Complex interactions between the steroid derivative RU 5135 and the GABAA-receptor complex. The modulation of [35S]t-butylbicyclophosporothionate ([35S]TBPS) binding was used to evaluate the actions of the steroid derivative RU 5135 at the gamma-aminobutyric acidA (GABAA) receptor complex. The inhibition of [35S]TBPS binding by GABA in the presence of various concentrations of RU 5135 was consistent with the hypothesis that RU 5135 is a competitive antagonist at the GABAA receptor. Despite common structural features (i.e., 3 alpha-hydroxylated, 5 beta-reduced A ring) with GABAA receptor-active neurosteroids, RU 5135 did not appear to be competitive at the putative steroid site on the GABAA receptor-active, as demonstrated by Schild analysis of 5 alpha-pregnane-3 alpha-ol-20-one (3 alpha,5 alpha-P) modulation of [35S]TBPS binding in the presence of different concentrations of RU 5135. On the other hand, the reduced potency of 3 alpha,5 alpha-P as an inhibitor of [35S]TBPS binding in the presence of RU 5135, as well as blockade of 5 alpha-pregnane-3 alpha-20 alpha-diol (5 alpha-pregnanediol) inhibition of [35S]TBPS binding by RU 5135 provide further support for the GABAA receptor antagonist properties of RU 5135. Moreover, this amidine steroid was able to partially inhibit [35S]TBPS binding independent of GABA with nanomolar potency; yet the mechanism by which this occurs remains to be determined.


Genomic organization, coding sequence and functional expression of human 5-HT2 and 5-HT1A receptor genes. The family of serotonin receptors consists of at least eight distinct subtypes, divided into four classes based on their pharmacological and functional characteristics. Here we report the cloning and expression in Swiss 3T3 cells of the human 5-HT2 and 5-HT1A receptor subtypes. Both genes encode functional receptors for 5-HT, that differ considerably in genomic structure, primary amino acid sequence, pharmacology and signal transduction. The 5-HT1A receptor transfectants displayed a single high affinity site for the agonist [3H](+/-)-8-hydroxy-2-(di-n-propylamino)tetralin HBr ([3H]8-OH-DPAT) and a pharmacological profile specific for the 5-HT1A receptor. In these transfectants, 5-HT mediated a dose-dependent inhibition of forskolin-stimulated cAMP levels. Cells expressing the 5-HT2 receptor exhibited high affinity binding for the antagonist [3H]ketanserin with a 5-HT2 receptor specific pharmacological profile. In these cells 5-HT activated phospholipase C in a dose-dependent manner. The 5-HT2 receptor displayed a genomic organization quite different from the 5-HT1A, 5-HT1B and 5-HT1D receptor subtypes. While these receptors are encoded by one single exon, the 5-HT2 receptor is encoded by three exons separated by two introns. The latter finding adds and additional molecular criterion for receptor classification.


[125I]His-neurokinin A binds selectively to NK2 receptors of the B-type in rat small intestine smooth muscle membranes. The high-affinity, reversible binding of [125I]His-neurokinin A (NKA) to rat small intestine smooth muscle membranes was investigated. Endogenous neurokinin agonists, selective neurokinin analogues, both agonist and antagonist, were used to define the selectivity of the binding. Both the endogenous and selective neurokinin analogue agonists displayed orders of potency indicating that [125I]His-NKA was binding to NK2 receptors. The use of recently developed NK2-selective antagonists indicated that the NK2 receptors present in this preparation were similar to those described in hamster trachea preparations (NK2B), and not endothelium-denuded rabbit pulmonary artery (NK2A). The absence of NK2A receptors and the predominance of NK2B was confirmed by blocking experiments using MEN10376 and L659877. Low-affinity binding of NKA was also observed with this preparation, which was not sensitive to the NK2-selective agonist, [beta-Ala8]NKA4-10. This was shown not to be due to the presence of NK1 or NK3 receptors by using selective agonists for NK1 and NK3 to block any such receptors. (No evidence for the presence of these receptors was obtained during these blocking experiments.) Guanylylimidodiphosphate appears to discriminate between the high- and low-affinity binding sites for NKA. It was thus concluded that high-affinity binding of [125I]His-NKA to rat small intestine smooth muscle membranes was selective for NK2B receptors. No evidence was found for the binding of [125I]His-NKA to NK1, NK3 or NK2A receptors.


Modulation of Ca2+ influx by protein phosphorylation in single intact clonal pituitary cells. In pituitary cells, electrical activity generates characteristic oscillations of the cytosolic free Ca2+ concentration, [Ca2+]i. These oscillations are controlled by activators as well as by inhibitors of secretion. We studied, in single fura-2-loaded cells, the role of protein phosphorylation in modulating [Ca2+]i oscillations, using either okadaic acid, an inhibitor of protein phosphatases, or activators of protein kinases A and C. Okadaic acid always increased rapidly both the frequency and amplitude of [Ca2+]i oscillations. In contrast, activation of protein kinases A or C generated more complex kinetic [Ca2+]i patterns: phosphorylation due to both kinases resulted in a sustained activation of [Ca2+]i oscillations in about one-third of the cells, whereas two-thirds of the cells responded by an arrest of [Ca2+]i oscillations. This transient phase of arrest was followed, after a few minutes, by a recovery of [Ca2+]i oscillations, often with enhanced frequency. During the arrest, depolarizing the cells with an external microelectrode could not trigger an increase in [Ca2+]i. We conclude that: (i) the fine regulation between phosphorylation/dephosphorylation events is crucial for the modulation of [Ca2+]i oscillations, and (ii) protein kinases A and C can control Ca2+ influx bidirectionally.


Lack of intrinsic activity and significant subtype selectivity of SR 95639A at muscarinic receptors. We assessed the intrinsic activity of the purported selective muscarinic M1 receptor agonist SR 95639A (morpholinoethylamino-3-benzocyclohepta-(5,6-c)-pyridazine) in inducing several receptor-mediated signals. Our results indicate that SR 95639A lacks the ability to activate phosphoinositide hydrolysis in rat cerebral cortex or in Chinese hamster ovary cells transfected with the genes of the muscarinic m1 and m3 receptors. Similarly, this compound did not exhibit intrinsic activity in stimulating muscarinic receptors which inhibit cyclic AMP synthesis and did not suppress acetylcholine release in rat striatum. In addition, SR 95639A did not show a marked selectivity at the level of the ligand recognition site at the muscarinic M1, M2 and M3 receptors, since it bound to these receptor subtypes with equilibrium dissociation constants of 4, 6 and 11 microM, respectively.


Differential effects on neutrophil activation of staurosporin and its protein kinase C-selective derivative cgp 41231. The relative insensitivity of the chemoattractant-induced respiratory burst to non-specific kinase inhibitors, such as staurosporin, is widely considered as evidence against the involvement of protein kinase C in signal transduction by chemoattractants. In this study we compared the effect on neutrophil activation of the non-specific kinase inhibitor staurosporin with the effect of its protein kinase C-selective derivative cgp 41251. Staurosporin activates secondary granule release by itself and enhances chemoattractant-induced primary granule release; it inhibits superoxide production in response to phorbol esters at low concentrations, but superoxide production in response to chemoattractants only at considerably higher concentrations. In contrast, cgp 41251 did not interfere with granule release, but inhibited phorbol ester- and chemoattractant-induced superoxide production with similar potency. These results suggest that many of the staurosporin effects, including its low potency to inhibit chemoattractant-induced superoxide production, are due to protein kinase C-independent effects. The results obtained with cgp 41251 are compatible with a role of protein kinase C in the mediation of the chemoattractant-induced respiratory burst of human neutrophils.


Proposed antagonists at GABAB receptors that inhibit adenylyl cyclase in cerebellar granule cell cultures of rat. The effects of various proposed GABAB receptor antagonists on baclofen-mediated inhibition of adenylyl cyclase were studied in cultured cerebellar granule cells from rat. (+/-)-Baclofen maximally inhibited adenylyl cyclase by approximately 60% of the basal enzyme activity with an EC50 value of 10 microM. 3-Aminopropane sulfonic acid (3-APS) and 5-aminovaleric acid (5-AVA) produced similar responses to that seen with (+/-)-baclofen. Saclofen reversed the action of (+/-)-baclofen, 50 microM, with a half maximal inhibitory concentration (IC50) of about 1.0 mM. The most effective antagonist in blocking the action of (+/-)-baclofen was 3-aminopropyl-diethoxy-methyl-phosphonic acid (CGP 35,348). In the presence of (+/-)-baclofen, 50 microM, the IC50 for CGP 35,348 was 290 microM and its inhibitory constant (KA) was 180 microM. The agonist-like actions of 3-APS and 5-AVA were antagonized by CGP 35,348 suggesting that 3-APS and 5-AVA may act as weak agonists at the GABAB receptor that inhibits adenylyl cyclase. All antagonists tested, except the new compound CGP 35,348, have very low potencies at GABAB receptors that inhibit adenylyl cyclase, though these compounds have been quite effective at other GABAB receptor-mediated events. Thus, the GABAB receptor which inhibits adenylyl cyclase differs pharmacologically from other reported GABAB receptor/effector systems and supports the existence of multiple receptor subtypes.


The effect of topoisomerase inhibitors on the expression of differentiation markers and cell cycle progression in human K-562 leukemia cells. Treatment of human K-562-J leukemia cells for 1 h with the topoisomerase II-reactive drugs VP-16, VM-26, or mAMSA resulted in a dose-dependent inhibition of proliferation and in an increase in the percentage of cells staining positive for hemoglobin, a marker of erythroid differentiation. Staining for hemoglobin of up to about 60% of the cells was observed at 20 microM VP-16, 1 microM VM-26, and 8 microM mAMSA. Such treatment also caused a G2/M arrest in the cell cycle. Incubation of the cells with radiolabeled VP-16 indicated that the induced erythroid differentiation was not due to continuous cell exposure to a residual amount of the drug. VP-16-induced erythroid differentiation was also not affected by DNA, RNA, or protein synthesis inhibitors. Differentiation induction and the G2/M arrest evoked by VP-16, VM-26, and mAMSA were, however, reduced in the presence of novobiocin. Our results indicate that topo-reactive drugs that cause G2/M arrest in the K-562-J cell cycle can induce in these cells erythroid differentiation after a short and irreversible interaction with their target molecule(s).


