Localization of severa G-protein subunits to the apical and basolateral membranes of cortical tubular cells from the rat kidney

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_α total, G_αs and Gβ over the two membranes. G_o 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.     

Muscarine modulation by a G-protein alpha-subunit of delayed rectifier K+ current in rat ventromedial hypothalamic neurones.

1. Rat cultured ventromedial hypothalamic (VMH) neurones obtained from embryonic hypothalamus were used to study the muscarinic (carbachol) modulation of voltage-gated K+ currents with the whole-cell patch-clamp technique. 2. Carbachol produced a potent and concentration-dependent (100 fM to 100 microM) decrease of the outward delayed rectifier K+ current (IK) with an IC50 of 44 pM and a Hill coefficient of 0.4. The carbachol-induced depression of IK was reduced by pirenzepine (1-10 microM) and atropine (1 microM). Carbachol had no effect on the transient outward K+ current (IA). 3. Intracellular dialysis with guanosine 5'-O-(2-thiodiophosphate) (GDP-beta-S, 500 microM) significantly diminished the carbachol-induced depression of IK, suggesting GTP-binding protein (G-protein) involvement. Pre-incubation of VMH neurones with pertussis toxin (200-400 ng ml-1) or cholera toxin (1 microgram ml-1) for 24-48 h had no effect on the carbachol-induced depression of IK. This suggested that the G alpha o, G alpha i, and G alpha s G-protein alpha-subunits were not involved in mediating the carbachol-induced depression of IK in VMH neurones. 4. Treatment (24-48 h) of VMH neurones with antisense phosphothio-oligodeoxynucleotides to the G alpha 11 G-protein subunit (10 microM) significantly diminished the carbachol-induced depression of IK. Treatment with 10 microM of either G alpha 11 sense or antisense to G alpha q had no effect. 5. These results demonstrate a novel and potent muscarinic depression of IK in VMN neurones, and that this depression is specifically mediated by the G alpha 11 G-protein subunit.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibitory modulation of fast and slow Ca(2+)-currents in neuroblastoma x glioma cells during differentiation.

Mouse neuroblastoma x rat glioma hybrid cells (N x G, 108CC15) were used to study the inhibitory effects of the synthetic opioid D-Ala2-D-Leu5-enkephalin (DADLE), somatostatin, adrenaline-alpha 2 and angiotensin II on voltage-dependent Ca(2+)-currents (ICa) using the patch-clamp technique in the whole-cell configuration mode. The inhibitory effects could be abolished by pretreatment of N x G cells with pertussis toxin or intracellular infusion of GDP beta S indicating an involvement of a pertussis toxin sensitive GTP-binding protein (G-protein), presumably Go. The effect of DADLE, the strongest inhibitor of ICa, was studied during dibutyryl cyclic AMP (dBcAMP) induced differentiation. Using omega-conotoxin GVIA (omega-CTX) and methoxyverapamil (D600) as specific Ca(2+)-channel blockers of the N- and L-type Ca(2+)-channels, it was found that in N x G cells DADLE predominantly induces inhibition of T- and N-type Ca(2+)-channels.     

Limited distribution of pertussis toxin in rat brain after injection into the lateral cerebral ventricles.

In vivo administration of pertussis toxin is often used to study the involvement of guanine nucleotide binding proteins in signal transduction. Especially when it is administered in the brain the effect is often poor. This could be due to the fact that pertussis toxin does not reach the area of interest. To evaluate the extent to which pertussis toxin is distributed in rat brain after intraventricular injection, different techniques were used. Immunohistochemical studies with an antibody against pertussis toxin showed that immunoreactivity was limited to periventricular brain structures less than 0.5 mm from the lumen. The highest immunoreactivity was seen 16-24 h after injection. After 96 h the labeling was very weak. The proportion of guanine nucleotide binding proteins that were ADP-ribosylated by in vivo injection of pertussis toxin into the ventricles as assessed by in vitro [32P]-back-ADP-ribosylation was very low 48 h after the injection, in all regions studied. Direct injection of pertussis toxin into the brain caused a marked ADP-ribosylation localized to the region injected that was maximal at 72 h after injection. At 96 h there were also effects after control injections, indicating non-specific effects. Synaptosomal membranes and other membranes were equally affected by pertussis toxin. The results suggest that in studies regarding the effect of pertussis toxin treatment on signal transduction, the toxin must be injected very close to the brain region of interest and, furthermore, that the rats should be killed 48-72 h after injection. In case of lack of effect on the response of interest one should examine whether the ADP-ribosylation of pertussis toxin-sensitive guanine nucleotide binding proteins in the area of concern has been affected.     

