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Yeast GTP-binding protein (YPT1 protein) has been reported to function in the early stages of the secretory pathway. Particularly, YPT1 protein is observed to regulate both the endoplasmic reticulum-to-Golgi transport and the autophagy. Therefore, the YPT1 protein overexpressed in yeast vacuoles is expected to enhance antimicrobial and anticancer activity. The enhancement of yeast vacuolar activity under the overexpression of YPT1 was evaluated by the analysis of lysozyme activity, antimicrobial activity against Escherichia coli and Staphylococcus aureus, and MTT assay against HeLa cell lines. Additionally, the rise in concentration of some important proteinases inside the vacuole, such as proteinase A, proteinase B, and vacuolar carboxypeptidase Y (CPY) were also recorded using a 2DE technique. All results imply YPT1 involvement in the recruitment of some specific proteinases into vacuoles, which leads to the enhancement of vacuolar activity. Since these there proteinases belong to the CPY pathway, YPT1 is even believed to up-regulate this trafficking pathway in yeast cells. Future studies, however, should be carried out to discover the mechanisms that allow YPT1 to recruit these proteins into yeast vacuoles. [ABSTRACT FROM AUTHOR]

Key message: Isolation, cloning, and expression characterization of an ARF gene from S. alterniflora, demonstrating its involvement in abiotic stress tolerance. Abstract: Adenosine diphosphate-ribosylation factors (ARFs) are small guanine nucleotide-binding proteins that play an important role in intracellular protein trafficking necessary for undertaking multiple physiological functions in plant growth and developmental processes. However, little is known about the mechanism of ARF functioning at the molecular level, as well as its involvement in abiotic stress tolerance. In this study, we demonstrated the direct involvement of an ARF gene SaARF from a grass halophyte Spartina alterniflora in abiotic stress adaptation for the first time. SaARF, which encodes a protein with predicted molecular mass of 21 kDa, revealed highest identity with ARF of Oryza sativa. The SaARF gene is transcriptionally regulated by salt, drought, cold, and ABA in the leaves and roots of S. alterniflora. Arabidopsis plants overexpressing SaARF showed improved seed germination and survival of seedlings under salinity stress. Similarly, SaARF transgenic Arabidopsis plants were more tolerant to drought stress, compared to wild-type plants, by maintaining chlorophyll synthesis, increasing osmolyte synthesis, and stabilizing membrane integrity. Oxidative damage due to moisture stress in transgenic Arabidopsis was also reduced possibly by activating antioxidant genes, AtSOD1 and AtCAT. Our results suggest that enhanced drought and salinity tolerance conferred by the SaARF gene may be due to its role in mediating multiple abiotic stress tolerance mechanisms. [ABSTRACT FROM AUTHOR]

cDNA clone designated arf1 was isolated from a physic nut ( Jatropha curcas L.) endosperm cDNA library which encodes a small GTP-binding protein and has significant homology to ADP-ribosylation factors (ARF) in plants, animals and microbes. The cDNA contains an open reading frame that encodes a polypeptide of 181 amino acids with a calculated molecular mass of 20.7 kDa. The deduced amino acid sequence showed high homology to known ARFs from other organisms. The products of the arf1 obtained by overexpression in E. coli revealed the specific binding activity toward GTP. The expression of arf1 was observed in flowers, roots, stems and leaves as analyzed by RT-PCR, and its transcriptional level was highest in flowers. In particular, the accumulation of arf1 transcripts was different under various environmental stresses in seedlings. The results suggest that arf1 plays distinct physiological roles in Jatropha curcas cells. [ABSTRACT FROM AUTHOR]