Cooperativity of SV40 T antigen and ras in progressive stages of transformation of human fibroblasts. Human diploid fibroblasts immortalized by SV40 T antigen provide an experimental system for studying the progression and synergism in transformation by secondary oncogenes. We have utilized the human fibroblast line HAL, which was immortalized with an orgin-defective SV40 genome encoding a temperature-sensitive T antigen, to study the cooperativity between SV40 T antigen and the ras oncogene in the progression of transformation. This study demonstrates that HAL cells possess characteristic growth patterns, requiring 10% serum, are anchorage dependent, and express a temperature-sensitive T antigen. HAL cells rely on the normal functioning of T antigen for continual growth and therefore do not proliferate at 39 degrees C. Three new derivatives of the HAL cell line were generated by microinjection of the ras oncogene. The cell line v-ras-HAL was derived by microinjection of HAL cells with v-Ha-ras DNA. The cell lines c-rasSVneo-HAL and c-rasLTRhygro-HAL were established by microinjection of HAL cells with the plasmids pSV2neoT24 or fpHVT24, respectively, wherein the ras gene is transcriptionally regulated by the cellular promoter and driven by either the SV40 enhancer or an upstream LTR enhancer. The three ras containing cell lines grow in reduced serum concentrations (0 to 5%), are anchorage independent, and express both T antigen and ras p21. The v-ras-HAL and c-rasSVneo-HAL cell lines are still dependent upon the normal functioning of T antigen for continual growth at 39 degrees C, however the c-rasLTRhygro-HAL cell line does proliferate at 39 degrees C in 10% serum-containing medium. Therefore, we propose that neither v-Ha-ras nor c-ras can replace T antigen at 39 degrees C; rather T antigen and ras cooperate in progressive stages of transformation of human fibroblasts.


Modulation of potassium transport in cultured retinal pigment epithelium and retinal glial cells by serum and epidermal growth factor. The ionic environment of retinal photoreceptors is partially controlled by potassium transporters on retinal glial and retinal pigment epithelial cells (RPE). In this study, serum and epidermal growth factor (EGF) were examined as modulators of potassium transport in confluent cultures of human RPE and rabbit retinal glia. EGF is a known mitogen for confluent RPE cultures and was shown here to also stimulate [3H]thymidine incorporation in cultures of retinal glia. For potassium transport studies 86Rb was used as a tracer during a 17-min incubation. For both retinal cell types the mean total 86Rb uptake in 10% serum was approximately 60% above basal, serum-free controls. For EGF, tested in several experiments in a concentration range from 1 to 100 ng/ml, maximal total uptake was 33 and 24% above controls for RPE and glia, respectively. Inhibitor studies suggested that basal and serum-stimulated uptake for both cell types occurred by the ouabain-sensitive Na-K ATPase pump and by the furosemide- or bumetanide-sensitive Na-K-Cl cotransporter. EGF-stimulated uptake appeared to be due predominantly to the cotransporter. The data suggest that serum components and EGF, which may be available to retina-derived cells under pathologic conditions, may not only stimulate proliferation but may also act as short-term modulators of potassium ion movement and thus affect physiologic processes that are sensitive to ion homeostasis.


Characterization of a panel of rat ventral prostate epithelial cell lines immortalized in the presence or absence of androgens. We have transfected rat ventral prostate (RVP) epithelial cells with a plasmid containing the SV40 large T-antigen in an attempt to establish a panel of cell lines that will be useful in molecular genetic studies of prostate cell function. Since the distribution of cell types in the RVP is dramatically affected by androgen withdrawal and replacement, cells isolated from normal, castrated, or castrated rats that were given daily injections of testosterone were used in these experiments. Cell lines were established in media that were supplemented or depleted of androgens to accommodate the possible requirements of different prostate cell types. Numerous cell lines were isolated which retain characteristics of RVP epithelial cells and five of these cell lines were studied in detail. All five cell lines express the SV40 large T-antigen, supporting the role of this viral protein in immortalization. The RVP cell lines were shown to contain high levels of functional glucocorticoid receptors, but very low levels of androgen binding activity even though androgen receptor RNA could be detected. It was determined that the decreased androgen receptor activity in the RVP cells was apparently due to low receptor expression based on the results of transient transfection assays using androgen receptor cDNA. Taken together, the biochemical, cytological, and morphological characterizations of the RVP cell lines suggest that they may all have been derived from basal prostate epithelial cells despite the initial differences in androgen status of the animal and the level of androgens in the culture media.


Connexins and the vacuolar proteolipid-like 16-kDa protein are not directly associated with each other but may be components of similar or the same gap junctional complexes. Gap junction preparations made from mouse liver plasma membranes by alkali extraction contain variable proportions of connexins (Cx32 and Cx26) and the 16-kDa protein which is closely related or may be identical to the 16-kDa proteolipid (subunit c) of the vacuolar H(+)-ATPase and the mediatophore complex. The absence of a stoichiometric relationship suggests that connexins and the 16-kDa protein are not subunits of the same channel complex, but analysis of alkali preparations by isopycnic centrifugation shows both types of protein are in membrane structures of the same buoyant density. Electron microscopic analysis of alkali preparations shows a homogeneous population of gap junctions of uniform morphology and width, suggesting the proteins are in the same or similar structures. The structures containing connexins and the 16-kDa protein can be separated by treatment of the plasma membranes with Triton X-100. After such treatment, the connexins remain associated with dense cellular or extracellular material and the gap junctional structures, after further extraction with N-lauroyl sarcosine and urea, contain only the 16-kDa protein. These detergent-extracted gap junctions are thinner (14.1 nm) than those in alkali preparations (18.4 nm).


Induction of c-jun independent of PKC, pertussis toxin-sensitive G protein, and polyamines in quiescent SV40-transformed 3T3 T cells. CSV3 clones of simian virus 40 large T antigen-transformed murine 3T3 T cells can be made quiescent as part of a differentiation process. In these quiescent cells, insulin- and vanadate-induced mitogenesis are both associated with the induction of the c-jun proto-oncogene (Wang and Scott 1991 J. Cell. Physiol. 147, 102-110; Wang et al. 1991 Cell Growth Differ. 2, 645-652). The current studies were therefore designed to compare the early signal transduction pathways employed by insulin and vanadate to regulate c-jun expression. In quiescent CSV3-1 cells, down-regulation of protein kinase C by prolonged exposure to 12-O-tetra-decanoylphorbol-13-acetate or inhibition of protein kinase C activity by treatment with the protein kinase C antagonist staurosporine is shown not to affect c-jun induction by insulin or vanadate. This suggests that both insulin and vanadate act in a protein kinase C-independent manner. Insulin's effect on c-jun induction does, however, involve a G protein because insulin's effect can be inhibited by pertussis toxin. In contrast, vanadate induction of c-jun is not affected by pertussis toxin. Genistein, a general tyrosine kinase inhibitor, can inhibit the ability of vanadate to induce c-jun but it does not inhibit insulin's effect. Finally, the depletion of polyamines, particularly spermidine, by DL-alpha-difluoromethylornithine treatment also prevents c-jun induction by insulin but DL-alpha-difluoromethylornithine treatment has no effect on c-jun induction by vanadate. These observations indicate that the c-jun induction by insulin and vanadate in CSV3-1 cells is mediated by different signal transduction mechanisms. Together with our previously published data, these results suggest that c-jun can be induced independent of protein kinase C activation, without involvement of pertussis toxin-sensitive G protein, independent of induction of c-fos, and without expression of high levels of intracellular polyamines.


Enzymatic activity and in vivo distribution of 5'-nucleotidase, an extracellular matrix binding glycoprotein, during the development of chicken striated muscle. The ecto-enzyme 5'-nucleotidase isolated from chicken gizzard has previously been shown to be a potent ligand of two glycoproteins of the extracellular matrix, namely fibronectin and laminin. Using immunofluorescent labeling techniques we observed that 5'-nucleotidase codistributed with laminin during the development of chicken striated muscle. In contrast, ecto-5'-nucleotidase was only faintly detectable on cells surrounded by a matrix expressing high levels of fibronectin. This distribution pattern distinguished 5'-nucleotidase from the pluripotent extracellular matrix receptors, chicken beta 1-integrins, which are expressed equally well in muscle and connective tissue. In addition, the specific activity of striated muscle ecto-5'-nucleotidase was stable during development and increased markedly posthatching. At each age considered, this specific activity corresponded to an 80-kDa enzyme which was inhibited by alpha,beta-methyleneadenosine diphosphate or by a monoclonal antibody directed against the smooth muscle isoform of the enzyme. Previous in vitro studies have revealed that 5'-nucleotidase is involved in the spreading of various mesenchyme-derived cells, such as chicken embryonic fibroblasts and myoblasts, on a laminin substrate. A prerequisite to examining a potential in vivo role for 5'-nucleotidase as an extracellular matrix ligand was to study its distribution. In adult muscle, 5'-nucleotidase displayed a more restricted distribution than in embryo. Results show that, in vivo, 5'-nucleotidase is revealed by immunofluorescent labeling using poly- and monoclonal antibodies to chicken gizzard 5'-nucleotidase in two structures, the costameres and myotendinous junctions, which are closely related to the focal adhesion sites observed in cell culture.


Adriamycin promotes neurite outgrowth in the "neurite-minus" N1A-103 mouse neuroblastoma cell line. Adriamycin, an anticancer agent acting on topoisomerase II, promotes the arrest of cell division and neurite extension in a "neurite-minus" murine neuroblastoma cell line, N1A-103. This morphological differentiation is accompanied by a blockade in the S phase of the cell cycle, modification of the amount of peripherin, and appearance of the beta 7-tubulin isoform. Yet, adriamycin-induced N1A-103 cells fail to express other neuronal markers, such as long-lasting Ca2+ channels, synaptophysin, and the shift in the proportion of the beta'1 tubulin isoform to the beta'2 isoform, whose appearance parallels the terminal differentiation of the wild type neuroblastoma cell line N1E-115. Hence, a comparison of the behavior of these two cell lines leads to the proposal that there are two programs of neuroblastoma differentiation: one where expression is triggered by the arrest of cell division and which is observed in adriamycin-induced N1A-103 variant cells, and the other, presumably occurring further downstream, which would involve further changes in morphogenesis and acquisition of new electrophysiological properties.


