Immunocytochemical characterization of porcine zona pellucida during follicular development

Sections of bovine ovaries fixed in Bouin's fluid or methanol-acetic acid and embedded in paraffin were incubated with chicken polyclonal antibodies to HPLC-purified zona glycoproteins ZP3 and ZP3. Oocytes of primordial follicles as well as of primary follicles showed weak labelling with anti-ZP3 and anti-ZP3. No immunostaining could be observed in the follicle cells. The ZP of primary follicles displayed distinct immunoreactivity for both ZP3 and ZP3. In secondary follicles, distinct labelling with anti-ZP3 and weak labelling with anti-ZP3 could be seen in the oocyte. The ZP showed immunoreactivity with antibodies to ZP3 and ZP3. Both antibodies labelled single follicle cells. In tertiary follicles, the oocytes were weakly labelled with anti-ZP3 and anti-ZP3. Some granulosa cells showed staining for ZP3 and ZP3. The ZP displayed strong immunoreactivity for ZP3 and ZP3. Cells of the corona radiata were strongly immunopositive for ZP3 and ZP3. Similar histotopography of immunoreactive cells could be seen in preovulatory follicles. The characteristic pattern observed for the distribution of ZP3 and ZP3 strongly suggests that in the porcine ovary both the oocyte and the follicle cells contribute to the synthesis of the ZP, perhaps in sequence.     

Down-regulation of tumor necrosis factor α activity by acute ethanol treatment in human peripheral blood monocytes

As the most commonly used drug that can modulate both metabolic and immune pathways, ethanol is evaluated in this report as a regulator of tumor necrosis factor (TNF) production in human peripheral blood monocytes (M) in combination with a variety of stimuli. While acute ethanol treatment did not induce TNF in M, it was a potent down-regulator of M TNF production whether induced by the combination of interferon- plus muramyl dipeptide (MDP) (P<0.001), lipopolysaccharide (LPS) alone (P<0.01), or interferon- plus LPS. Down-regulation of M TNF by ethanol was dose dependent and statistically significant in the biologically relevant, 25–150 mM, ethanol concentration range. We also demonstrate that these ethanol concentrations did not affect M viability. TNF down-regulation by ethanol was most effective when ethanol was administered 4 hr prior to MDP stimulation; however, it was also effective—though to a lesser extent—if it was added at the time of MDP stimulation. Furthermore, ethanol also down-regulated TNF production of thein vivo preactivated M of trauma patients, which produce hyperelevated levels of TNF. We have previously shown that the majority of posttrauma elevated M TNF is produced by the M subpopulation expressing high-affinity type I Fc receptors (FcRI). When the FcRI cross-linking-stimulated M subpopulation was treated with acute ethanol, TNF production was suppressed again both inin vivo preactivated M of trauma patients and in M of normal controls. In experiments utilizing cyclooxygenase inhibitor, we also demonstrate that ethanol has a direct, prostaglandin E₂-independent, effect on M TNF production. These results demonstrate that acute ethanol exposure has the potential to down-regulate M production of TNF significantly regardless of the TNF-inducing stimulus. Decreased capacity of M to produce TNF might, therefore, contribute to the immunological and metabolic abnormalities described after ethanol uptake.     

Altered synthesis of interferon-γ and expression of interferon-γ receptor by peripheral blood mononuclear cells from patients with IgA nephropathy and non-IgA proliferative glomerulonephritis