Sensitivity of the adenylyl cyclase signaling system (ACSS) to polypeptide hormones and biogenic amines is studied in testes and ovaries of rats after the 2- and 4-day fasting as compared with control animals. In tissues of the fasted rats there is shown a decrease in the basal activity of adenylyl cyclase (AC) and of the basal level of the GTP binding of heterotrimeric G proteins. An increase in duration of fasting from 2 to 4 days led to intensification of these changes. In the fasted rats, the AC-stimulating effects of chorionic gonadotropin, PACAP-38, and isoproterenol, realized via G protein of stimulatory type are enhanced, whereas the inhibitory effects of somatostatin on the AC activity realized via G protein of the inhibitory type are reduced. In testes of the fasted rats the stimulating effect of serotonin on AC via both types of G proteins increased, whereas the inhibitory effects of the hormone decreased. Thus, under conditions of fasting, in rat testes and ovaries the ACSS sensitivity to regulatory effects of hormones is changing: their stimulatory effects are increased, while its inhibitory effects, on the contrary, are decreased. We suggest that these changes are one of the key mechanisms of adaptation of organism to deficiency of nutritional resources to be aimed at intensifying the tissues catabolic processes, preferably lypolysis. [ABSTRACT FROM AUTHOR]

Reactive oxygen intermediates (ROIs) such as hydrogen peroxide (H2O2) and the superoxide anion radical (O2·-) accumulate in many plants during attack by microbial pathogens. Despite a huge number of studies, the complete picture of the role of ROIs in the host–pathogen interaction is not yet fully understood. This situation is reflected by the controversially discussed question as to whether ROIs are key factors in the establishment and maintenance of either host cell inaccessibility or accessibility for fungal pathogens. On the one hand, ROIs have been implicated in signal transduction as well as in the execution of defence reactions such as cell wall strengthening and a rapid host cell death (hypersensitive reaction). On the other hand, ROIs accumulate in compatible interactions, and there are reports suggesting a function of ROIs in restricting the spread of leaf lesions and thus in suppressing cell death. Moreover, in situ analyses have demonstrated that different ROIs may trigger opposite effects in plants depending on their spatiotemporal distribution and subcellular concentrations. This demonstrates the need to determine the particular role of individual ROIs in distinct stages of pathogen development. The well-studied interaction of cereals with fungi from the genus Blumeria is an excellent model system in which signal transduction and defence reactions can be further elucidated in planta. This review article gives a synopsis of the role of ROI accumulation, with particular emphasis on the pathosystem Hordeum vulgare L.–Blumeria graminis. [ABSTRACT FROM AUTHOR]

The putative functions of NDP (nucleoside diaphosphate) kinases from various organisms focusing to fungi and plants are described. The biochemical reactions catalyzed by NDP kinase are as follows. (i) Phosphotransferring activity from mainly ATP to cognate NDPs generating nucleoside triphosphates (NTPs). (ii) Autophosphorylation activity from ATP and GTP. (iii) Protein kinase (phosphotransferring) activity phosphorylating such as myelin basic protein. NDP kinase could function to provide NTPs as a housekeeping enzyme. However, recent works proved possible functions of the NDP kinases in the processes of signal transduction in various organisms, as described below. By use of the extracts of the mycelia of a filamentous fungus Neurospora crassa blue-light irradiation could increase the phosphorylation of a 15-kDa protein, which was purified and identified to be NDP kinase (NDK-1). By use of the etiolated seedlings of Pisum sativum cv Alaska and Oryza sativa red-light irradiation of intact plants increased the phosphorylation of NDP kinase. However, successive irradiation by red–far-red reversed the reaction, indicating that phytochrome-mediated light signals are transduced to the phosphorylation of NDP kinase. NDP kinase localizing in mitochondria is encoded by nuclear genome and different from those localized in cytoplasm. NDP kinase in mitochondria formed a complex with succinyl CoA synthetase. In Spinicia oleraceae two different NDP kinases were detected in the chloroplast, and in Pisum sativum two forms of NDP kinase originated from single species of mRNA could be detected in the choloroplast. However, the function of NDP kinases in the choloroplast is not yet known. In Neurospora crassa a Pro72His mutation in NDP kinase ( ndk-1 Pro72His) deficient in the autophosphorylation and protein kinase activity resulted in lacking the light-induced polarity of perithecia. In wild-type directional light irradiation parallel to the solid medium resulted in the formation of the perithecial beak at the top of perithecia, which was designated as “light-induced polarity of perithecia.” In wild-type in darkness the beak was formed at random places on perithecia, and in ndk Pro72His mutant the perithecial beak was formed at random places even under directional light illumination. The introduction of genomic DNA and cDNA for ndk-1 demonstrated that the wild-type DNAs suppressed the mutant phenotype. With all these results except for the demonstration in Neurospora, most of the phenomena are elusive and should be solved in the molecular levels concerning with NDP kinases. [ABSTRACT FROM AUTHOR]