Study of the Ca2+/Na+ exchange mechanism in vesicles isolated from apical membranes of lens epithelium of spiny dogfish (Squalus acanthias) and bovine eye. Apical membrane vesicles of dogfish and bovine lens epithelium were prepared by differential centrifugation and Mg2+ precipitation. Enrichment of the apical enzyme markers, for Na+,K(+)-ATPase was achieved. Na+,K(+)-ATPase was enriched 25.9-fold and 23.6-fold for dogfish and bovine lens epithelia, respectively, and acid phosphatase was enriched 10.4-fold and 12.6-fold, respectively. There was no enrichment of the cytoplasmic marker, NADH reductase. The majority of the apical membrane vesicles isolated from both dogfish and bovine lens epithelia were oriented right-side out. Electron micrographs of the purified vesicles show the presence of circular sealed vesicles with an average size of approximately 0.2-0.5 microns. Incubation of the vesicles with either the acetoxymethyl ester of SBFI, a new indicator for sodium, or with Fura-2, the calcium-specific indicator, leads to a large accumulation of the de-esterified SBFI or Fura-2 in the lens epithelial apical vesicles as determined by fluorescence measurements. When an outwardly direct Ca2+ gradient is formed across the vesicular membranes, the influx of Na+ is stimulated 77.8 and 63.0% for dogfish and bovine lens epithelia, respectively. When an outwardly directed Na+ gradient is formed across the vesicular membranes, the Ca2+ influx is also greatly enhanced. Both cases indicate that there is a bidirectional Ca2+/Na+ exchanger present in the apical side of the lens epithelial cell. The exchanger is inhibited by 50 microM bepridil or by 200 microM La3+. The stimulatory effects are also observed in membrane vesicles that are 'short-circuited' with valinomycin and high concentrations of K+.(ABSTRACT TRUNCATED AT 250 WORDS)


A Na+/H+ exchanger and its relation to oxidative effects in plasma membrane vesicles from lens fibers. The existence of a Na+/H+ exchange mechanism and its role in the regulation of lens fiber cell intracellular pH, as well as the effect of oxidants and antioxidants, have been examined in vesicular preparations from spiny dogfish and bovine eyes. The fluorescent probes, SBFI (sodium-binding benzofuran isophthalate) for Na+ and SNARF-1 (seminaphthorhodafluor-1) for H+, were used to determine fluorescent ratios in a dual-wavelength spectrofluorimeter. The results show that: (1) the plasma membrane vesicles can be purified from lens fiber (52.5% of the vesicles were in the right-side out orientation for the fish eyes and 56.3% for the bovine eyes, show enrichment for the membrane marker activities and have an average size of 0.2-0.5 microns); (2) the influx of Na+ was dependent on an outwardly directed pH gradient with the uptake of Na+ sensitive to 500 microM amiloride but not affected by 50 microM valinomycin and 50 mM K gluconate; (3) when the pHo (extravesicular pH) of the vesicles was set at 8.1 and pHi (intravesicular pH) to 6.1, an inwardly directed Na+ gradient caused an increase in intravesicular pH by about 0.3 pH unit, and in bovine lens fiber vesicles the Na+ influx is highly dependent on the intravesicular pH (or on the pHo/pHi gradient); (4) 50 microM H2O2 increases the Na+/H+ exchange rate by 174% in the vesicles derived from fish lens fibers. Similarly, 100 microM H2O2 stimulates the Na+/H+ exchange rate by 194% in bovine eye fibers. This activation is prevented by preincubation with GSH (reduced glutathione) but not with GSSG (oxidizing glutathione). We conclude that a Na+/H+ exchange mechanism is present in the lens fiber membranes. This exchange possibly regulates intracellular pH and controls the intracellular Na+ concentration. The sensitivity of the Na+/H+ exchanger to the oxidant, hydrogen peroxide (H2O2) and the protection by the antioxidant GSH suggests a possible role for the Na+/H+ exchange mechanism in the formation of cataracts.


Evidence for an ATP-driven H(+)-pump in the plasma membrane of the bovine corneal epithelium. In a highly enriched plasma membrane fraction isolated from the bovine corneal epithelium, MgATP dependent intravesicular acidification was identified by measuring Acridine Orange quenching. The rate of acidification was increased 2.7-fold by pre-exposure of the membranes to 1% cholate which was subsequently removed by Sephadex G-50 column chromatography. However, in a lysosomal fraction whose enrichment with respect to the homogenate was 82-fold in N-acetyl-beta-D-glucosaminidase, cholate pre-exposure had no significant effect on the rate of intralysosomal acidification. This difference is assumed to reflect reorientation by cholate of the H(+)-pump's normally inaccessible ATP-binding site in right-side-out vesicles of the plasma membrane-enriched fraction to a configuration in which this site becomes accessible to externally added ATP. In contrast, the ATP-binding site of the H(+)-pump in the lysosomal fraction is completely exposed to the exterior even in the absence of cholate treatment. The characteristics of the H(+)-pump in the plasma membrane fraction was subsequently determined using cholate-pretreated membrane vesicles. The rank order of nucleotide support of the H(+)-pump activity was: ATP >> GTP > ITP. However, UTP and CTP were totally inactive. The pump is electrogenic because the activity of the pump was enhanced in voltage-clamped membrane vesicles. Substitution of Mg2+ with Mn2+ did not change the acidification rate but Co2+ only partly activated whereas Ca2+ and Zn2+ were ineffective as activators. The H(+)-pump was relatively unaffected by oligomycin, azide or vanadate but completely inhibited by 10 microM NEM or NBD-Cl and 92% inhibited by 20 microM DCCD.(ABSTRACT TRUNCATED AT 250 WORDS)


Rubidium transport in cultured monkey retinal pigment epithelium. Rb+ influx was used to assess Na-K-Cl cotransport and Na,K-ATPase activities in cultured monkey retinal pigment epithelium. Bumetanide-sensitive (Na-K-Cl cotransport-mediated) Rb+ influx exceeds ouabain-sensitive (Na,K-ATPase-mediated) Rb+ influx, with these two transporters accounting for approximately 95% of total Rb+ uptake. Half-maximal inhibition of Rb+ influx by bumetanide is attained at 75 nM bumetanide. The bumetanide-sensitive Rb+ influx depends on both extracellular Na+ and Cl-, and is activated by extracellular Rb+ with a relatively high affinity. Na-K-Cl cotransport activity is stimulated (2.5-fold) by increased extracellular osmolarity. Elevated cAMP content and glycolytic inhibition both depress cotransport activity. Cyanide application, however, had very little effect on Na-K-Cl cotransport activity. Monkey retinal pigment epithelial cells, maintained in culture, provide a system in which the activity and regulation of cation transport mechanisms can be examined.


Human retinal pigment epithelial cells possess beta 2-adrenergic receptors. Beta-adrenergic receptors on cultured human retinal pigment epithelium were demonstrated by the binding of [125I]cyanopindolol. Its pharmacologic specificity was also examined. Specific [125I]cyanopindolol binding was saturable, with a dissociation constant of 130 pM and a receptor density of 12 fmol per one-half million cells, which is equivalent to 14,000 receptor sites per retinal pigment epithelial cell. Agonists competed for specific [125I]cyanopindolol binding, with the following rank order of potencies: (-)-isoproterenol > (-)-epinephrine > (-)-norepinephrine. Beta 2-selective antagonist ICI-118551 was approximately 3 log orders more potent than the beta 1-selective antagonist, betaxolol. These receptors were also coupled to an adenylate cyclase. These results suggest strongly that cultured human retinal pigment epithelial cells possess beta 2-adrenergic receptors. The potential significance of these findings with regard to retinal pigment epithelial functions is discussed herein.


Sodium activity in the aqueous humor and corneal stroma of the rabbit. The present study examined the free sodium concentration of the aqueous humor and corneal stroma of both transparent and non-transparent corneas to assess the transendothelial activity gradient for sodium. In the transparent cornea of the adult rabbit, the sodium activity was higher in the aqueous humor than the stroma. This difference in sodium activity would cause water to diffuse down its concentration gradient from stroma to aqueous humor. In this way corneal transparency and the deturgesced state are maintained. Removal of the corneal endothelium in the adult rabbit produced an opaque swollen cornea. Under these conditions the sodium activity was higher in the stroma than the aqueous humor. However, an osmotic gradient was not produced by the Na+ activity gradient because the endothelium was not present to act as a semi-permeable membrane. The corneal endothelium was no longer present to establish and sustain the activity gradient for sodium that is necessary for corneal transparency in the mature rabbit. The transendothelial sodium activity gradient was also measured in 13-day-old rabbits. At this age, the cornea was not yet transparent, nevertheless the free sodium concentration of the aqueous humor was higher than that of the stroma, similar to the adult transparent cornea. This suggests that forces other than the establishment of the proper transendothelial sodium gradient are responsible for the lack of corneal transparency in the young rabbit.


Alpha 1-adrenoceptors in the corneal endothelium. The potent alpha 1-adrenoceptor antagonist, [3H]prazosin, exhibited high affinity, specific and reversible binding to intact rabbit, bovine and human corneal endothelial cells in culture. The binding of 1 nM [3H]prazosin to rabbit cells reached a steady-state level within 10 min at 37 degrees C. Under these conditions, approximately 50% of the [3H]prazosin bound was specific. The level of specific [3H]prazosin binding was concentration-dependent, but Rosenthal analysis indicated that [3H]prazosin bound to at least two sites. One site exhibited a high affinity for [3H]prazosin (Kd = 0.2 nM), but a relatively low binding capacity (Bmax = 175 fmol bound mg-1 protein); the other site showed a relatively low affinity for the radioligand (Kd = 85 nM), but a much higher binding capacity (1280 fmol mg-1). Several known alpha 1-adrenoceptor antagonists and agonists competitively inhibited [3H]prazosin binding at the high affinity site when incubated with the radioligand. The relative potencies of these competing ligands were generally consistent with their binding affinities for alpha 1-adrenoceptors in other tissues. Phenylephrine stimulated the rate of hydrolysis of phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate by 63% in these cells. This stimulation was inhibited by 52% if phentolamine was also present during the incubation. These data indicate that corneal endothelial cells have alpha 1-adrenoceptors which can modulate polyphosphoinositide turnover in this tissue.


Immunohistochemical localization of Na,K-ATPase in the in situ lens, cultured human lens epithelium and lentoid. The localization of Na,K-ATPase in the lens is quite controversial. We explored this problem through immunoelectron microscopic examination of rat and human lens. Unlike previously reported results, we have found that Na,K-ATPase is localized in the basal plasma membrane, but not in the lateral or apical plasma membrane of both rat and human lens epithelium. The lens fiber lacked immunoreaction. Localization of Na,K-ATPase was also investigated in the cultured human lens epithelium and in lentoid. Immunoreaction was detected in the apical (facing the media) plasma membrane of the lens epithelium cultured on the lens capsule, whereas the reaction was observable in both apical and basal plasma membrane of the lens epithelium cultured on the biopore membrane filters. Immunoreaction in lentoid was observed in the surface plasma membrane. These data indicate that the polarized distribution seen in the in situ lens epithelium changes when these cells are cultured, and that Na,K-ATPase in the cultured lens cells including lentoid is located in the plasma membrane which is in contact with the growth media. This change in polarity of Na,K-ATPase distribution in cultured epithelial cells may be dictated by the need to maintain ion homeostasis by extrusion of sodium ions across the cell membrane facing the media.


Epidermal growth factor potentiates the transmitter-induced stimulation of C-AMP and inositol phosphates in human pigment epithelial cells in culture. Salbutamol, isoproterenol and dopamine stimulate C-AMP production in human retinal pigment epithelium (RPE) cells by activation of beta 2-type receptors. Epidermal growth factor (EGF) in contrast does not alter basal levels of C-AMP but elevates in an apparently dose-dependent manner the isoproterenol-induced stimulation of C-AMP. EGF also potentiates the forskolin-induced stimulation of C-AMP but has no effect on the elevation of C-AMP caused by NECA (5'-[N-ethyl]-carboxamido adenosine), an adenosine A2-receptor agonist. EGF, isoproterenol and NECA have no effect on basal levels of inositol phosphates (InsPs) in human RPE cells, but EGF specifically elevates the carbachol-induced stimulation of InsPs. The carbachol effect on InsPs is attenuated by the phorbol ester PMA (4 beta-phorbol 12 myrisate 13-acetate). PMA did not, however, affect the stimulation of C-AMP caused by isoproterenol. The interaction of EGF and C-AMP is further demonstrated in experiments where the incorporation of [3H]thymidine into RPE cells was studied, as an index for proliferation. EGF stimulates RPE cell proliferation while isoproterenol and dibutyryl C-AMP nullify the EGF effect. Dibutyryl C-AMP has a negative effect on RPE cell proliferation while isoproterenol is ineffective. The data presented here suggest that after stimulation of EGF receptors, tyrosine-kinase-activated products can influence secondary messenger products produced from activation of beta 2-type (linked with C-AMP formation) and muscarinic (linked with InsPs production) receptors in RPE cells. We could find no evidence of an interaction between receptors associated with C-AMP and InsPs/diacylglycerol production.