Previously we reported disease-specific interaction between interferon- (IFN-) and interleukin-4 (IL-4) in patients with IgA nephropathy (IgAN), suggesting the existence of unusual T cell behavior in this disease. In the present study, we investigated characteristic synthesis of interferon- (IFN-) and expression of IFN- receptor (IFN-R) in the peripheral blood mononuclear cells (PBMC) from patients with IgAN and other chronic proliferative glomerulonephritis (PGN). Heparinized peripheral blood samples were obtained from 38 patients with chronic mesangial proliferative glomerulonephritis (CGN; including 24 with IgA nephropathy) and 20 healthy controls. PBMC were isolated by gradient centrifugation and fragments were cultured in Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% fetal calf serum (FCS) for 72 hr. IFN- concentrations in supernatants were evaluated by the enzyme-linked immunosorbent assay (ELISA). Other parts of PBMC pellets were reacted with anti-human IFN-R monoclonal antibody and FITC-labeled anti-mouse second antibody for analysis of IFN-R expression on these cells by FACScan. The remaining PBMC were fractionated into CD4⁺ T cells, CD8⁺ T cells, B cells, NK, cells and macrophages using the MACS cell sorting system. The isolated cells were evaluated for IFN- or IFN-R mRNA expression by the semiquantitative RT-PCR method.In vitro IFN- synthesis was enhanced in patients with CGN, and NK cells were revealed to be responsible for such enhancement. On the other hand, the expression of IFN-R on macrophages was suppressed in CGN patients. These results suggest that impairment of regulation of the IFN- system might be involved in the development of CGN.     

Differential effects of interleukin-1α and β on the arachidonic acid cascade in human synovial cells and chondrocytes in culture

The effects of interleukin-1 and were tested on the [³H]-arachidonic acid release and the prostaglandin synthesis by human cultured synovial cells and chondrocytes. Both forms of interleukin-1 stimulated the arachidonic acid release but interleukin-1 was more potent than IL-1. Human synovial cells and chondrocytes synthesized three types of prostaglandins upon stimulation with interleukin-1 or : prostaglandin E₂, F₂ and 6-keto-prostaglandin F₁. Regarding the synthesis of these prostaglandins, IL-1 was again more potent than IL-1. A comparison between interleukin-1-stimulated synovial cells and chondrocytes revealed neither significant quantitative nor qualitative differences in both the arachidonic acid release and the prostaglandin synthesis.     

GM1-gangliosidosis: Chromosome 3 assignment of theβ-galactosidase-A gene (β GAL A )

The structural gene (GAL_A) coding for lysosomal -galactosidase- A (EC 3.2.1.23) has been assigned to human chromosome 3 using man-mouse somatic cell hybrids. Human -galactosidase-A was identified in cell hybrids with a species-specific antiserum to human liver -galactosidase-A. The antiserum precipitates -galactosidase-A from human tissues, cultured cells, and cell hybrids, and recognizes cross-reacting material from a patient with G_M1 gangliosidosis. We have analyzed 90 primary man-mouse hybrids derived from 12 separate fusion experiments utilizing cells from 9 individuals. Enzyme segregation analysis excluded all chromosomes for GAL_A assignment except chromosome 3. Concordant segregation of chromosomes and enzymes in 16 cell hybrids demonstrated assignment of GAL_A to chromosome 3; all other chromosomes were excluded. The evidence suggests that G_M1 gangliosidosis is a consequence of mutation at this GAL_A locus on chromosome 3.     

β-Subunits co-determine the sensitivity of rat neuronal nicotinic receptors to antagonists

We have investigated the effect of 4 ganglionic cholinergic antagonists (hexamethonium, mecamylamine, pentolinium, trimetaphan) on rat 32 and 34 neuronal nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes. Current responses were elicited by fast application of acetylcholine on voltage-clamped oocytes (holding potential Vinh = -80mV). Concentration-inhibition curves were used to get estimates of IC₅₀, the antagonist concentration yielding 50% reduction of the peak current. The K_B's of the antagonists were calculated using estimates of the apparent K_D of acetylcholine. The order of affinity of the antagonists was similar for both receptor subtypes: mecamylamine pentolinium > hexamethonium > trimetaphan. However, 34 neuronal nAChRs were 9 to 22 times more sensitive to each of the 4 antagonists than 32 receptors. These results further underline the importance of the -subunit as co-determinant of the functional properties of neuronal nAChRs.     