We studied cAMP-mediated exocytosis in rat pancreatic acinar cells. We monitored changes in the membrane capacitance (ΔC), which reflects the granule fusion/retrieval process, with whole-cell patch-clamp capacitance measurement. The rise in cellular cAMP, caused indirectly by receptor activation by vasoactive intestinal polypeptide or directly by dibutyryl cyclic AMP, was able to induce an increase in ΔC independently of cellular Ca2+. Using the latter stimulation, we estimated the magnitude of the response to internal GTPγS [guanosine 5′-(γ-thio)trisphosphate] and/or GDPβS [guanosine 5′-(β-thio)diphosphate]. The internal GTPγS and GDPβS amplified and depressed the response, respectively. Thus, the cellular cAMP alone can trigger granule insertion independently of cellular Ca2+ and it can be controlled by cellular GTP-binding proteins, presumably those belonging to the Rab family. [ABSTRACT FROM AUTHOR]

The Trichoderma reesei gene, rho3, encoding the functional homologue of the Saccharomyces cerevisiae small GTP-binding protein Rho3p was cloned as a suppressor of the secretion-deficient mutation sec15-1 in yeast. The encoded protein showed 61% amino acid identity to the Rho3 protein. Rescue of the growth defect of a RHO3 disruption strain by an expression vector carrying rho3 cDNA confirmed the functional homology with the S. cerevisiae RHO3 gene. In addition, overproduction of T. reesei RHOIII in this yeast strain appeared to improve the actin organization and chitin localization of the cells. Three putative mutant alleles of the T. reesei rho3 gene (rho3Gly20Val, rho3Thr25Asn, rho3Asp126Ala) were introduced into the wild-type yeast, in yeast with sec15 mutation and in yeast with Rho3p depletion. Cells expressing rho3Gly20Val displayed wild-type growth and those expressing rho3Thr25Asn and rho3Asp126Ala had a loss-of-function phenotype. [ABSTRACT FROM AUTHOR]

To investigate the mechanisms of receptor-mediated regulation of heart muscle contraction, we developed a tension-recording system using β-escin-skinned single cardiac cells of rats and studied the effects of agonists on myofibrillar Ca2+ sensitivity and Ca2+ release from the sarcoplasmic reticulum (SR). In pCa/tension relations, 1 µM isoproterenol plus 100 µM guanosine 5′-triphosphate (GTP) decreased the myofibrillar Ca2+ sensitivity (pCa50, the [Ca2+] required for half-maximal tension, as an indicator of the sensitivity; from 6.07 to 5.92); this effect was blocked by 1 µM metoprolol or 1 mM guanosine 5′-O-(2-thiodiphosphate) (GDPβS). Phenylephrine (10 µM) plus 100 µM GTP increased the Ca2+ sensitivity (pCa50; from 6.12 to 6.28), and this effect was blocked by 1 µM phentolamine or 1 mM GDPβS. After Ca2+ loading into the SR, 10 µM phenylephrine plus 100 µM GTP in a low-ethylene- glycol-bis(β-aminoethylether)- N, N, N′, N′-tetraacetic acid (EGTA, 0.1 mM) relaxing solution induced oscillatory contractions that were attenuated by either 1 µM phentolamine or pre-treatment with 10 µM inositol 1,4,5-trisphosphate. Our results demonstrate that β1-adrenergic stimulation decreases myofibrillar Ca2+ sensitivity and that α1-adrenergic stimulation both increases the Ca2+ sensitivity and activates Ca2+ release from the agonist-sensitive SR through GTP-binding protein-related mechanisms. [ABSTRACT FROM AUTHOR]