Respiratory burst in peritoneal exudate cells in response to a modified tuftsin. Intraperitoneal administration of tuftsin-M [Thr-Lys-Pro-Arg-NH-(CH2)2-NH-CO-C15H31] to Balb/C mice has been shown to induce a respiratory burst in the peritoneal exudate cells. The macrophages exhibited enhanced levels of O2-, H2O2, NADPH oxidase and myeloperoxidase, but the activities of superoxide dismutase, catalase and glutathione peroxidase remained virtually unchanged. The magnitude of the oxidative burst depended directly on the dose of tuftsin-M; higher activity was observed at higher doses of the peptide. Tuftsin-M enhanced the generation of both O2- and H2O2 under in vitro conditions, as did phorbol myristate acetate. These results suggest that tuftsin-M could enhance non-specific defence against infections by activating the macrophages.


A simple assay to detect endothelial cell injury; measurement of released thrombomodulin from cells. A simple assay to determine the degree of endothelial cell injury has been developed using released thrombomodulin as the index. Thrombomodulin is a cell surface protein on endothelial cells which is released from the cell upon injury. In this assay, bovine arterial endothelial cells were cultured in serum free medium with the test substances and the amount of thrombomodulin released into the culture medium was measured by enzyme immunoassay. Substances which are known to injure cells such as H2O2, prostaglandin A2, lipopolysaccharide, and elastase released significant amounts of thrombomodulin. The sensitivity of this assay for mild injury was superior or at least equal to the traditional 51Cr release method. Since this method does not require the use of radioisotopes, it seems to be advantageous and suitable for the detection of endothelial cell injury during routine examination.


Interaction between alcohol consumption and positivity for antibodies to hepatitis C virus on the risk of liver cirrhosis: a case-control study. Provincial Group for the Study of Chronic Liver Disease. To assess the risk of developing liver cirrhosis associated with alcohol consumption, HBV and HCV infection markers, we carried out a case-control study involving 115 patients admitted to the medical departments of the general hospitals in the province of L'Aquila (Abruzzo, Italy) who received for the first time the diagnosis of liver cirrhosis, and 167 controls randomly selected among patients admitted to the same hospitals as the cases. Alcohol intake was measured in all 282 subjects using an already validated standardized questionnaire, and expressed as mean lifetime daily alcohol intake in grams. The mean lifetime daily alcohol intake showed a dose-dependent effect on the risk of cirrhosis: the relative risk significantly rose to 3.8 (95% CI: 2.0-7.3) for a mean daily intake of > or = 101 g alcohol; for HBsAg positivity the relative risk of cirrhosis was 23.0 (95% CI: 4.9-107.8) and for anti-HCV positivity it was 8.7 (95% CI: 4.3-17.6). After applying a multiple logistic regression analysis in a multivariate model including mean lifetime alcohol intake and anti-HCV status, both variables were significantly associated with the risk of cirrhosis (relative risks = 5.3-95% CI: 2.3-12.2 and 9.9-95% CI: 4.4-22.0, respectively). The combination of these two variables was found to fit an additive--but not multiplicative--model relative to the risk of cirrhosis: furthermore, the interaction of the anti-HCV status with the presence or absence of cirrhosis did not result in a significant source of variability for the mean lifetime daily alcohol intake.(ABSTRACT TRUNCATED AT 250 WORDS)


Infection by hepatitis B and C virus in non-intravenous drug using female prostitutes in Spain. We have studied the prevalence of hepatitis B (HBV) and hepatitis C virus (HCV) serologic markers in female blood donors and in female prostitutes and the relationship of antibodies to hepatitis B core antigen (anti-HBc) and of antibodies to HCV (anti-HCV) with the presence of treponemal antibodies (FTA-ABS) in non-intravenous drug using female prostitutes. Hepatitis B surface antigen (HBsAg) was found in 1.0% of the female blood donors, anti-HBc in 15.6% and anti-HCV in 0.7%. In the prostitutes, the prevalence of HBsAg was 6.1%, anti-HBc was positive in 29.0% and anti-HCV in 8.8%. No significant statistical association between the prevalence of anti-HBc or anti-HCV and the age of prostitutes (p = 0.9111 and p = 0.8254 respectively) or the length of time as prostitutes (p = 0.3583 and p = 0.5770) was found. FTA-ABS positive prostitutes had a significantly higher prevalence of anti-HCV than FTA-ABS negative prostitutes (p < 0.001). No statistical association was found between anti-HBc antibodies and positive FTA-ABS prostitutes (p = 0.336).


Under-registration of gestational trophoblastic disease in the Swedish Cancer Registry. The paper presents a study of completeness of registration of hydatidiform mole and gestational trophoblastic neoplasia in the Swedish Cancer Registry. The study is based on patients treated in Stockholm County, Sweden from 1971 through 1986. Non-notified cases were identified through a computerized register covering nearly all hospital admissions in the region and local hospital patient register. The results show that 25% of the cases with a diagnosis of hydatidiform mole were not included in the cancer register. Of all patients treated for trophoblastic malignancy, 66% were not recorded in the Cancer Registry. The frequent absence of histopathological confirmation in cases with malignant trophoblastic disease was probably the main factor contributing to the observed under-registration. The study indicates that the Swedish Cancer Registry alone does not provide sufficient data for studies on the incidence of gestational trophoblastic disease.


Re-expression of senescent markers in deinduced reversibly immortalized cells. We have developed a simian virus 40 (SV40) T-antigen immortalized human cell line, 1MR90-D305.2H4 (IDH4), in which the expression of T-antigen is controlled by the mouse mammary tumor virus (MMTV) promoter and thus regulated by steroids such as dexamethasone. Studies on the regulation of proliferation by T-antigen led to the formulation of a two-stage model for human cell immortalization, in which a mortality stage 1 mechanism (M1) was the target of T-antigen action, and an independent mortality stage 2 mechanism (M2) produced crisis and prevented T-antigen from directly immortalizing cells. Rarely, a cell expressing T-antigen escaped crisis (e.g., M2) and was capable of indefinite proliferation. This model predicted that the deinduction of T-antigen in IDH4 cells would lead to the reexpression of the M1 mechanism, and thus a reexpression of the senescent phenotype. Our study confirms the prediction that, in the absence of steroids, IDH4 cells express a variety of morphological and biochemical markers characteristic of normal senescent human fibroblasts.


Dose duration of nebulized nedocromil sodium in exercise-induced asthma. The dose-duration effect of nebulized nedocromil sodium was studied in ten patients with exercise-induced asthma (7 males mean (SEM) age 30.1 (3.5) yrs and predicted forced expiratory volume in one second (FEV1) 102%). All of these patients showed > 40% protection of their exercise asthma with 4 mg of nedocromil sodium delivered via metered dose inhaler. Three concentrations of nedocromil sodium (0.5, 2.5 and 10 mg.ml-1) and placebo were administered in double-blind, randomized manner. One ml of each solution was nebulized via a Wright nebulizer. Effects were assessed from the mean maximal percentage fall in FEV1 after 6-8 min treadmill exercise at 15, 135 and 255 min following each treatment and expressed as percentage protection. The mean baseline FEV1 values before and after treatments were comparable on four days of testing. Nedocromil sodium inhibited exercise-induced fall in FEV1 at all concentrations (p < 0.001) and the inhibitory effect was still present at 255 min. No differences were observed between active treatments.


Lung function in relation to silicosis and silica exposure in granite workers. Lung function tests (forced expiratory volume in one second (FEV1), forced vital capacity (FVC) and FEV1/FVC %) were related to silica exposure and the extent of radiological opacities in a study of 206 active and 132 previously employed granite workers from two quarries. The investigations included detailed personal interviews, spirometric testing and radiographic examination of the chest. The chest X-ray films were read randomly and independently by three readers, using International Labour Office (ILO) standard films. Cumulative exposure to respirable silica (mg.m-3-yr) and total granite dust (million particles per cubic foot (mppcf-yr)) were estimated for each subject based on his years of employment at various jobs and historical and current measurements of quarry-, period- and job-specific exposures. The results suggest that chronic simple silicosis, especially for profusion category 2 and 3, was associated with significant lung function loss. As expected, mixed dust fibrosis was associated with little or no functional disturbance. Massive fibrosis was associated with significant obstructive and restrictive impairment. No additional effect of exposure to respirable silica on lung function loss was found after allowing for the presence of "silicosis". However, exposure to total dust (mppcf-yr) appeared to be associated with some lung function loss independent of silicosis. Our results indicate that chronic simple silicosis is not a benign disease; silica exposure is the primary cause, but the lung function loss in silicotics is directly attributable to the fibrotic lung disease. However, exposure to total granite dust beyond the respirable size range may separately produce additional lung function loss.


Effect of inhaled beclomethasone and nedocromil sodium on bronchial hyperresponsiveness to histamine and distilled water. In a randomized, cross-over study we compared the effects of inhaled nedocromil sodium, 4 mg q.i.d., with inhaled beclomethasone dipropionate, 200 micrograms q.i.d. in 23 atopic asthmatic patients. After a 3 week single-blind placebo period, regarded as the baseline, and after 4 and 8 weeks of active treatment, drug effects were assessed with regard to bronchial hyperresponsiveness to histamine and distilled water, lung function and beta 2-agonist use. After 4 and 8 weeks of treatment, nedocromil sodium reduced the histamine responsiveness (p < 0.005 and p < 0.0005), but not the distilled water responsiveness, and did not improve lung function and peakflow measurements compared to baseline. After 4 and 8 weeks of treatment, beclomethasone caused a significant increase in lung function (p < 0.005) and decrease in bronchial hyperresponsiveness to histamine (p < 0.0005) and distilled water (p < 0.0005) as compared to baseline. beta 2-agonist use was significantly diminished after an 8 week treatment with beclomethasone, whereas nedocromil sodium had no effect. Treatment with beclomethasone was superior to treatment with nedocromil sodium with regard to bronchial hyperresponsiveness to histamine and distilled water (p < 0.0005 and p < 0.005), lung function (p = 0.003), peakflow measurements (p < 0.05) and beta 2-agonist use (p < 0.005).