TRP channels: a TR(I)P through a world of multifunctional cation channels

The transient receptor potential (TRP) family of ion channels comprises more than 50 cation-permeable channels expressed from yeast to man. On the basis of structural homology, the TRP family can be subdivided in to seven main subfamilies: the TRPC (Canonical) group, the TRPV (Vanilloid) group, the TRPM (Melastatin) group, the TRPP (Polycystin), the TRPML (Mucolipin), the TRPA (Ankyrin) and the TRPN (NOMP) family. The cloning and characterization of members of this cation channel family has exploded during recent years, leading to a plethora of data concerning TRPs in a variety of cell types, tissues and species. This paper briefly reviews the TRP superfamily and the basic properties of its many members as a readers guide in this Special Issue. Hopefully, a better understanding of TRP channel physiology will provide important insight into the relationship between TRP channel dysfunction and human diseases.     

The sensitivity to Zn2+ discriminates between typical and atypical mast cells

The effects of zinc gluconate have been studied on rat peritoneal mast cells and rat basophilic leukemia cells (RBL 2H3) stimulated by various secretagogues. The IC₅₀'s of zinc gluconate on peritoneal cells were (M)1.6, 1.9, 5.4 and 18 for ionophore A23187, phorbol 12-myristate 13-acetate, substance P and immunoglobulin E-antigen, respectively. Higher concentrations of zinc gluconate were required to inhibit histamine secretion from RBL 2H3 cells, i.e. 12 M (ionophore A23187) and 140 M (immunoglobulin E-antigen). Zinc gluconate (10^–4 to 10^–3 M) also inhibited the IgE-dependent contraction of guinea pig trachea but was unable to affect that induced by exogenous histamine. These results suggest that zinc gluconate acts intracellularly and is selective of typical or connective tissue mast cells.     

Distribution of renal integrin receptors and their ligands in congenital nephrotic syndrome of the finnish type

The aim of this study was to examine the distribution of 1 and v integrins (Ints) and some of their ligands in the kidneys of patients with congenital nephrotic syndrome of the Finnish type (CNF) and in controls using indirect immunofluorescence with monoclonal antibodies. The mesangial reactivity of Int 1 and Int 1 subunits was more variable and an increased glomerular reactivity with Int 3 and Int-6 antibodies was found in CNF kidneys than in controls. Int 2 subunit was either completely missing from or found in significantly lesser amounts in CNF kidney glomeruli. The immunoreactivity for Int v was more variable, fainter and also more granular in CNF samples than in control kidneys. The glomerular reactivity for Int 5 was more diffuse and weaker, and in sclerotic Bowman's capsules more intense in CNF kidneys than in controls. Immunoreactivity for Int 6 was restricted and was comparable in extent in CNF and control kidneys. Of the extracellular matrix components studied, the expression of EDAFn, EDBFn, OncFn, Ln 2 chain, Ln 1 chain and tenascin was increased. This is also seen in several glomerular diseases with inflammation and sclerosis. Immunoreactivity for vitronectin was decreased. Several differences were found in the intensity or location of the immunostaining for the 1 and v Ints and their ligands in CNF kidneys compared with controls, which have not been found in any other proteinuric disease. Disturbed Int expression pattern in CNF may specifically reflect the disturbance of glomerular function caused by the primary defect in this disease.     

Inhibition of calmodulin expression prevents low-pH-induced gap junction uncoupling inXenopus oocytes