Heterogeneous distribution of monomeric elements from the globular domain (NC1) of type IV collagen in renal basement membranes as revealed by high resolution quantitative immunocytochemistry.

Heterogeneity in the distribution of major components, namely laminin, entactin/nidogen, heparan sulfate proteoglycan and type IV collagen among renal basement membranes, was previously demonstrated (Desjardins and Bendayan, J Histochem Cytochem 37:885-897, 1989). To further investigate the nature of basement membranes, we applied high resolution immunocytochemistry to reveal monomers M1, M2* and M3 composing the NC1 domain of type IV collagen of various rat renal basement membranes. Labeling for the three monomers was confined to basement membranes. Quantitative morphometrical analysis demonstrated that the labeling for each monomer was not evenly distributed among the various basement membranes. Labeling for M1 was intense over the proximal tubule and the Bowman's capsule basement membranes. In the glomerulus, the mesangial matrix was highly labeled, whereas the glomerular basement membrane was labeled with a lower intensity. In contrast, labeling for M2* and M3 were high in the glomerular basement membrane, while very low in the mesangial matrix. Labeling intensities were intermediate in the other renal basement membranes. The ultrastructural distribution analysis made over the glomerular basement membrane showed a preferential subendothelial localization of M1 monomers. M2 and M3 on the other hand, were found throughout the entire thickness of the basement membrane. M1 monomers being associated to alpha 1(IV) and alpha 2(IV) chains of type IV collagen, and M2* and M3 to alpha 3(IV) and alpha 4(IV) chains respectively, our results thus demonstrate the existence of an heterogeneity in the collagenous nature of renal basement membranes, particularly the glomerular one. This heterogeneity must reflect variations in the structural arrangement of basement membranes and therefore in their functional properties.     

Inorganic anion transport in kidney and intestinal brush border and basolateral membranes

The efflux of inorganic anions from purified brush border and basolateral membrane vesicles from dog kidney cortex was measured under equilibrium exchange conditions. Marked differences in temperature sensitivity and effects of inhibitors were found between the Cl and SO4 transport pathways and between the two types of membranes. SO4 transport in both brush border and basolateral membranes was markedly reduced by cooling, but significant inhibition by 4,4'-diisothiocyano-2,2'-disulfonic stilbene (DIDS) was only observed in basolateral vesicles. In contrast, Cl efflux from both types of vesicles was neither substantially inhibited by DIDS nor by lowering the temperature to 0 degrees C. Phosphate efflux from basolateral membrane vesicles was found to be only partially sensitive to DIDS. Attempts to label the stilbene-sensitive SO4 pathway in basolateral vesicles using [3H2]DIDS as a marker were unsuccessful due to the nonspecific labeling of many membrane components. The asymmetry in inorganic anion transport behavior exhibited by brush border and basolateral membrane vesicles from dog renal proximal tubule was also observed in equivalent vesicles prepared from rat small intestine.     

Effect of pertussis toxin and N-ethylmaleimide on voltage-dependent and -independent calcium current modulation in serotonergic neurons