Protein phosphorylation is involved in the regulation of chromatin condensation during interphase. Loci affecting the condensation state of interphase chromatin have been previously identified from analysis of suppression and enhancement of position effect variegation (PEV) in Drosophila. Here we show that Su-var(3)6 and an allelic mutant, e078, which both show suppression of PEV in the heterozygous state, have point mutations (Gly220-->Ser and Gly220-->Asp, respectively) in a protein phosphatase 1 catalytic subunit located at 87B (PP1 87B). The mutated glycine is conserved in all known protein serine/threonine phosphatases in the same gene family, and its substitution decreases PP1 activity. We conclude that protein dephosphorylation by PP1 87B regulates the condensation state of chromatin during interphase.


Effect of perturbing diacylglycerol metabolism on cytosolic free Ca2+ oscillations induced in single hepatocytes. Single rat hepatocytes microinjected with aequorin show free Ca oscillations when stimulated with Ca(2+)-mobilizing hormones. We show here that an inhibitor of diacylglycerol kinase (R 59 022) and an analogue of native diacylglycerol (diC8) inhibit free Ca oscillations induced by phenylephrine and vasopressin. These results agree with a negative feedback effect of protein kinase C on free Ca oscillations.


Escherichia coli DNA gyrase: genetic analysis of gyrA and gyrB mutations responsible for thermosensitive enzyme activity. Escherichia coli gyrA43 and gyrB203 alleles conferring temperature-sensitive (ts) growth encoded Gly751-->Asp and Pro171-->Ser substitutions in the DNA gyrase A and B subunits, respectively. A plasmid-borne gyrA43 allele was genetically dominant over a chromosomal quinolone-resistant gyrA gene at 30 degrees C but not at 42 degrees C. These results and others confirm the ts phenotype of the mutation, the first to be identified in the C-terminal DNA binding/complex stabilizing domain of gyrase A protein. By contrast, the Pro171-->Ser mutation is located near the ATP-binding site of gyrase B protein and could interfere with energy coupling during DNA supercoiling. These data are discussed in regard to recently described gyrA(ts) mutations that affect the control of chromosome segregation.


Neuronal-type nicotinic receptors in human neuroblastoma and small-cell lung carcinoma cell lines. A beta subunit of the neuronal nicotinic receptor, sharing 88% homology with the rat beta 4 subunit, has been cloned from a human neuroblastoma cell line. The gene encoding the human beta 4 subunit is expressed in association with the alpha 3 gene in neuroblastoma and small-cell lung carcinoma cell lines. Patch-clamp experiments and radioligand binding assays confirm that these neuroendocrine tumor cell lines express functional neuronal nicotinic receptors. We suggest that these receptors might play a crucial role in the control of neurotransmitter and hormone secretion from neurosecretory human tumors.


pH changes associated with cytochrome c oxidase reaction with H2O2. Protonation state of the peroxy and oxoferryl intermediates. pH changes associated with the mitochondrial cytochrome oxidase reaction with H2O2 have been studied. In the presence of ferricyanide or Tris-phenanthroline complex of CoIII as electron acceptors, reaction of H2O2 with the oxidized cytochrome oxidase is accompanied by a steady proton release with a rate constant of ca. 3 M-1.s-1 at pH 6.8. The acidification is completely inhibited by superoxide dismutase and its pre-steady-state kinetics correlates with that of the oxoferryl compound (F) accumulation. Apparently, the proton release is linked to superoxide generation by cytochrome oxidase under these conditions. In the presence of superoxide dismutase and without the electron acceptors, the H2O2-induced transitions of cytochrome oxidase from the oxidized to the peroxy (P) and from the peroxy to the oxoferryl state are not associated with any significant proton release or uptake. The results point to the following mechanism of O2- generation and protonation states of the cytochrome oxidase compounds P and F: [formula: see text]


Changes in Na+,K(+)-ATPase structure induced by cation binding. Approach by Raman spectroscopy. Raman analysis of Na+,K(+)-ATPase structural changes induced by cation binding reveals a slight decrease ( < 10%) of the alpha-helical content upon E1-E2 transition. Pronounced conformational changes of the enzyme are unlikely as the character of the environment of tyrosine residues remains unaltered. However, local changes can take place as evidenced by changes in tryptophan vibration at about 880 cm-1.


Inhibition by acidic phospholipids of protein degradation by ER-60 protease, a novel cysteine protease, of endoplasmic reticulum. A protein (ER60) with sequence similarity to phosphoinositide-specific phospholipase C-alpha purified from rat liver endoplasmic reticulum (ER) degraded ER resident proteins and is really a protease [(1992) J. Biol. Chem. 265, 15152-15159]. Therefore, ER60 is called ER-60 protease. We now show that negatively charged phospholipids, phosphatidylinositol, phosphatidylinositol 4,5-bisphosphate and phosphatidylserine inhibit ER protein degradation by ER-60 protease. Phosphatidylcholine and phosphatidylethanolamine show no effect on the activity of ER-60 protease. With the use of protease inhibitors, ER-60 protease is shown to be a novel cysteine protease distinct from those of the cytosol and lysosomes.


Interferon-gamma induces cell surface expression for both types of tumor necrosis factor receptors. Interferons are known to potentiate various biological effects of tumor necrosis factor (TNF). Recently, two different types of TNF receptors with molecular masses of 60 kDa (p60) and 80 kDa (p80), primarily expressed by epithelial cells and myeloid cells, respectively, have been identified. In the present report, we examined the effect of interferon-gamma (IFN-gamma) on each type of TNF receptor. Our results indicate that IFN-gamma induces TNF receptors on both myeloid (e.g. HL-60) and epithelial cells (e.g. HeLa). Furthermore, by using antibodies specific to each type of receptor, we demonstrate that both TNF receptors are equally inducible by IFN-alpha, IFN-beta and IFN-gamma. Thus, the increase in TNF receptors by interferons may play a role in their synergistic cellular response.


p42 MAP kinase phosphorylation sites in microtubule-associated protein tau are dephosphorylated by protein phosphatase 2A1. Implications for Alzheimer's disease [corrected]. The paired helical filament (PHF), which comprises the major fibrous element of the neurofibrillary tangle of Alzheimer's disease, is composed of abnormally phosphorylated microtubule-associated protein tau. Here we show that p42 MAP kinase phosphorylates recombinant tau and converts it to a form which is similar to PHF tau. Of the major serine/threonine protein phosphatases found in mammalian tissues only protein phosphatase 2A (PP2A) could dephosphorylate tau phosphorylated in this manner, with PP2A1 being the most effective form of the enzyme.


Stable expression of human D3 dopamine receptors in GH4C1 pituitary cells. Human D3 dopamine receptor DNA was stably transfected into GH4C1 pituitary cells. Displacement of iodosulpiride binding in hD3 transfected cells (Kd = 0.3 nM, Bmax = 89 fmol/mg protein) by dopaminergic ligands was indistinguishable from that of hD3 receptors in CHO cells. Only two clonal cell lines exhibited weak GppNHp-dependent shifts in [3H]N-0437 binding, and these were used for functional assays. Neither arachidonic acid metabolism, cAMP levels, inositol phosphate turnover, intracellular calcium, or potassium currents were consistently affected by dopamine (1-10 microM). The paucity of responses indicates that human D3 receptors do not couple efficiently to these second messengers in GH4C1 cells.


A complex between replication factor A (SSB) and DNA helicase stimulates DNA synthesis of DNA polymerase alpha on double-stranded DNA. A helicase-like DNA unwinding activity was found in highly purified fractions of the calf thymus single-stranded DNA binding protein (ctSSB), also known as replication protein A (RP-A) or replication factor A (RF-A). This activity depended on the hydrolysis of ATP or dATP, and used CTP with a lower efficiency. ctSSB promoted the homologous DNA polymerase alpha to perform DNA synthesis on double-stranded templates containing replication fork-like structures. The rate and amount of DNA synthesis was found to be dependent on the concentration of ctSSB. At a 10-fold mass excess of ctSSB over double-stranded DNA, products of 200-600 nucleotides in length were obtained. This comprises or even exceeds the length of a eukaryotic Okazaki fragment. The ctSSB-associated DNA helicase activity is most likely a distinct protein rather than an inherent property of SSB, as inferred from titration experiments between SSB and DNA. The association of a helicase with SSB and the stimulatory action of this complex to the DNA polymerase alpha-catalyzed synthesis of double-stranded DNA suggests a cooperative function of the three enzymatic activities in the process of eukaryotic DNA replication.


A voltage-gated calcium channel is linked to the antigen receptor in Jurkat T lymphocytes. Activation of T lymphocytes results in an increase in intracellular Ca2+ due in large part to influx of extracellular Ca2+. Using the patch clamp technique, an inward current in Jurkat T lymphocytes was observed upon depolarization from a holding potential of -90 mV but not from -60 mV. This whole-cell current was insensitive to tetrodotoxin, carried by Ba2+, and blocked by Ni2+. Occupancy of the T lymphocyte antigen receptor increased the current's magnitude. These data suggest that antigen receptor-induced Ca2+ entry in T lymphocytes may be mediated by a voltage-regulated Ca channel.


A carboxyl-terminal truncated version of the activin receptor mediates activin signals in early Xenopus embryos. The function of a carboxyl-terminal truncated version of the Xenopus activin receptor, encoded by a previously isolated gene XSTK2, was investigated in early embryos. The transcript corresponding to the truncated receptor gene was detected throughout embryonic development although the temporal expression pattern was different from that of an intact receptor. Injection of XSTK2 mRNA into early embryos resulted in the formation of a duplicated body axis. Mesoderm induction as evaluated by the activation of the alpha-actin gene in presumptive ectoderm (animal cap) treated with exogenous activin was significantly enhanced by the injection of XSTK2 mRNA. These results suggest that the truncated receptor is capable of transmitting the activin signal to the same extent as the native receptor.


Expression of the ras-related rab3a gene in insulinoma-derived cell lines. This study was designed to search for the expression of the small-molecular-weight GTP-binding protein rab3a in endocrine pancreatic cell lines. Total RNA was isolated from five different cell lines (RINm5F, RIN 104836, beta-TC1, HIT-15, and INRI-G9) and from whole rat brain. The expression of rab3a was analyzed by Northern blots. Similar as in brain two transcripts of 1300 and 1800 bp were detected in RIN-cells at low stringency conditions with the predominant signal at 1300 bp. At high stringency the stronger signal was at 1800 bp. When a 300 bp PstI fragment derived from the coding region of rab3a was utilized as probe the 1800 bp signal was predominant under each condition. Only a faint band at 1800 bp occurred in preparations from beta TC1-cells and no signal at all was found in HIT-15 and INRI-G9-cells. In conclusion, rab3a is expressed in rat insulin-releasing insulinoma-derived RIN-cells with a specific 1800 bp transcription product.