The relationship among intracellular pH (pH_i), –log₁₀ intracellular Ca²⁺ concentration (pCa_i) and gap junctional conductance, the participation of Ca²⁺ stores, and the role of calmodulin in channel regulation have been studied inXenopus oocytes, expressing the native connexin (Cx38), exposed to external solutions bubbled with 100% CO₂. The time courses of pH_i [measured with 2,7-bis(2-carboxyethyl)-5,6-carboxylluorscein (BCECF)], (pCa_i) (measured with the membrane-associated fura-C₁₈) and junctional conductance (measured with a double voltage-clamp protocol) were compared. The data obtained confirm previous evidence for a closer relationship of junctional conductance with (pCa_i) than with pH_i. Evidence for an inhibitory effect of intracellularly injected ruthenium red or 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA) on CO₂-induced uncoupling, coupled to negative results with Ca²⁺-free external solutions, point to a low-pH_i-induced Ca²⁺ release from internal stores, likely to be primarily mitochondria. The hypothesis proposing a participation of calmodulin in channel gating was tested by studying the effects of calmodulin expression inhibition by intracellular injection of oligonucleotides antisense to the two calmodulin mRNAs expressed in the oocytes. Calmodulin mRNA was permanently eliminated in 5 h. The oocytes injected with the antisense nucleotides progressively lost the capacity to uncouple with CO₂ within 72 h. The effect of CO₂ on junctional conductance was reduced by 60% in 24 h, by 76% in 48 h and by 93% in 72 h. Oocytes that had lost gating sensitivity to CO₂. partially recovered gating competency following calmodulin injection. The data suggest that lowered pH_i uncouples gap junctions by a Ca²⁺-calmodulin-mediated mechanism.     

Convergent inputs from articular,cutaneous and muscle receptors onto ascending tract cells in the cat spinal cord

Summary 1.Responses were recorded from 160 ascending tract cells in segments L4 to L6 of the spinal cord in chloralose anaesthetized, spinalized cats. The tract cells were identified by antidromic activation following stimulation of pathways in the lateral and ventral funiculi at the level of the spinal cord transection at the thoracolumbar junction. Axonal conduction velocities ranged from 9 to 114 m/s. 2. A sample of 152 of the neurones examined could be subdivided according to the distribution of their receptive fields into 49 cells activated just from receptors located in skin (s cells), 17 neurones excited by receptors in deep tissues (d cells), 15 units with a convergent input from receptors in skin and deep tissues (sd cells), and 25 neurones with a convergent input from the knee joint and either skin (sj cells), deep tissues (dj cells) or both (sdj cells). No receptive fields could be demonstrated for the remaining 46 neurones. 3. S and sj cells were found almost exclusively in the dorsal horn, whereas many d, sd, sdj and dj units were in the ventral horn. Almost all of the cells that lacked receptive fields were in the ventral horn or intermediate grey. 4. Ninety-one of 158 cells (56%) demonstrated no background activity. Of these, 43 cells (27%) lacked receptive fields. Many of the silent neurones were in the ventral horn, but some were in the dorsal horn. Of 25 cells having knee joint input, 18 (72%) had background activity. 5. All of the neurones that had a receptive field in the knee joint also had a convergent input from receptors in other tissues. In 3 cases, there was a receptive field in the skin over the foot (sj cells). For 16 cells, receptive fields included not only the knee joint but also skin and deep tissue (sdj cells). Usually, the cutaneous receptive field was near the knee joint, but sometimes it was remote, such as on the foot. The deep receptive fields were chiefly in the muscles of the thigh and/or leg. For 6 dj cells, the receptive fields included not only the knee joint but also deep fields like those of sdj cells. 6. Cutaneous receptive fields were classified as low threshold (cells excited best by innocuous intensities of mechanical stimulation), wide dynamic range (cells activated by weak mechanical stimuli, but the best responses were to noxious stimuli) or high threshold (innocuous stimuli had little effect, but noxious mechanical stimuli produced a vigorous discharge). Similarly, stimulation of the knee joint with weak mechanical stimuli could excite some neurones, while others could be activated by weak or strong articular stimuli but were excited best by noxious stimuli, and still other neurones were activated by knee joint stimuli only if the intensity was noxious. 7. In several instances, contralateral receptive fields were noted. These were generally in deep tissue or in the knee joint. 8. It was concluded that many of the responses to articular stimulation of the spinal cord ascending tract cells examined in this study could have been mediated by the fine afferent fibres that supply the knee joint. Although further work will be required to determine which particular ascending tracts transmit nociceptive information concerning the knee joint, it can be proposed that many of the responses demonstrated here were likely to play a role in either joint pain of in triggering responses associated with joint pain.     