Introduction of GTP-gamma-S into a neuronal cell spontaneously results in G-protein activation. A possible contribution to this mechanism is that some receptors have a constitutive activity that stimulates GDP/GTP exchange resulting in increased GTPase activity of G-protein alpha subunits, leading to a facilitation of GTP-gamma-S binding. It follows that partial or complete uncoupling of receptors and G-proteins could inhibit Ca(2+) current modulation by GTP-gamma-S. This possibility was tested in acutely isolated rat dorsal raphe neurons by uncoupling the receptor and G-protein using N-ethylmaleimide and pertussis toxin. Since these compounds have been suggested to differentially block voltage-dependent inhibition, relative to voltage-independent, we investigated whether the apparent voltage-independent component of Ca(2+) channel modulation by 5-hydroxytryptamine (5-HT) shares the same mechanism as the voltage-dependent component. N-ethylmaleimide inhibited the response to 5-HT by about 50% but had no effect on the response to GTP-gamma-S. In dorsal raphe neurons 28.9% of the total response to 5-HT was voltage-independent. N-ethylmaleimide had identical effects on the voltage-dependent and -independent components as measured by tail current inhibition. The response to 5-HT was completely sensitive to pertussis toxin, and completely uncoupling the receptors and G-proteins did not affect the maximal response to GTP-gamma-S.Our results suggest that the apparent voltage-independent component of Ca(2+) channel modulation by 5-HT in dorsal raphe neurons might share the same mechanism as does the voltage-dependent component. In addition, these experiments provided evidence that partial or even complete uncoupling of receptors and G-proteins did not affect Ca(2+) current modulation by direct activators of G-proteins.     

Membrane localization of the S1 subunit of pertussis toxin in Bordetella pertussis and implications for pertussis toxin secretion

Pertussis toxin is secreted from Bordetella pertussis with the assistance of the Ptl transport system, a member of the type IV family of macromolecular transporters. The S1 subunit and the B oligomer combine to form the holotoxin prior to export from the bacterial cell, although the site of assembly is not known. To better understand the pathway of pertussis toxin assembly and secretion, we examined the subcellular location of the S1 subunit, expressed with or without the B oligomer and the Ptl proteins. In wild-type B. pertussis, the majority of the S1 subunit that remained cell associated localized to the bacterial membranes. In mutants of B. pertussis that do not express pertussis toxin and/or the Ptl proteins, full-length S1, expressed from a plasmid, partitioned almost entirely to the bacterial membranes. Several lines of evidence strongly suggest that the S1 subunit localizes to the outer membrane of B. pertussis. First, we found that membrane-bound full-length S1 was almost completely insoluble in Triton X-100. Second, recombinant S1 previously has been shown to localize to the outer membrane of Escherichia coli (J. T. Barbieri, M. Pizza, G. Cortina, and R. Rappuoli, Infect. Immun. 58:999-1003, 1990). Third, the S1 subunit possesses a distinctive amino acid motif at its carboxy terminus, including a terminal phenylalanine, which is highly conserved among bacterial outer membrane proteins. By using site-directed mutagenesis, we determined that the terminal phenylalanine is critical for stable expression of the S1 subunit. Our findings provide evidence that prior to assembly with the B oligomer and independent of the Ptl proteins, the S1 subunit localizes to the outer membrane of B. pertussis. Thus, outer membrane-bound S1 may serve as a nucleation site for assembly with the B oligomer and for interactions with the Ptl transport system.     

Peptide mapping studies of the pertussis toxin substrate in human neutrophils, platelets and erythrocytes

Protease digestion of the ADP-ribosylated pertussis toxin substrate (PTS) protein was carried out after solubilization with SDS (Cleveland gels) and in the intact membrane. Cleveland gel analysis showed substantial similarities in the maps for the PTS component in neutrophils, platelets and erythrocytes and also in the S49 AC-lymphoma cell line. In the intact membrane ADP-ribosylation followed by digestion showed limited access of proteases to the PTS component. Of eight proteases tested, only papain and Staphylococcus aureus gave substantial digestion. This pattern was observed in the human platelet, erythrocyte and neutrophil plasma membranes. When the sequence was reversed and ADP-ribosylation was carried out after protease digestion, a very different pattern was observed with much greater susceptibility to digestion being noted with several proteases. By contrast, analysis of the murine AC-membrane showed some minor variations in the digest patterns. In addition, under all three conditions tested, maps of the cholera toxin substrate for the human platelet showed remarkable similarities to those obtained with the pertussis toxin substrate. Our results indicate that the protease sensitive sites of the alpha subunit of PTS and protection from proteolysis after ADP-ribosylation are properties which are shared by the PTS components of human platelets, erythrocytes and neutrophils.     