1. Antidepressants have been used clinically for many years; however, the neurochemical mechanism for their therapeutic effect has not been clarified yet. Recent reports indicate that chronic antidepressant treatment directly affects the postsynaptic membrane to increase the coupling between the stimulatory GTP-binding (G) protein, Gs, and adenylyl cyclase. Tubulin, a cytoskeletal element, is involved in the stimulatory and inhibitory regulation of adenylyl cyclase in rat cerebral cortex via direct transfer of GTP to G proteins. In this study, we investigated whether the functional change of the adenylyl cyclase system caused by chronic antidepressant treatment involves an alteration of tubulin function in the regulation of adenylyl cyclase activity. 2. Male Sprague–Dawley rats were treated once daily with amitriptyline or saline by intraperitoneal injection (10 mg/kg) for 21 days, and their cerebral cortex membranes and GppNHp-liganded tubulin (tubulin-GppNHp) were prepared for what. 3. GppNHp-stimulated adenylyl cyclase activity in cortex membranes from amitriptyline-treated rats was significantly higher than that in control membranes. Furthermore, tubulin–GppNHp prepared from amitriptyline-treated rats was more potent than that from control rats in the stimulation of adenylyl cyclase activity in the cortex membranes of the controls. However, there was no significant difference in manganese-stimulated adenylyl cyclase activity between control and amitriptyline-treated rats. 4. The present results suggest that chronic antidepressant treatment enhances not only the coupling between Gs and the catalytic subunit of adenylyl cyclase but also tubulin interaction with Gs in the cerebral cortex of the rat. [ABSTRACT FROM AUTHOR]

The interaction of various hormones and regucalcin on (Ca−Mg)-ATPase activity in rat liver plasma membranes was investigated. The presence of epinephrine (10-10 M), and insulin (10-10 M) in the reaction mixture produced a significant increase in (Ca−Mg)-ATPase activity, while the enzyme activity was decreased significantly by calcitonin, (3×10-3×10 M). These hormonal effects, except for calcitonin, were clearly inhibited by the presence of vanadate (10 M) which can inhibit the Ca-dependent phosphorylation of enzyme. Meanwhile, regucalcin (0.25 and 0.50 μM), isolated from rat liver cytosol, elevated significantly (Ca−Mg)-ATPase activity in the plasma membranes, although this elevation was not inhibited by vanadate (10 M). the epinephrine (10 M) or phenylephrine (10 M)-induced increase in (Ca−Mg)-ATPase activity was disappeared in the presence of regucalcin; in this case the effect of regucalcin was also weakened. However, the inhibitory effect of calcitonin (3×10 M) was not weakened by the presence of regucalcin (0.5 μM). Moreover, GTP (10 and 10 M)-induced increase in (Ca−Mg)-ATPase activity was not seen in the presence of regucalcin (0.25 μM). The present finding suggests that the activating mechanism of regucalcin on (Ca−Mg)-ATPase is not involved on GTP-binding protein which modulates the receptor-mediated hormonal effect in rat liver plasma membranes. [ABSTRACT FROM AUTHOR]