Changes of the elastin compartment in the human meniscus

Summary We report on the elastin, collagen and ground substance compartments in the human meniscus and their interrelationships. Elastin is found in neonates and shows both an arrangement parallel to the collagen fibre and into net-like structures. Branching, caliber inconstancy, rupture and the phenomenon of the rubber band are findings within the different forms of the meniscopathy. The function of the elastin compartment cannot be visualised without suggesting puncta fixa. The morphology of the collagen-elastin-junctions has been described and their possible mode of function is discussed with the help of a model.     

Targeted gene replacement at the endogenousAPRT locus in CHO cells

We demonstrate the feasibility of targeted gene replacement at an endogenous, chromosomal gene locus in cultured mammalian cells, employing a two-step strategy similar to an approach routinely used for genetic manipulation in yeast. Utilizing an APRT⁺ recombinant generated by targeted integration of plasmid sequences (including a functional copy of the gpt gene) at the CHO APRT locus, we have been able to select gpt^– pop-out recombinants that have arisen by intrachromosomal recombination between APRT direct repeats at the targeted integration site. Reciprocal exchanges leading to pop-out of integrated plasmid/gpt gene sequences occur at a rate of 6.3×10^–6 per cell generation. Depending on the site of crossover, such pop-out events result in either replacement or restoration of the original APRT target gene sequence.     

Durchblutung der arterio-venösen Anastomosen und Wärmeaustausch an der Hundeextremität

Summary A certain amount of plasma of low (room) temperature is injected into the femoral artery of the experimental animal, the time course of the passage of the haemodilution and of the temperature change in the femoral vein are recorded. A maximal value for the fraction of the injected that has not left the blood during its passage through the vascular system of the limb is calculated from the records. This value—the maximal residual heat fraction—is compared to the fraction of the blood flow that passes through the arterio-venous anastomoses, measured as the recovery fraction of injected 22 spheres.In denervated hind limbs of anaesthetized dogs the maximal residual heat fraction was 9 per cent, the fraction of blood flow passing through the arterio-venous anastomoses 54 per cent, on the average. No positive correlation was found between the maximal residual heat fraction and the blood flow passing through the arterio-venous anastomoses.It is concluded from these results that the blood that passes through the arterio-venous anatomoses in the limbs of the dog has an extensive heat exchange with adjacent tissues—a prerequisite for the postulated thermoregulatory function of the arterio-venous anastomoses.

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Herrn Prof. Dr. Karl Thomas in Dankbarkeit zum 75. Geburtstag gewidmet.     

The role ofγδ T cells in the normal and disordered immune system

Summary A small population of T cells does not express the conventional T cell receptor characterized by the and polypeptide chains (TCR) but instead, two polypeptides termed and (TCR). This alternative receptor is able to recognize antigen. It appears early in T cell ontogeny, but its role in the thymus prior to the availability of TCR remains unclear. In selected sites such as skin or gut TCR predominates in mice which might suggest a role of T cells in the first line of defense against infection, T cells secrete lymphokines and display cytotoxic activity. However, their activation requirements may differ from what is known for T cells since MHC-nonrestricted and also CD4 and CD8 negative T cells have been described. Preferential activation by mycobacterial antigens possibly indicates a special repertoire of the T cells. In various diseases slightly increased numbers of T cells were found, but these preliminary studies have not yet provided evidence for a major pathogenetic role of T cells.List of abbreviations C constant region (immunoglobulin or TCR gene segment) - CD4 cluster of differentiation 4 (mainly on helper cells) - CD8 cluster of differentiation 8 (mainly on cytotoxic cells) - D diversity region (immunoglobulin or TCR gene segment) - DNA desoxyribonucleic acid - IL2 interleukin 2 - J joining region (immunoglobulin or TCR gene segment) - kD kiloDalton - MHC major histocompatibility complex - NK natural killer (cells) - RA rheumatoid arthritis - TCR T cell receptor - V variable region (immunoglobulin or TCR gene segment)     