The C terminus of the human C5a receptor (CD88) is required for normal ligand-dependent receptor internalization

The biological effects of the potent inflammatory mediator C5a, a complement split product, on human neutrophils and monocytes are limited by the rapid internalization of its specific receptor (C5aR, CD88). The C terminus of the C5aR is phosphorylated after stimulation with C5a of phorbol ester, and this phosphorylation might lead to receptor internalization. In this context, we have studied the effects on C5aR internalization of C5a, phorbol 12-myristate 13-acetate (PMA), the protein kinase inhibitor staurosporine, and pertussis toxin on rat basophilic RBL.2H3 cells stably transfected with the human wild-type or mutant C5aR. C5aR mutants lacked either part of the cytosolic C terminus, including suggested major phosphorylation sites, or a putative phosphorylation motif for protein kinase C in the third cytosolic loop. Additionally, agonist-induced internalization was analyzed on HEK293 cells co-transfected with C5aR and the pertussis toxin-resistant G protein alpha subunit, Gα16. Staurosporine-sensitive agonist-dependent C5aR internalization could be detected, suggesting that C5aR phosphorylation, most likely of the C terminus, participates in this type of internalization. In contrast, PMA-induced C5aR internalization seems to be independent of putative phosphorylation sites in either the truncated section of the C terminus or the third cytosolic loop. The phorbol ester-induced C5aR internalization may, therefore, be caused by an indirect and less specific effect of protein kinase C on the internalization machinery. Manipulation of the pertussis toxin-sensitive or -resistant G protein-dependent signal transduction had no effect on ligand-induced internalization.     

Eicosanoid production and phospholipase A2 secretion by peritoneal macrophages from rats with adjuvant-induced arthritis

The molecular mechanism of action of several non-immunological histamine releasers has been investigated using pertussis toxin which interfers, via ADP-ribosylation, with some G-proteins. Pertussis toxin (100 ng/ml) inhibited histamine release induced by compound 48/80, substance P, mastoparan, peptide 401, bradykinin and spermine showing that a G-protein sensitive to pertussis toxin was involved in the non-immunological histamine release. All these compounds directly activate purified G-proteins. The sensitivity to pertussis toxin of this direct stimulatory effect was demonstrated for compound 48/80, mastoparan and substance P. Altogether these results suggest that a direct activation of G-protein might be the molecular mechanism of action of histamine secretagogues acting through a pertussis toxin sensitive G-protein and in this way mimic agonist-ligand receptor interaction.     

Impaired inhibitory G-protein function contributes to increased calcium currents in rats with diabetic neuropathy

There is a growing body of evidence that sensory neuropathy in diabetes is associated with abnormal calcium signaling in dorsal root ganglion (DRG) neurons. Enhanced influx of calcium via multiple high-threshold calcium currents is present in sensory neurons of several models of diabetes mellitus, including the spontaneously diabetic BioBred/Worchester (BB/W) rat and the chemical streptozotocin (STZ)-induced rat. We believe that abnormal calcium signaling in diabetes has pathologic significance as elevation of calcium influx and cytosolic calcium release has been implicated in other neurodegenerative conditions characterized by neuronal dysfunction and death. Using electrophysiologic and pharmacologic techniques, the present study provides evidence that significant impairment of G-protein-coupled modulation of calcium channel function may underlie the enhanced calcium entry in diabetes. N- and P-type voltage-activated, high-threshold calcium channels in DRGs are coupled to mu opiate receptors via inhibitory G(o)-type G proteins. The responsiveness of this receptor coupled model was tested in dorsal root ganglion (DRG) neurons from spontaneously-diabetic BB/W rats, and streptozotocin-induced (STZ) diabetic rats. Intracellular dialysis with GTPgammaS decreased calcium current amplitude in diabetic BB/W DRG neurons compared with those of age-matched, nondiabetic controls, suggesting that inhibitory G-protein activity was diminished in diabetes, resulting in larger calcium currents. Facilitation of calcium current density (I(DCa)) by large-amplitude depolarizing prepulses (proposed to transiently inactivate G proteins), was significantly less effective in neurons from BB/W and STZ-induced diabetic DRGs. Facilitation was enhanced by intracellular dialysis with GTPgammaS, decreased by pertussis toxin, and abolished by GDPbetaS within 5 min. Direct measurement of GTPase activity using opiate-mediated GTPgamma[(35)S] binding, confirmed that G-protein activity was significantly diminished in STZ-induced diabetic neurons compared with age-matched nondiabetic controls. Diabetes did not alter the level of expression of mu opiate receptors and G-protein alpha subunits. These studies indicate that impaired regulation of calcium channels by G proteins is an important mechanism contributing to enhanced calcium influx in diabetes.     