Three G proteins from human brain membranes were purified to near homogeneity by conventional techniques including preparative electrophoresis. These G proteins were characterized by their ability to bind GTP, GDP and GTP analogs. Two of these proteins have molecular weights of 50,000 (G) and 36,000 (G), as determined on SDS-gels. G was ADP-ribosylated by pertussis toxin. Thus, G could represent a Gα subunit, whereas G could be Gα or Gα. G was phosphorylated by cAMP dependent protein kinase and protein kinase C. G was phosphorylated by a protein kinase independent of calcium and phospholipid, a proteolytic product of protein kinase C, analogous to protein kinase M. Phosphorylation of G by this protein kinase induced a dramatic decrease in its GTPase activity. The third G protein, of molecular weight 22,000 probably belongs to the group of monomeric G proteins possessing functional similarities with ras gene products. The regulation of G proteins involving calcium-dependent and independent pathways is delineated. [ABSTRACT FROM AUTHOR]

A cDNA clone was selected from a cDNA library constructed using mRNA from ABA-treated Fagus sylvatica L. dormant seeds as a template. The clone is highly expressed in the presence of ABA and tends to disappear in stratified seeds. A search of sequence databases showed that the clone encodes a small GTP-binding protein. By means of in situ hybridization, the mRNA has been located in the apical meristem of the embryonic axis and in the central vascular cylinder. Its possible involvement in growth regulation in the embryonic axis of F. sylvatica is discussed. [ABSTRACT FROM AUTHOR]

A cDNA encoding for a 68 kDa GTP-binding protein was isolated from Arabidopsis thaliana (aG68). This clone is a member of a gene family that codes for a class of large GTP-binding proteins. This includes the mammalian dynamin, yeast Vps1p and the vertebrate Mx proteins. The predicted amino acid sequence was found to have high sequence conservation in the N-terminal GTP-binding domain sharing 54% identity to yeast Vps1p, 56% amino acid identity to rat dynamin and 38% identity to the murine Mx1 protein. The northern analysis shows expression in root, leaf, stem and flower tissues, but in mature leaves at lower levels. Southern analysis indicates that it may be a member of a small gene family or the gene may contain an intron. [ABSTRACT FROM AUTHOR]

To investigate the functions of GTP-binding protein(s) in the melanosome-aggregating response in fish melanophores, the effects of activators of G-proteins, namely, mastoparan and compound 48/80, were examined in cultured melanophores of the balck-moor goldfish, Carassius auratus. Both mastoparan and compound 48/80 induced an approximately 40% increase in the GTP-hydrolyzing activity in the melanophore membranes compared to the basal level. In intact melanophores, these compounds inhibited the effect of 3-isobutyl-1-methylxanthine, which induced the accumulation of intracellular cAMP. Pretreatment of melanophores with pertussis toxin at 1 μg·ml for 15 h attenuated the inhibitory effect of mastoparan on the accumulation of cAMP. However, pretreatment with the toxin only slightly attenuated the inhibitory effect of compound 48/80 on the accumulation of cAMP. In addition, compound 48/80 at 1 mg·ml induced full aggregation of the melanosomes in melanophores, though mastoparan at 5 μmol·l induced only 10-20% aggregation of melanophores. These results suggest that mastoparan and compound 48/80 can each activate the inhibitory G-protein in goldfish melanophores, which results in inhibition of adenylate cyclase activity. This signal-transduction pathway is involved in the aggregation of melanosomes in these cells. [ABSTRACT FROM AUTHOR]

Concentrations of the α-subunits of GTP-binding protein, Go (Goα) and of Gi 2 (Gi 2α) in 6 areas (the hippocampus, parahippocampus, putamen, caudate head, orbital frontal cortex, and lateral temporal cortex) of control and schizophrenic postmortem brains were investigated using the highly sensitive enzyme immunoassay method. There was a significant decrease in Goα in the hippocampus and caudate head of the right hemisphere in schizophrenic patients compared to controls; the ANOVA (a general linear model; SAS Type II) demonstrated a significant diagnosis × side interaction only in the hippocampus. In other areas of the brain, analysis by grouping under diagnosis, side, age, gender, and postmortem delay showed no significant deviations in Goα between controls and schizophrenics. The concentrations of Gi 2α did not differ significantly in any area. These findings contrasted with the results yielded by ADP-ribosylation, which showed decreased pertussis toxin ADP-ribosylated amounts in the hippocampus and putamen of the contralateral (left) hemisphere. Some abnormal receptor - Go or Gi 1 signalling in hippocampus, putamen or caudate head may be involved in the pathogenesis of schizophrenia. [ABSTRACT FROM AUTHOR]

