0.64 at pH 8.0. The channel activity was significantly inhibited by elevation of intracellular Ca2+. A small-conductance K+ channel was also observed mainly in excised patches with single-channel conductance of 48 pS in symmetrical KCl solutions. However, the activity of this channel was partially obscured by the intermediate-conductance K+ channel and further analysis was not possible. A physiological role of these channels in mediating K+ recycling through the monolayer is suggested.

Membrane topology of the rat kidney neutral and basic amino acid transporter.

A recently cloned rat kidney protein (NBAT) mediates the sodium-independent transport of neutral as well as basic amino acids and cystine when expressed in Xenopus laevis oocytes. The human equivalent of this transporter may be the one that is defective in cystinuria. Immunocytochemical studies have indicated that NBAT is primarily localized in the brush border membranes of rat kidney and intestinal epithelial cells, a localization consistent with its proposed role in amino acid transport. Two contrasting topological models have been proposed for NBAT: a four membrane-spanning domain (MSD) Nin-Cin model and a single MSD Nin-Cout model. We have investigated the topology of this membrane protein using two different approaches. One method was an immunofluorescent labeling technique in which intact or membrane-permeabilized cells expressing NBAT were probed with antibodies directed against putative extracellular and intracellular domains of the protein. In the second method, fragments generated by limited surface proteolysis of intact brush border membrane vesicles were subjected to immunoblot analysis using several site-specific antibodies. Both approaches yielded results consistent with a four MSD Nin-Cin topological model for NBAT.

Pig kidney (LLC-PK1) cell membrane fluidity during exposure to gentamicin or tobramycin

The surface membrane properties of LLC-PK1 cells grown with and without various amounts of gentamicin or tobramycin for various lengths of time were determined by measuring the diffusion coefficient of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)dipalmitoyl-L- alpha-phosphatidylethanolamine (NBD-PE) and the percentage of NBD-PE free to diffuse after photobleaching. One hour of exposure to tobramycin decreased the percentage that was free to diffuse. After 1 day or longer of exposure to either aminoglycoside the percentage that was free to diffuse returned to preexposure levels and the diffusion coefficient decreased.

Plasmolipin: The other myelin proteolipid. A review of studies on its structure, expression, and function

Proteolipids were discovered by Marjorie B. Lees and Jordi Folch-Pi in 1951 (1) when they isolated the myelin proteolipid protein (PLP). PLP is a major constituent of CNS myelin where it represents at least 40% of the total myelin protein. Since then, proteolipids have been associated with a variety of other membrane domains and organelles (2-4). However with few exceptions (5) all proteolipids described thus far show little homology with each other. In this review we describe the properties of a proteolipid, plasmolipin, which is restricted to specific plasma membranes of kidney and brain (6,7). Plasmolipin is particularly relevant to a Neurochemical Research issue dedicated to Marjorie Lees, since the studies on this protein were inspired by her work on PLP and, like PLP, it is a major myelin protein (8). This review covers our knowledge to date of plasmolipin and describes its structure, localization, devel-

Characterization of a chymotrypsin-like hydrolytic activity in the opossum kidney cell.

To characterize a chymotrypsin-like hydrolytic activity in the cell surface membranes of intact opossum kidney (OK) cells, we partially purified a protease from the membrane fractions of OK cells using Suc-Leu-Leu-Val-Tyr-MCA (Suc, succinyl; MCA, 4-methylcoumaryl-7-amide), a synthetic substrate for chymotrypsin, as the substrate. The semipure enzyme showed seryl chymotrypsin-like characteristics such as preferential hydrolysis of Suc-Leu-Leu-Val-Tyr-MCA and inhibition by phenylmethylsulfonyl fluoride, diisopropylfluorophosphate, and chymostatin. However, it clearly differed from alpha-chymotrypsin in its weak ability to hydrolyze Suc-Ala-Ala-Pro-Phe-MCA and in its high molecular mass (250-300 kDa). The enzyme also had an endopeptidase-like activity in that it cleaved human parathyroid hormone(1-84) at the Leu(37)-Gly(38) and Arg(52)-Lys(53) bonds. These results suggest that a high molecular mass chymotrypsin-like endopeptidase with unique characters is present in the membrane fractions of OK cells.

Non-classical receptors for aldosterone in plasma membranes from pig kidneys

Rapid, nongenomic in vitro effects of aldosterone on intracellular electrolytes, cell volume and the sodium-pro- ton antiporter have been found in human mononuclear leukocytes (HML), as have related membrane receptors. In the present study, binding of ^I-labeled aldosterone to plasma membrane preparations from pig kidneys was studied, since nongenomic in vitro effects of aldosterone have also been described in cultured kidney cells. In this preparation, binding of aldosterone shares important features with both functional and binding data in HML. These include a very low apparent K, of ~ 0.1 nM for aldosterone, a high turnover rate and binding selectivity for aldosterone and fludrocortisone. Desoxycorticosterone acetate and corticosterone show intermediate affinity, with apparent K^ values of ~ 1 and 100 nM, with hydrocortisone even less active. Thus binding of aldosterone to kidney plasma membranes is compatible with the major features of its nongenomic renal effects.

Platelet-derived growth factor stimulates the release of protein kinase A from the cell membrane.

The mitogenic action of growth factors involves the stimulation of intracellular protein kinases. In this report we have characterized the major protein kinase released from Balb/c 3T3 and normal rat kidney plasma membranes by the action of platelet-derived growth factor (PDGF). PDGF appears to stimulate the release of approximately 10 proteins, at least one of which is a kinase capable of phosphorylating proteins on Ser or Thr (as determined by the lability of the phosphate to alkali treatment). More than 90% of the Ser/Thr kinase activity was inhibited by PKI5-22, a specific peptide inhibitor of the cAMP-dependent protein kinase (PKA). We used immunoblotting to confirm that the kinase released in response to PDGF was PKA. cAMP also stimulated the release of PKA, and the set of protein substrates phosphorylated was similar following PDGF or cAMP stimulation. Interestingly, in the presence of a cAMP analogue ((Rp)-cAMPS), cAMP could not induce dissociation of PKA from the membranes, whereas stimulation by PDGF increased the level of PKA activation. Furthermore, unlike Swiss 3T3 cells, neither Balb/c 3T3 fibroblasts nor normal rat kidney cells accumulate cAMP in response to PDGF, yet the level of PKA in the cytosol of these intact cells increases in response to PDGF. Thus, it appears as though PDGF activation of the membrane-associated form of the PKA holoenzyme occurs by a mechanism independent of an elevation in cAMP levels.