Requirements for assembly of PtlH with the pertussis toxin transporter apparatus of Bordetella pertussis

PtlH is an essential component of the Ptl system, the type IV transporter responsible for secretion of pertussis toxin (PT) across the outer membrane of Bordetella pertussis. The nine Ptl proteins are believed to interact to form a membrane-spanning apparatus through which the toxin is secreted. In this study, we monitored the subcellular localization of PtlH in strains of B. pertussis lacking PT, lacking other Ptl proteins, or from which ATP has been depleted in order to gain insight into the requirements for assembly of PtlH with the remainder of the Ptl transporter complex that is thought to be tightly embedded in the membrane. We found that PtlH is exclusively localized to the inner membrane fraction of the cell in a wild-type strain of B. pertussis. In contrast, PtlH localized to both the cytoplasmic and inner membrane fractions of a mutant strain of B. pertussis that does not produce PT. In comparison to how it localized in wild-type strains of B. pertussis, PtlH exhibited aberrant localization in strains lacking PtlD, PtlE, PtlF, and PtlG. We also found that localization of PtlH was perturbed in B. pertussis strains that were treated with carbonyl cyanide m-chlorophenylhydrazone and sodium arsenate, which are capable of depleting cellular ATP levels, and in strains of B. pertussis that produce an altered form of PtlH that lacks ATPase activity. When taken together, these results indicate that tight association of PtlH with the membrane, likely through interactions with components of the transporter-PT complex, requires the toxin substrate, a specific subset of the Ptl proteins, and ATP. Based on these data, a model for the assembly of the Ptl transporter-PT complex is presented.     

Prosaptide D5 reverses hyperalgesia: inhibition of calcium channels through a pertussis toxin-sensitive G-protein mechanism in the rat

A retro-inverso 11-mer peptidomimetic of prosaposin, Prosaptide D5, induced neurite outgrowth in NS20Y neuroblastoma cells and enhanced [35S]GTPgammaS binding to rat synaptosomal membrane at low nanomolar concentrations similar to prosaposin. Intramuscular injection of D5 ameliorated thermal hyperalgesia in the Seltzer rat model of neuropathic pain, returning paw withdrawal latency to control levels within 3 h after treatment. The effect was sustained for at least 48 h after injection. Prosaposin and D5 inhibited K+-stimulated synaptosomal 45Ca2+ uptake similar to omega-conotoxin MVIIC, demonstrating that both effectors modulated voltage-dependent calcium channels (VDCC); inhibition was largely abolished by pretreatment with pertussis toxin before D5 treatment. The results suggest a mechanism whereby VDCC are modulated by a pertussis toxin-sensitive G-protein coupled receptor; D5 binds to this receptor and thereby ameliorates hyperalgesia in the Seltzer model of neuropathic pain.     

Postsynaptic action mechanism of somatostatin on the membrane excitability in spinal substantia gelatinosa neurons of juvenile rats