High level expression of transfected G protein alpha i3 subunit is required for plasma membrane targeting and adenylyl cyclase inhibition in NIH 3T3 fibroblasts. The alpha subunits of pertussis toxin-sensitive G proteins Gi1, Gi2 and Gi3 have been shown to inhibit adenylyl cyclase in transfected cells. However, Gi3 has recently been associated with protein transport and localized to the Golgi apparatus in a number of cell lines, rather than to the plasma membrane. We studied NIH 3T3 clones stably expressing different levels of a constitutively activated mutant of the alpha subunit of Gi3 (alpha i3-Q204L). Transfected alpha i3 subunits were localized to the Golgi apparatus in all NIH 3T3 clones. In clones expressing alpha i3-Q204L at high levels, alpha i3 subunits were also localized to the plasma membrane. Those clones which demonstrated expression of alpha i3 at the plasma membrane showed a 40% to 60% inhibition of forskolin-induced cAMP accumulation. Transfected NIH 3T3 clones in which plasma membrane alpha i3 was undetectable, did not show inhibition of forskolin-induced cAMP accumulation. These data suggest that, unless high expression is achieved in transfected cells, alpha i3 is targeted predominantly to the Golgi, not to the plasma membrane, and does not control adenylyl cyclase activity in NIH 3T3 fibroblasts.


Primary structure and distribution of a novel ryanodine receptor/calcium release channel from rabbit brain. The complete amino acid sequence of a novel ryanodine receptor/calcium release channel from rabbit brain has been deduced by cloning and sequence analysis of the cDNA. This protein is composed of 4872 amino acids and shares characteristic structural features with the skeletal muscle and cardiac ryanodine receptors. RNA blot hybridization analysis shows that the brain ryanodine receptor is abundantly expressed in corpus striatum, thalamus and hippocampus, whereas the cardiac ryanodine receptor is more uniformly expressed in the brain. The brain ryanodine receptor gene is transcribed also in smooth muscle.


CDP-diacylglycerol synthesis in rat liver mitochondria. CDP-diacylglycerol for polyglycerophosphatide biogenesis can be synthesized within rat liver mitochondria. This membrane-associated enzyme was predominantly located in the inner mitochondrial membrane. GTP had a significant effect in activating the microsomal CDP-diacylglycerol synthase, especially if the microsomes were preincubated with GTP in the presence of phosphatidic acid. This stimulatory effect of GTP on the microsomal enzyme was not detected in the mitochondrial fractions. The enzymes could be solubilized from the membrane fractions using CHAPS, and the detergent-soluble activity partially restored by addition of phospholipids. Mitochondrial and microsomal CDP-diacylglycerol synthase activity could be completely separated by anion-exchange column chromatography. The mitochondrial and microsomal CDP-diacylglycerol synthases appear to be two distinct enzymes with different localization and regulatory characteristics.


Rabbit fast skeletal muscle phospholipase C. Molecular weight determination by renaturation after polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. Phosphoinositide specific phospholipase C from rabbit fast skeletal muscle has been enriched ca. 1,000-fold with a specific activity of 40 mumol x min-1 x mg-1. Following SDS-PAGE, renaturation of the enzyme protein in the presence of deoxycholate allowed the determination of an apparent molecular weight of 110 kDa. Gel-filtration of the native enzyme resulted in a very similar apparent molecular weight of 115 kDa, however, associated proteins of higher molecular weight were also found. Free Ca2+ concentrations needed for half-maximal activation of PtdIns(4,5)P2, PtdIns4P and PtdIns hydrolysis are 6.3 microM, 85 microM and 1.8 mM, and the Km values for these substrates 102, 340 and 937 microM, respectively.


Formation of a covalent Hg-Cys-bond during mercurial activation of PMNL procollagenase gives evidence of a cysteine-switch mechanism. A common method for the activation of mammalian metalloproteinases is the use of mercurial compounds. Activation of PMNL procollagenase by soluble mercurials takes place as a three-step mechanism with a final intermolecular loss of the PRCGVPD autoinhibitor region. In this study covalently bound mercury in the form of mercurial agarose was chosen to probe activation of PMNL procollagenase. Activation was not achieved, since the final intermolecular cleavage with removal of the PRCGVPD motif could not take place. An intermediate form of the enzyme was bound to the column. Its N-terminal sequence determination proved cleavage of the Asp64-Met65 peptide bond leaving the cysteine of the propeptide domain for covalent attachment to the mercurial agarose. This gives further evidence of a cysteine-switch mechanism involving Cys71.


Photoinhibition affects the non-heme iron center in photosystem II. Effects on the PS II acceptor side caused by exposure to strong white light (180 W/m2) of PS II membrane fragments (spinach) at pH 6.5 and 0 degrees C were analyzed by measuring low temperature EPR signals and flash-induced transient changes of the fluorescence quantum yield. The following results were obtained: (a) the extent of the light induced g = 1.9 EPR signal as a measure of photochemical Fe2+QA- formation declines with progressing photoinhibition. The half-life of this effect is independent of the absence or presence of an exogenous electron acceptor during the photoinhibitory treatment; (b) in samples photoinhibited in the absence of an electron acceptor and subsequently incubated with K3[Fe(CN)6] in the dark, the extent of the g = 8 EPR signal (reflecting the oxidized Fe3+ form of the endogenous non-heme iron center) and of the flash-induced change of the fluorescence yield (as a measure of fast electron transfer from QA- to Fe3+ after the first flash; [see (1992) Photosynth. Res. 31, 113-126] exhibits the same dependence on photoinhibition time as the g = 1.9 EPR signal; (c) in samples photoinhibited in the presence of an exogenous electron acceptor, the signals reflecting Fe(3+)-formation and fast electron transfer from QA- to Fe3+ decline faster than the g = 1.9 EPR signal. These results provide for the first time direct evidence that the endogenous non-heme iron center located between QA and QB is susceptible to modifications by light stress. The implications of this finding will be discussed.


Accumulation of the pre-assembled membrane arm of NADH:ubiquinone oxidoreductase in mitochondria of manganese-limited grown Neurospora crassa. The NADH:ubiquinone oxidoreductase (complex I) of mitochondria is constructed from two arms arranged perpendicular to each other. The peripheral arm protruding into the matrix contains the proximal section of the electron pathway, and the membrane arm with all mitochondrially encoded subunits contains the distal section of the electron pathway. When Neurospora crassa is grown under manganese limitation the formation of the peripheral arm is disturbed, but the membrane arm containing the iron-sulfur cluster N-2, is accumulated. An extra-polypeptide, assumed to be a chaperone, is found to be associated with this pre-assembled membrane arm.


Angiotensin II induces formation of the early growth response gene-1 protein in rat vascular smooth muscle cells. The effect of angiotensin II (Ang II) on the early growth response gene-1 (Egr-1) mRNA, on the Egr-1 protein and on the phosphoinositide PI turnover signalling system was investigated in the presence and absence of EXP3174, a potent non-peptide Ang II receptor antagonist. Ang II induced an accumulation of 3.4 kb Egr-1 mRNA and the 80 kDa Egr-1 protein, with a maximum at 30 min and 60 min, respectively. EXP3174 blocked the Ang II-induced increase of inositol phosphates, Egr-1 mRNA and the Egr-1 protein, suggesting the involvement of the PI signalling system by the expression of the Egr-1 gene.


Ethanol potentiation of GABAA receptors requires phosphorylation of the alternatively spliced variant of the gamma 2 subunit. The mammalian GABAA receptor is a multisubunit protein containing a variety of binding sites for psychotropic agents. One of the most widely used of these drugs, ethanol, enhances the function of GABAA receptors in certain circumstances but not others. Previous studies have demonstrated that alternative splicing of the gamma 2L GABA subunit results in an ethanol sensitive and an ethanol-insensitive form, when combined with alpha and beta subunits. We have used in vitro mutagenesis and expression in Xenopus oocytes to show that the consensus site for phosphorylation by protein kinase C contained in the gamma 2L insert is critical for modulation by ethanol but not benzodiazepines, and manipulation of the phosphorylating enzymes in oocytes containing alpha 1 beta 1 gamma 2L can prevent ethanol enhancement. It is likely that phosphorylation or dephosphorylation of a specific site on the GABAA receptor protein can act as a control mechanism for neuronal responses to alcohol exposure.


Synthesis of tissue inhibitor of metalloproteinase-1 (TIMP-1) in human hepatoma cells (HepG2). Up-regulation by interleukin-6 and transforming growth factor beta 1. Metalloproteinases and their specific inhibitors, believed to play a role in extracellular matrix metabolism, are regulated by inflammatory cytokines. Here we have addressed the question of whether liver, the major site of synthesis of plasma proteinase inhibitors, is also capable of synthesizing the tissue inhibitor of metalloproteinase-1 (TIMP-1). We show at mRNA and protein levels that TIMP-1 is expressed in differentiated human hepatoma cells (HepG2) and that its synthesis is up-regulated by interleukin-6 (IL-6), transforming growth factor beta 1 and phorbol 12-myristate 13-acetate. The physiological role of this phenomenon is underlined by the fact that lipopolysaccharide administration into rats in vivo, as well as IL-6-stimulation of rat hepatocytes in primary culture, also leads to an increase of TIMP-1 mRNA in liver cells.


Effect of dibutyryl cyclic AMP and isoproterenol on 7 beta-hydroxycholesterol cytotoxicity and esterification in spontaneous transformed cell lines derived from astrocyte primary cultures. Incubation of spontaneous transformed cells derived from astrocyte primary cultures with 30 microM 7 beta-hydroxycholesterol (7 beta-OH-CH) which is lethal to the cells or with 150 microM isoproterenol reduces the intracellular level of cAMP (4- and 2-fold respectively). Treatment of the cultures with 0.5 mM dibutyryl (db)-cAMP and 7 beta-OH-CH increases 3-fold the intracellular level of cAMP and both, db-cAMP and isoproterenol, raise the lethal effect of 7 beta-OH-CH and its esterification on C-3-OH by naturally occurring fatty acids (metabolite). Kinetic studies of net steryl-3-esters hydrolysis revealed that db-cAMP and isoproterenol lower that of cholesteryl-3-esters (2-fold) whereas the opposite is found for the metabolite. These data demonstrate that (i) high cAMP intracellular levels modulate differently the net hydrolysis of cholesteryl-3-esters and metabolite, (ii) isoproterenol acts otherwise than cAMP on 7 beta-OH-CH esterification, (iii) the cytotoxicity of 7 beta-OH-CH is linked to its own esterification. The accumulation of metabolite subsequent to db-cAMP or isoproterenol treatment as a result of acyl-CoA:cholesterol acyl transferase activation is discussed.


Transport protons do not participate in ATP synthesis/hydrolysis at the nucleotide binding site of the H(+)-ATPase from chloroplasts. The H(+)-ATPase from chloroplasts CFoF1, was brought into the active, reduced state by illumination of thylakoids in the presence of thioredoxin and dithiothreitol. Uni-site ATP synthesis was initiated by the addition of 20 nM [alpha-32P]ADP, and enzyme-bound and free nucleotides were separated by a pressure column. The ratio of enzyme-bound ADP to ATP was 0.55 +/- 0.05. In a second experiment, uni-site ATP hydrolysis under energized conditions was initiated by the addition of 36 nM [alpha-32P]ATP; enzyme-bound and free nucleotides were separated by a pressure column. Both procedures were carried out under continuous illumination. The ratio of enzyme-bound ADP to ATP was 0.46 +/- 0.04. In a third experiment, uni-site ATP hydrolysis under de-energized conditions was initiated by the addition of 39 nM [alpha-32P]ATP and NH4Cl/valinomycin in the absence of illumination. Free and enzyme-bound nucleotides were separated also by a pressure column. The ratio of enzyme-bound ADP to ATP was 0.43 +/- 0.02. This ratio was always the same irrespective of whether the reaction runs in the synthesis or the hydrolysis direction. Furthermore, the ratio does not depend on the membrane energization. We conclude, therefore, that the protons are not directly involved in the reaction at the catalytic site.