„Leptospira Mini“ ein neuer Serotyp pathogener Leptospiren

Zusammenfassung Auf Grund ihres immunbiologischen Verhaltens gehören die Leptospirenstämme Sari, Ghidorsi und Szwajizak zu demselben serologischen Leptospirentyp, für den der Name Leptospira Mini vorgeschlagen wird.Der Stamm Sari wurde 1942 vonMino sowieVercelli in Italien isoliert. Der Stamm Ghidorsi wurde von uns im Zuge unserer Leptospirenforschungen bei einer Reisfeldarbeiterin der Po-Ebene nachgewiesen. Der Stamm Szwajizak, der vonSmith, Brown, Tonge u. Mitarb. im Jahre 1954 beschrieben wurde, ist in Nord-Qeensland gefunden worden. Der Stamm Sari und Ghidorsi gehören dem kompletten Biotyp (AB), der Stamm Szwajizak dem inkompletten (A) an.Leptospira Mini gehört zur Serogruppe hebdomadis. Ihre Virulenz ist schwach und ihre Bedeutung als Erreger menschlicher Leptospiren-infektionen scheint gering zu sein.     

Expansion of CD14+CD16+Monocytes in Critically Ill Cardiac Surgery Patients

We have asked whether critically ill cardiac valve surgery patients identified by a high APACHE II score exhibit an increase in the number of proin-flammatory CD14⁺ CD16⁺ monocytes. A group of 12 patients was studied over a period of 5 days post cardiac valve surgery for changes in blood monocyte populations. Patients were selected on day 1 post surgery to either be in good clinical condition (APACHE II Score of 14; N = 9) or to be critically ill (APACHE II score of 24; N = 3). The 14 patients had an uneventful course and could leave the ICU after 2–3 days. Among the 24 patients two showed a decrease of the score to 14 within the 5 days of observation and they could leave the ICU thereafter. One 24 patient (patient #2) had a persistently high score and finally died on day 28. Analysis of blood monocytes on day 1 post surgery revealed that the 14 patients had normal values of CD14⁺CD16⁺ monocytes (44 ± 9/l). By contrast the 24 patients had increased values of these cells with 243 ± 106 cells per 1 on day 1. The numbers of CD14⁺CD16⁺ monocytes returned to the control range over the 5 days of observation in 2 of the 24 patients concomitant with the improvement of the APACHE II score. CD14⁺CD16⁺ monocytes remained, however, at a high level in patient #2, the patient with persistently high APACHE II score.     

Identification and quantitation of cortisol metabolites in rat liver homogenate and -slices

Summary Cortisol-1, 2-H³ was incubated with rat liver homogenate and/or rat liver slices in the presence of a NADPH-generating system. The following metabolites could be identified in adult male rats: -cortol, allo--cortol, 3-allo--cortol, 20-hydroxy-cortisol, 11, 17, 20, 21-tetrahydroxy-5-pregnan-3-one, 3-allotetrahydrocortisol, tetrahydrocortisol, trace amounts of allotetrahydrocortisol and two highly polar metabolites only partly identified. In female rats only tetrahydrocortisol, allotetrahydrocortisol and allodihydrocortisol could be detected in significant amounts.The radioactive metabolites mentioned above were localized and quantitated on paper chromatograms by a 4-radiochromatogram scanner. A nearly perfect correlation was found between these results so obtained and those given by liquid-scintillation counting of each metabolite after its elution from the paper.Part of this work was supported by grant n° 695 of the National Fonds voor Wetenschappelijk Geneeskundig Onderzoek.Stagiair of the Nationaal Fonds voor Wetenschappelijk Onderzoek.     