This short review summarizes recent results and hypotheses about the activation mechanism of photoreceptor enzymes via photoexcitation of rhodopsin. [ABSTRACT FROM AUTHOR]

We studied the effects of the binary Clostridium botulinum C2 toxin on stimulated [3H]serotonin release and protein tyrosine phosphorylation in RBL 2H3 hm1 cells. Actin was specifically ADP-ribosylated by C2 toxin in intact cells resulting in a 2–3 fold increase in antigen- or calcium ionophore (A23187)-induced degranulation. The effects of C2 toxin were time- and concentration-dependent. Toxin treatment, which dramatically changes the morphology of RBL cells, was not sufficient to induce mediator release in the absence of activators of secretion. Antigen- and A23187-stimulated tyrosine phosphorylation of 60–80kDa and 110–120kDa proteins was reduced or blocked after C2 toxin incubation. Treatment of RBL cells with the tyrosine phosphatase inhibitor pervanadate reversed the inhibitory effect of C2 toxin on stimulated protein tyrosine phosphorylation indicating activation of phosphatases by C2 toxin. The data indicate that disassembly of the actin cytoskeleton by C2 toxin facilitates FcɛRI-mediated signal-secretion coupling and suggest a role of the actin cytoskeleton in phosphatase regulation in RBL cells. [ABSTRACT FROM AUTHOR]

The effect of intracerebroventricular treatment of mice with pertussis toxin (PTX) on pain perception and H-nitrendipine binding was examined to study a possible change in the GTP-binding proteins in morphine tolerant rodents. It was observed that both PTX treatment and chronic administration of morphine cause hyperalgesia in the acetic acid-induced writhing test. Analgesic effects brought by the acute administration of morphine or nifedipine, a calcium antagonist, were not affected by PTX treatment. In synaptic membrane fractions prepared from mice treated with PTX or morphine chronically, specific binding of H-nitrendipine was enhanced approximately 41.8% and 35.7%, respectively, without alteration in its affinity. Chronic administration of morphine followed by PTX treatment did not display further increases in H-nitrendipine binding. These results suggest that the PTX-sensitive GTP-binding proteins may not be involved in the manifestation of the analgesic effect of morphine in mice. [ABSTRACT FROM AUTHOR]

Endothelium-dependent relaxation of the guinea pig pulmonary artery induced by histamine was inhibited by preincubation of the tissue with 10 μM N-ethylmaleimide (NEM) for 10 min, whereas the endothelium-dependent relaxation induced by the calcium ionophore A 23187 was not affected by NEM. Pretreatment of the preparations with 0.2-1 μg/ml pertussis toxin for 120 min inhibited concentration-dependently the histamine-induced relaxation. In contrast, endothelium-dependent relaxation in response to the calcium ionophore A 23187 was not affected by pertussis toxin. Since NEM and pertussis toxin are thought to interfere with membrane located GTP binding proteins, it is suggested that such a coupling protein is involved in the signal transduction of the histamine receptor leading to endothelium-dependent relaxation. [ABSTRACT FROM AUTHOR]