Salivary gland tumours: is colour Doppler imaging of added value in their preoperative assessment? Using a Toshiba SSA-270A Colour Duplex Scanner, 15 patients with suspected parotid and submandibular gland tumours had preoperative duplex scanning done. Peak systolic doppler shifts (fmax) were recorded and their vascularity was also subjectively scored. There were no arteries identified within the substance of contralateral normal glands. All recordings were analysed retrospectively and an independent histological assessment of tumours was made. Pleomorphic adenomas (n = 9) had a median peak systolic frequency (fmax) of 0.8 kHz, while adenolymphomas (n = 4) had a fmax of 1.65 and the two malignant tumours had fmax of 4.5 (P = 0.007 Kruskal-Wallis test). There appears to be a strong correlation between the fmax and the subjective vascular score (Corr 0.92). Colour duplex scanning is a non-invasive procedure which may be of help in the preoperative assessment of salivary gland tumours.


[The role of progesterone and its receptors in the development of the uterotropic reaction in ovariectomized rats]. In intact rats, the progesterone level in cytosolic fractions of various organs did not correspond to the level of the progesterone receptors. In ovariectomised animals receiving progesterone, the hormone accumulation in the uterus was fulfilled without participation of progesterone receptors. Over half of the progesterone in the rat uterine tissues exists in receptor-unbound "free" form or, possibly, in the form of complexes with small molecular weight. The uterotropic effect of progesterone in ovariectomised animals seems to be determined by the presence of the hormone in the "free" form in the uterus cells or, possibly, in the form of low-molecular complexes.


[The functional characteristics of the hypophyseal-adrenal system in female mice with a hereditary autoimmune pathology]. Functional activity of the pituitary-adrenal axis was studied in genetically autoimmune mice (NZB strain). Young and healthy females (2-3-month old) had high total plasma corticosteroids which sharply decreased by the 6-7th month when the first anti-erythrocyte autoantibodies appeared. At the same time, their ACHT plasma concentration significantly increased compensating, probably, for the low level of corticosteroids. At 6-7 month of age, the amount of corticosteroid-binding globulin was significantly increased therefore decreasing the pool of the active hormones. Low doses of dexamethasone increased the pool of free active glucocorticoids and prevented development of autoimmune processes. The data obtained suggest a possible role of endogenous glucocorticoids in the protection against autoimmune disease.


[Sexual dimorphism in the role of the left and right hemispheres of rats in controlling pain sensitivity]. The effect of unilateral cortical spreading depression on the threshold and latency of pain vocalisation was studied in male and female Wistar rats. The effect involved a reduction of the latency both in male and female rats. However, the left inactivation latency was shorter than the right one in male rats. Thresholds were also reduced but only in male rats and for the left inactivation alone. The data obtained suggest an inter-hemisphere asymmetry in the cortical control of pain sensitivity in male Wistar rats.


[Changes in the central reacting to an interoceptive signal in experimental neurosis]. The EEG arousal response to gastric distension, was studied. Neurotization reduced the response to interoceptive stimulation. This seems to be due to disturbance of the central analysis of interoceptive stimuli including the disturbance of normal activity of the activation-deactivation mechanism.


[The neuroendocrine functions and behavior of dogs after corticoliberin administration into the caudate nucleus]. Within 30-60 min after corticoliberin administration into the dog caudate nucleus, a considerable activation of hypophyseal-adrenocortical system occurred. The motor behavior of the dogs also changed as well as their orienting behaviour, emotional tension, their blood pressure being elevated. Along with its effect on the adrenocortical function, corticoliberin seems to activate behavioural as well as vegetative processes by means of a direct action upon the caudate nucleus' neurons.


[The functional asymmetry of the olfactory control of visceral functions]. In amphibia (frogs) and mammals (rats, guinea pigs, cats), unilateral electrical stimulation of olfactory lobes or section of olfactory tracts resulted in considerable functional changes in respiratory and cardiovascular systems, the olfactory influences being functionally asymmetric. Possible interactions of asymmetric influences at the level of truncal visceral centers, are discussed.


[The effect of the lateral hypothalamic area on the pulmonary surfactant system]. The level of lipids, lipase activity and lungs' blood capacity were studied on 62 rats. Dispersal cobalt was infused into the hypothalamic lateral area of rats with adrenalectomy or injected with droperidol, obsidan, reserpine. Their effect upon the hypothalamus involved a decrease in surfactant phospholipids and lung's blood capacity. Possible adrenergic mechanisms of affecting the lung surfactant system are discussed.


[Changes in the saccharase and lactase activities of the small intestine in suckling rats prematurely weaned from the nursing dam]. In suckling rats prematurely weaned on the 15th, 17th, 19th or 21st day of life, the body weight and the weight of small intestine were studied as well as the activity of enteral sucrase and lactase. A delay in the gain of body weight and small intestine weight was the greater the earlier the sucklings had been weaned. The sucrase activity did not depend on the term of weaning. Whereas weaning on the 15th or 17th days of life considerably delayed the decrease in lactase activity. The latter seems to be due to the changes in the suckling's thyroid state because of thyroid depression under these conditions and absence of thyroid hormones intake from maternal milk.


[The effect of stress on the enzyme systems of the enterocyte microvilli in rat pups and the possible mechanism of the transmission of stress effects from dam to progeny]. In suckling rats whose mothers had had an immobilisation stress or been injected HC and T4 at the last day of their pregnancy, the sucrase induction decreased in the small intestine, if the HC was injected on the 10th day of life. The hormone injection to 1-day suckling rats did not affect the induction. These effects seem to be exerted not by the direct permeation of hormone from mother to litter but to be mediated rather by a special factor "stressin". The latter is transferred from mother to litter and decreases the sensitivity of intestinal enzymes to exogenous HC, thus exerting an antistressor effect.


[Heat transfer by the blood in the venous vessels of the rabbit under different temperature conditions]. The blood temperature in the main venous vessels reflects the formation of the heat balance in the body. In the rabbit posterior vena cava, the temperature increased by 0.19 degrees C-0.26 degrees C after confluence of the kidney and liver veins and decreased by 0.20 degrees C after confluence of the lumbal veins collecting the venous blood from the back muscles and skin vessels. The venous blood temperature in the right auricle was equal to blood temperature in the abdominal artery. When the ambient temperature changed from 22 degrees C-24 degrees C to 35 degrees C-40 degrees C, the temperature in the posterior vena cava and abdominal artery decreased by 0.25 degrees C-0.35 degrees C.


[The temperature gradient values in the skin and their significance for thermoregulation]. The ambient temperature decrease from 32-35 degrees C to 15-17 degrees C was shown to increase the temperature gradient between the skin surface and its deep layers (2-3 mm from the surface) from 0.13 +/- 0.1 degrees C to 1.29 +/- 0.13 degrees C in humans. Considering the localisation of thermoreceptors in different skin layers, such a considerable gradient can be measured by the thermoregulation system and serve for determination of direction and intensity of heat flow across the skin.


[Brain temperature in small birds and mammals]. The brain and body temperature were simultaneously recorded in bullfinches and mice at the ambient temperature 20-22 degrees C. The data obtained showed the existence of constant brain/body temperature difference in birds (the brain temperature was lower by 1.8 degrees C). The brain temperature in mice was higher than the rectal one by 0.4 degrees C. There were constant rapid oscillations of the brain temperature with the period of 1-2 min and the amplitude of 0.05-0.10 degrees C as well as slow oscillations with the period of 11-20 min and the amplitude of 0.5-1.5 degrees C both in bullfinches and mice.


[The characteristics of the energy support for muscular activity in different somatotypes]. 62 healthy untrained males of different somatotypes were given a 30-minute bicycle ergometry [correction of veloergometric] load at the threshold of aerobic exchange. Subjects with a higher percentage of muscular tissue and decreased percentage of fat had their ventilation threshold at a minimal distance from the point of anaerobic transition as compared with the subjects with a more developed fat component. The latters revealed a more active utilisation of lipids whereas the subjects with developed muscles used the carbohydrates rather as the energy substrate.


[The Na+,K(+)-ATPase activity in the brain structures of neuroticized rats differing by the threshold of nervous system excitability]. A 2-week neurotization of two rat lines formed a long-term stress and increased neuronal Na+,K(+)-ATPase activity in hippocampus, locus coeruleus and n. raphe dorsalis, whereas the glial enzyme activity was decreased in hippocampus and N. raphe dorsalis. (Line H). In rats with low threshold of excitability (line L), the decrease of the Na+,K(+)-ATPase activity occurred in n. raphe dorsalis and hippocampus. After a 15-day rest, the activity decreased in all the structures of the line rats, whereas in the line L rats the activity was still increased in hippocampal neurons. Differences between the rat lines and dependence of the enzyme activity on the functional state of the nervous system, are discussed.


[The content of sex steroid receptors and the activity of marker enzymes of estrogenic action in the uterus of androgen-sterile rats]. In rats with preserved ovaries, both in normal and in neonatally androgenized, the activity of enzymes kreatinphosphokinase and peroxidase cannot serve as the marker of the estradiol effect upon the uterus. In ovariectomised animals, the dynamics of activity of these enzymes corresponds to uterotropic responses to estradiol. but not to the dynamics of hormone receptor complexes. The data obtained makes doubtful the participation of the hormone receptor complexes in realisation of the estrogens effect upon the uterus.


[The size of the neuronal receptive fields of the cat prestriate cortex in the left and right hemispheres]. Sizes of neurons' receptive fields (RFs) were studied in area 21 using as a stimulus the light bar 2.5 x 45 degrees C. The RFs were found to be larger than in areas 17-19 and LS cortex. Means sizes of the RFs were found to be larger in the left hemisphere (45 degrees) as compared with the right one (30 degrees). The whole visual field seems to be formed in area 21 in the cats.


[The effect of stimulation of the vestibular area of the cortex on the neuronal activity of the lateral vestibular nucleus during vibration]. In rabbits, the effect of stimulation of cortical vestibular area upon neuronal activity in the lateral vestibular nucleus following vibration, was studied. The long-term vibration weakened the inhibitory and increased the activating effects of corticofugal influence upon neuronal activity in the lateral vestibular nucleus.


[The participation of the sensorimotor cortex in assessing the quality of food reinforcement against a background of thirst]. In trained cats, unilateral extirpation of the sigmoid gyri's cortex disinhibited the unreinforced runs while preserving the positive conditioned reflexes associated with differentiation of different salt food reinforcement. The bilateral extirpation induced a disorder of differentiated inhibition while preserving the conditioned runs reinforced with the highly salted food. The unilateral extirpation did not affect the food-motor conditioning against the background of the salt diet. The bilateral extirpation delayed the differentiation inhibition and facilitated the positive conditioning.