We used tight-seal, whole-cell recording in juvenile rat spinal slices to investigate the action of somatostatin on substantia gelatinosa neurons. Bath application of somatostatin caused a robust and repeatable hyperpolarization or outward current in substantia gelatinosa neurons. Somatostatin inhibited spontaneous action potentials in subpopulation of substantia gelatinosa neurons. The amplitude of dorsal root-evoked excitatory postsynaptic currents and the frequency of spontaneous excitatory postsynaptic currents were not affected by somatostatin. The current induced by somatostatin developed almost instantaneously and did not show any time-dependent inactivation. The current-voltage relationship exhibited inward rectification. The conductance of somatostatin-sensitive current increased with the concentration of external K(+). The reversal potentials in different external K(+) concentrations were close to the K(+) equilibrium potentials. The effect of somatostatin was dose-dependent, with an EC(50) of 113 nM. The somatostatin-sensitive current was blocked by low concentration of extracellular Ba(2+) but not by glibenclamide, an inhibitor of ATP-sensitive K(+) channels. Hyperpolarization-activated cation current in a subpopulation of substantia gelatinosa neurons was not affected by somatostatin. In neurons recorded with an internal solution containing GTPgammaS, somatostatin induced outward current and hyperpolarization that did not reverse on washing. When the spontaneous induction of outward current with GTPgammaS was greatest, somatostatin did not induce any outward currents. Furthermore, intracellular dialysis of GDPbetaS, a G-protein antagonist, abolished the effect of somatostatin. In addition, SST-sensitive neurons were fewer in slices incubated with pertussis toxin than in adjacent control slices incubated without pertussis toxin.These results suggest that somatostatin decreases the postsynaptic membrane excitability of substantia gelatinosa neurons by a pertussis toxin-sensitive G-protein-mediated activation of an inwardly rectifying K(+) conductance.     

Mechanism of action of maternal serum on the interleukin2 receptor expression.

Neoplastic Jurkat cells were submitted to a PHA stimulation test after a preincubation in maternal or nulliparous serum (10% dilution). The Il2R expression was significantly downregulated among maternal serum treated cells. Retroplacental serum was significantly more inhibitive than peripheral maternal serum (P less than 0.01). The maternal IgG fractions and mostly the retroplacental IgG fraction proved to contain a factor mainly responsible for the Il2R expression inhibitive property. The molecular mechanism of this phenomenon was further studied. It was shown that H7 (acting as a protein kinase inhibitor) could not influence the Il2R modulation. E.G.T.A., a calcium chelator, was not able to interfere with the inhibitive influence of maternal serum. It was suggested that the maternal serum mediated inhibition of the IL2R expression is not influenced by the hydrolysis of membrane bound phosphatidyl inositol. In contrast, pertussis toxin markedly enhanced, in a dose dependent way, the suppressive influence of maternal serum as compared to nulliparous serum. At low concentrations, pertussis toxin lost its stimulating property and retained its ability to ADP ribosylate the alpha subunit of G proteins inducing a release of adenylcyclase mediating cAMP synthesis. This mechanism has been further studied by the addition of dbc AMP or dbc GMP to Jurkat cells preparations stimulated by PHA. dbc AMP, in a dose-related way, induced a downregulation of the IL2R expression of stimulated neoplastic cells preincubated in nulliparous or maternal serum. dbc GMP did not influence the IL2R expression in the same experimental conditions. The maternal serum mediated cells showed the most pronounced IL2R inhibition. Finally, it was shown that the cAMP synthesis by PHA stimulated Jurkat cells was upregulated in a dose dependent way, after a previous cellular incubation in progressive concentrations of maternal serum. In contrast, among nulliparous serum pretreated cells, cAMP synthesis remained significantly lower, after a lectin stimulation, as compared to the cAMP production derived from retroplacental serum treated and stimulated cells. Taken together, these experiments suggest that the maternal serum dependent suppression of the IL2R expression is related to a protein G stimulation followed by an enhanced cAMP synthesis.     

Dopamine (D2) or gamma-aminobutyric acid (GABAB) receptor activation hyperpolarizes rat melanotrophs and pertussis toxin blocks these responses and the accompanying fall in [Ca2+]i

The effects, on membrane potential, of dopamine (DA) and gamma-aminobutyric acid (GABA), transmitters present in the secreto-inhibitor innervation to the melanotrophs, were monitored in primary cultures of rat melanotrophs with bis-oxonol. DA and GABA, acting through D2 and GABAB receptors, hyperpolarized the melanotrophs. Hyperpolarization was not suppressed by tetrodotoxin but was prevented by pertussis toxin and may thus be due to a G protein mediated mechanism. Pertussis toxin also blocked the effects mediated by the two receptors to reduce intracellular free Ca2+ ([Ca2+]i).     