Molecular specificity of the tubular resorption of “acidic” amino acids

Single sections of superficial proximal convolutions of rat kidney were microperfused in vivo and in situ. The perfusion fluids contained radioactively labelledl- ord-aspartate,l-glutamate,l-pyroglutamate, or N-methyl-d-aspartate.l--Carboxyglutamate as well as the other amino acids were added in the unlabelled from. Results.l- andd-Aspartate (0.073 mmol·1^–1) are quickly resorbed at about the same rate.d-Aspartate resorption was blocked byl-aspartate (5 mmol·1^–1) but not by -alanine (5 mmol·1^–1).l-Aspartate resorption was inhibited byl-glutamate (2 mmol·1^–1) but not byd-glutamate,l-asparagine,l-phenylalanine or by succinate (2 mmol·1^–1, each). The fast resorption ofl-glutamate (0.073 mmol·1^–1) was blocked byd-aspartate,l-cysteate (2 mmol·1^–1), but not by 3-mercaptopicolinic acid (0.15 mmol·1^–1),l-glutamine, 2-oxoglutarate, taurine, N-methyl-l-glutamate or kainic acid (2 mmol·1^–1, each).l--Carboxyglutamate (0.66 mmol·1^–1) and N-methyl-d-aspartate (2mol·1^–1) were found to be resorbed only at an extremely small rate.l-pyroglutamate (0.076 mmol·1^–1) resorption was not influenced byl-glutamate (1 mmol·1^–1). Fractional excretion of -carboxyglutamate was 7–25% (l-from) or 45–70% (d-form) at an artificially elevated plasma level of 12mol·1^–1.It is concluded thatl- andd-aspartate,l-glutamate,l-cysteate and, to a much smaller extent,l--carboxyglutamate, are accepted by the tubular resorption mechanism highly specific for acidic amino acids. N-Substitution, the amidation of the - or -carboxyl group, or the removal of the -amino moiety almost completely abolish the ability of such compounds to be resorbed via this carrier; N-methylated or -carboxylated derivatives of acidic amino acids are not resorbed at all from the proximal tubule. The resorption of glutamate, but not of aspartate, is highly stereospecific.Parts of this work were presented at meetings of the German Physiological Society in 1978 [28] and of the Gesellschaft für Nephrologie in 1980 [29] as well as at the VIIIth International Congress of Nephrology in Athens in 1981 [26]with technical assistance of Angelika Ascher and Gertaud Vetter     

Pertussis-toxin-sensitive inhibition by (-) baclofen of Ca signals in bovine chromaffin cells

Ca signals in bovine adrenal chromaffin cells were studied both in Fura-2/AM-loaded intact cells, and in voltage-clamped cells under whole-cell patch-clamp conditions. The effects of gamma-aminobutyric acid b subtype (GABA_b) receptor activation on K⁺-depolarization-induced signals and on voltage-activated Ca²⁺ currents were investigated. Both GABA (20 M) plus bicuculline (20 M) and (-)baclofen (20–100 M), effectively inhibited the Ca signal in intact cells. The effects caused by baclofen continued to develop during the time interval between two successive stimuli. The restoration of the Ca signal during washout of baclofen was also delayed and continued in some experiments for 10–20 min. The inhibitory effect of baclofen on the Ca signal was eliminated by pre-treatment of the cells with pertussis toxin (PTX, 1g/ml, for 4–6h at 37°C). Baclofen (50 M) inhibited Ca²⁺ current in whole-cell mode by at most 20%. The effect developed quickly and was reversible. Infusion into the cells of a non-hydrolyzable analogue of guanosine 5-triphosphate GTP S (100 M), led to complete inhibition of the Ca²⁺ conductance and of voltage-evoked intracellular Ca ([CA]_i) transients within 2 min. In paired cells intracellularly perfused with GTPS-free solution, the Ca²⁺ current amplitude decreased by only about 30% for 5–6 min. It is concluded that bovine chromaffin cells have functional GABA_b receptors the activation of which, mediated by a PTX-sensitive GTP-binding protein, inhibits the evoked increase in cytosolic free Ca²⁺. The small size of the effect on Ca²⁺ current in whole-cell mode as compared to that on the Ca signal in intact cells may be explained by washout of some regulatory element during cell dialysis, or by a relatively small contribution of the normal voltage-activated Ca²⁺ current to the Ca signal. Alternatively, it might indicate GABA_b effects on mechanisms other than Ca²⁺ channels.