[The functional plasticity of the cells of the paraventricular nucleus in surviving slices of the hypothalamus]. In surviving cuts of the hypothalamus, stimulation of the supraoptic nucleus induced focal potentials in the paraventricular nucleus. A repeated stimulation was followed by a reduction of the descending of the focal potentials. Tetanization of the supraoptic nucleus induced an additional wave at the descending phase of focal potentials preserving for 15 min. The data obtained suggest a functional plasticity of the hypothalamus paraventricular nucleus' cells.


[The stimulating influence of the raphe nuclei on the morphofunctional development of the hippocampus during their cocultivation]. During cocultivation in vitro, raphe nuclei, as distinct from cerebellum, were shown to stimulate the growth and morphofunctional development of hippocampus, including glia proliferation, neurite outgrowth, earlier formation of spontaneous unit activity, greater number of active neurons, and predominance of cells with a periodic type of activity. These findings coincide with the effects of exogenous serotonin and suggest that the raphe-hippocampal serotoninergic input may be important for hippocampal cells maturation in early ontogenesis.


[The superposition of neural vasoconstrictor and local vasodilator effects on skeletal muscle vessels]. In anesthetised cats' m. gastrocnemius, under conditions of stabilised perfusion pressure, preservation of the sympathetic effect against the background of reactive and working hyperemia was shown in both rhythmic muscle contractions and in single tetani. The reactive hyperemia practically did not develop during sympathetic stimulation whereas the working one turned out to be a short-term one and considerably weakened as compared with the control.


[The effect of the activation of the sympathetic-adrenal system on the pulmonary inactivation of catecholamines in rats]. Norepinephrine extraction was found to be decreased in the lungs of immobilised rats. Administration of epinephrine to intact rats up to the same level in the blood plasma as after stress, induced an increase in the norepinephrine extraction by the lungs. The data obtained suggest that norepinephrine inactivation due to immobilisation stress is unrelated to the plasma epinephrine level. The role of the lungs in the maintenance of increased plasma of norepinephrine by means of decreasing its metabolic inactivation, is discussed.


[The pulse trains of the splanchnic and vagus nerves during changes in the temperature of the gastric mucosa]. In anesthetised cats, impulse activity of splanchnic afferents increased after a temperature rise by 2 degrees C and a drop by 2 degrees C, then the firing rate decreased during heating and increased during cooling. The activity of vagal afferents increased during heating and decreased during cooling of the mucosa. The firing rate of splanchnic efferents changed similarly to the afferents. The magnitude of the responses to these stimuli sharply decreased during hypothermia. The data obtained suggest a secondary nature of gastric temperature reception and its relative specifics.


[Osmotic actions on the epithelial Na,K pump of the small intestine]. Intragastric administration of mannitol or NaCl hypotonic solution induced a significant shift of the electrolyte balance in the small intestine epithelium, and activated the enterocytes' Na,K pump in Wistar rats. De-ionised water caused the maximal enhancement of the Na,K-ATPase activity, whereas hypertonic solutions exerted the opposite effect, a substance-inhibitor appearing in the blood serum. Similar results were obtained in vitro. The possibility of the Na,K-pump regulation by enterocytes and the role of small intestine in secretion of an endogenous factor regulating this enzyme's activity, is discussed.


[The superslow bioelectrical activity of the brain in monkeys during the development of the high-pressure nervous syndrome]. In immersions over 20 ata, symptoms occur known as the high pressure neural syndrome (HPNS). Regular changes of the infraslow electrical activity were revealed in development of the HPNS: the waves of the 20-sec period start developing, the average oscillation amplitudes becoming considerably higher. The inter-lead correlation becomes more obvious.


[The regulation of respiration under the conditions of the altered density of a gaseous environment during muscular work]. In 10 young healthy subjects, both at resting and in muscular work, the substitution of air by a helium-oxygen mixture reduced the central inspiratory drive, the work of breathing, and electrical activity of inspiratory muscles, whereas the inhalation of elegas-oxygen mixture led to obvious opposite effects: the effort for even the least maximal inspiratory flow increased in the same lung ventilation as when breathing air and in constant chemoreceptor stimulus; the central inspiratory activity increased nearly three-fold. The mechanism of the load compensation and of the respiratory system's load seems to be mediated mainly through the afferents from the respiratory muscles' proprioreceptors.


[Animal motor behavior under increased pressure]. Changing pressure of the helium-oxygen mixture (6, 36, 70 and 100 Ata) induced a sharp increase of the grooming in guinea pigs. The greatest increase (up to 22.5 movements on the average) occurs during first days of the compression. During decompression, some instability of motor activity is revealed but the grooming still exceeds the control level (2.6). The changes of the grooming reflect physiological processes occurring in the CNS stem and spinal structures. The obvious pattern of the changes of grooming suggests some specific mechanisms of the high pressure effect upon the neural functions and reveals early symptoms of the high pressure neural syndrome.


[Sleep as a restorative process under extreme exposure conditions]. In 40 aquanauts, a prolonged stay under increased pressure (11 to 46 kgs/cm2) of the oxygen-helium-nitrogen mixture did not affect the average duration of sleep. Slow-wave sleep, mostly its 3 rd and 4 th stages, and paradoxical sleep were significantly decreased whereas the light sleep/profound sleep ratio increased. The cyclic structure of sleep became altered. The longer the exposure to high pressure led to an augmentation of the slow-wave sleep and REM-phase, but the normal cycles terminating with a REM-phase could not be formed during the experiment.


[The local blood supply of the brain in guinea pigs during the development of the high-pressure nervous syndrome]. Under continuous compression with normoxic helium-oxygen mixture up to 100 Ata with the velocity 1 Ata/min, guinea pigs developed successively tremor, myoclonias, seizures of clonic and tonic types. Blood supply of cerebral structures (cortex, black substance, caudate nucleus) during motor disorders increased depending on the stage of development of the high pressure neural syndrome. The role of cerebral circulation in the latter's pathogenesis is discussed.


[The oxygenation status of the brain in guinea pigs breathing high-density gaseous mixtures]. Compression of helium-oxygen mixture (HOM) and neon-oxygen one (NOM) led to changes in the guinea pig black substance, caudate nucleus and occipital cortex concerning the pO2. The pO2 level increased along with the increase in the HOM density to 15.56 milligrams and NOM to 76.61 milligrams. The pO2 changes were unequal in the cortex and deep structures of the brain. The increase in oxygenation of the brain under these conditions seems to occur because of compensatory responses of the cardiorespiratory system and redistribution of cerebral blood flow.


[The distribution of oxygen pressure in the pial arterioles of the rat under normobaric hyperoxia]. Breathing with air revealed that oxygen tension in arterioles practically does not depend on the diameter of vessels under study in rats. Whereas in breathing with oxygen, oxygen tension sharply decreased along the course of the microvessel bed. The above decrease seems to be due to exclusion of hemoglobin's dumping function and the effect of such factors as the ratio of arterioles surface's extension to the volume of their blood and the velocity of blood flow in pial arterioles.


[The central nervous system function of guinea pigs after a prolonged stay in an artificial gaseous environment of varying composition]. The pattern of statistical and correlational-spectral parameters of the heart rate characterising the CNS regulatory functions, was estimated in guinea pigs after their prolonged stay in an artificial gas milieu at the pressure 71 kGs/cm2. The obviousness of activation of subcortical centres was significantly lower after stay in the oxygen-helium-nitrogen mixture than in the oxygen-helium one. The increase of the oxygen partial pressure up to 0.3 kGs/cm2 exerted a favourable effect.


[Maximal pulmonary ventilation and the forced expiratory rate under hyperbarism]. In divers, breathing with artificial gas mixtures of 14.3 g/l density, the maximal lung ventilation and the maximal velocity of forced expiration decrease along with an increase in the mixture density. The decrease of these parameters is unrelated to nitrogen anesthesia or exhaustion of respiratory muscles. The findings suggest that the value of both these parameters is only limited by expiratory dynamic compression of respiratory pathways.


[The external respiration parameters in an environment under excessive pressure]. In 21 alert rabbits staying for 1 hr in normoxic nitrogen-oxygen mixture under the pressure 6 kgs/cm2 and 1/2 hr respiration in air at atmospheric pressure, the increase in loading the respiratory system's muscles induced compensatory responses directed to preservation of the necessary level of lung ventilation. Total electrical activity enhances in the inspiratory muscles, and the pattern of separate motor units' discharges changes: firing rate increases, doubling and shift of discharges in the expiratory phase occur as well as alternation of activity in different motor units, recruiting of additional units, which seems to aid to the increase of the contraction force.


[An analysis of respiratory muscle fatigue under a resistive load with the breathing of gas mixtures with different oxygen levels]. In anesthetized cats, the respiration muscles' fatigue under normoxia occurred in heavy mechanical load equal to 80% of the maximal inspiratory effort. Under hypoxia the force of the diaphragm contraction did never decrease whereas when breathing with hypoxic gas mixture weakening of the contractile ability occurred yet under moderate load. In breathing with additional mechanical load under hypoxia, mainly the peripheral part of the respiratory system's motor apparatus suffers, as the weakening of diaphragm's electrical activity and reduction of its contractions' force occurred during unfalling activity of the diaphragmatic nerve. The oxygen content in arterial blood affects the contractile ability of respiratory muscles. However, the increase in the energy supply level does not necessarily exclude the possibility of respiratory muscles' fatigue, but just decelerates its development.


[The electrophysiological and ultrastructural aspects of the effect of increased oxygen pressure on the sensorimotor cortex of rats]. Stay of the rats in barochamber under 0.3 MPa oxygen during 2 hrs induced separate sharp waves with no generalisation, the electron microscopic changes expressing activation of compensatory-adaptive processes. The generalisation of seizure activity over the cortex at 0.7 MPa oxygen affects a part of neuronal pool due, probably, to irreversible changes in inhibitory axosomatic synapses.


Management of retinal detachment associated with CMV retinitis in AIDS patients. In patients with AIDS, the most important ocular opportunistic infection, CMV retinitis, can now be treated effectively with virostatic agents. Associated retinal detachment is encountered frequently, and its management has become increasingly significant to quality of life as improvements in medical care have helped to preserve vision and extend life expectancy. Although retinal detachment in these eyes is typically rhegmatogenous, the pathophysiology is distinctive due to the association with CMV retinitis which, even in remission, is characterised by atrophic changes at all levels of affected retina and alterations of the vitreous. Despite initially successful surgical reattachment, multiple, small, often posterior holes may develop because of progressive CMV infection. For these reasons, vitrectomy and silicone oil injection with scleral buckling may currently provide the best overall means of maintaining retinal reattachment and restoring visual function. Nevertheless, management must be individualised in each case, with the realisation that progressive visual loss frequently ensues from retinitis progression.