Induction of interstitial nephritis in rats by basement membrane of human origin

A study was conducted to investigate nephritogenic tubular basement membrane antigens common to human and rat kidneys. Brown Norway (BN) rats were immunized with human renal basement membrane in complete Freund's adjuvant simultaneously with Bordetella pertussis vaccine. The immunized rats developed polyuria and increased levels of serum creatinine one week after the second immunization. Renal histology at this time revealed marked, acute tubulointerstitial nephritis with linear deposition of IgG and C3 along the tubular basement membrane and Bowman's capsule, but not along the glomerular basement membrane. Rats with this tubulointerstitial nephritis rapidly developed antibodies against renal antigens from normal BN rats such as tubular basement membrane and proximal tubule brush border, however antibodies to glomerular basement membrane appeared later. Western blotting using the same rat sera detected a 145-kDa antigen from 8 M urea-solubilized human renal basement membrane and 120-kDa, 135-kDa and 145-kDa antigens from 8 M urea-solubilized BN rat renal basement membrane. This suggests that renal basement membranes of human and rat origin have common antigens involved in the pathogenesis of tubulointerstitial nephritis.     

Subcellular distribution of nucleotide cyclases in rat intestinal epithelium.

The subcellular distributions of adenylate cyclase and guanylate cyclase were determined for the mature enterocyte from the rat duodenum. Brush-border and basolateral membranes were prepared from isolated cells by an analytical isolation procedure, and multiple linear regression analysis was used to obtain a quantitative estimate of the distribution of recovered cyclase activities between the brush borders and basolateral membranes. Adenylate cyclase was largely confined to the basolateral surface of the epithelium, whereas guanylate cyclase was found on the brush-border and basolateral membrane fractions in the ratio 2.4:1. There was no evidence for the presence of nucleotide cyclases in the cytosol. Guanylate cyclase in both the brush-border and basolateral membranes was stimulated by epinephrine, insulin, and Triton X-100, but not by carbachol. Adenylate cyclase was not influenced by epinephrine, but was markedly stimulated by NaF and vasoactive intestinal peptide. These results are discussed in relation to the effects of hormones on transport across the small intestine.     

Transport of sulphate in rat jejunal and rat proximal tubular basolateral membrane vesicles

Basolateral membrane vesicles were isolated by a Percoll density gradient centrifugation method from small intestinal and renal proximal tubular epithelial cells. Transport of sulphate across the basolateral membrane was analyzed by measuring the uptake of tracer sulphate.In both membrane preparations, preloading the vesicles with sulphate-or hydroxyl-anions stimulated tracer sulphate uptake (trans-stimulation); an inwardly directed sodium gradient did not stimulate sulphate influx whether in the absence or in the presence of sulphate- or hydroxyl-iontrans-stimulation. Under sulphate trans-stimulation conditions, DIDS (10^–4 mol/l) inhibited sulphate influx.In intestinal membranes, trans-stimulation of sulphate influx was obtained by preloading the vesicles with chloride, in renal membranes by preloading with bicarbonate. Under sulphate trans-stimulation conditions, in intestinal membranes, sulphate influx was strongly inhibited by chloride, in renal membranes, chloride inhibition was absent. Under bicarbonate trans-stimulation conditions, in renal membranes, sulphate transport was inhibited by lactate.It is concluded that small intestinal and renal proximal tubular basolateral membrane vesicles contain a transport mechanism for sulphate that cannot be energized by a sodium gradient. The transport system in small intestinal basolateral membranes seems to be different from that in renal membranes. It is suggested that the observed interaction between inorganic and organic anion transport in renal basolateral membranes is indirect.Abbreviations DIDS 4,4-Diisothiocyano-2,2-disulfonic acid stilbene - DTT dithiothreitol - HEPES N-2-hydroxyethylpiperazine-N-ethanesulfonic acid - MES N-morpholinoethanesulfonic acid - PAH p-aminohippuric acid - PMSF phenylmethylsulfonyl fluoride - SITS 4-acetamido-4-isothiocyanostilbene-2,2-disulfonate - TMA tetramethylammonium - Tris tris(hydroxymethyl)aminomethane