T cell activation and proliferation via CD-TCR complex were investigated by lymphocyte DNA synthesis in vitro. Several interfering factors were also discussed. The result indicated that lymphocyte activation and proliferation are calciumdependent. A rise of cytoplasmic free Ca quickly following activation with CD McAb is mainly due to intracellular mobilization of Ca, while lymphocyte proliferation needs both intracellular mobilization of Ca as well as influx of extracellular Ca. It was confirmed that CTX sensitive G protein plays a role in regulating T cell proliferation by pretreatment with CTX suppressing lymphocyteH-TdR incorporation obviously. PLC and PKC inhibitor neomycin and P. S. S could also decrease T cell proliferation. [ABSTRACT FROM AUTHOR]

We examined the effects of riluzole, a neuroprotective drug, on voltage–dependent Na channels, nicotinic receptors, and voltage-dependent Ca channels, as well as catecholamine secretion, in comparison with those of verapamil and nicardipine, in primary cultures of bovine adrenal chromaffin cells. Riluzole inhibited veratridine-induced 22Na influx via voltage-dependent Na channels even in the presence of ouabain, an inhibitor of Na,K-ATPase. Blockade of Na channels by riluzole was concentration-dependent with an IC50 of 5.3 µM. It was associated with a similar concentration-related reduction of veratridine-induced 45Ca influx via voltage-dependent Ca channels, and of catecholamine secretion. Riluzole had no effect on 45Ca influx caused by high K, which directly activates voltage-dependent Ca channels, and on nicotine-induced 22Na influx, which passes through the nicotinic receptors. Verapamil and nicardipine attenuated 22Na influx caused by veratridine or nicotine at the same concentrations as they suppressed high K-induced 45Ca influx. The inhibitory effect of riluzole on veratridine-induced 22Na influx disappeared at high concentrations of veratridine. A potentiation of veratridine (site 2 toxin)-induced 22Na influx caused by α-scorpion venom (site 3 toxin), β-scorpion venom (site 4 toxin), or brevetoxin PbTx-3 (site 5 toxin), occurred in the presence of riluzole in the same manner as in control cells. These results suggest that riluzole binds to the veratridine site in voltage–dependent Na channels. It does not impair the cooperative interaction between the functional peptide segments of Na channels, but selectively inhibits gating of Na channels, thereby reducing Ca influx via Ca channels and catecholamine secretion. In contrast, verapamil and nicardipine suppress Na influx both Na channels and nicotinic receptors. [ABSTRACT FROM AUTHOR]

In the present study, we have examined the direct actions of angiotensin II (AII) in rabbit renal brush border membrane (BBM) where binding sites for AII exist. Addition of AII (10-10 m) was found to stimulateNa uptake by the isolated BBM vesicles directly. AII did not affect the Na-dependent BBM glucose uptake, and the effect of AII on BBMNa uptake was inhibited by amiloride, suggesting the involvement of Na/H exchange mechanism. BBM proton permeability as assessed by acridine orange quenching was not affected by AII, indicating the direct effect of AII on Na/H antiport system. In search of the signal transduction mechanism, it was found that AII activated BBM phospholipase A (PLA) and that BBM contains a 42-kDa guanine nucleotide-binding regulatory protein (G-protein) that underwent pertussis toxin (PTX)-catalyzed ADP-ribosylation. Addition of GTP potentiated, while GDP-ßS or PTX abolished, the effects of AII on BBM PLA andNa uptake, suggesting the involvement of G-protein in AII's actions. On the other hand, inhibition of PLA by mepacrine prevented AII's effect on BBMNa uptake, and activation of PLA by mellitin or addition of arachidonic acid similarly enhanced BBMNa uptake, suggesting the role of PLA activation in mediating AII's effect on BBMNa uptake. In summary, results of the present study show a direct stimulatory effect of AII on BBM Na/H antiport system, and suggest the presence of a local signal transduction system involving G-protein mediated PLA activation. [ABSTRACT FROM AUTHOR]

Modulation of the voltage-gated K conductance in T-lymphocytes by substance P was examined. Whole-cell recordings from Jurkat E6-1 human T-lymphocytes revealed two components of substance P action on the outward K current: (i) dose- and time-dependent reduction of current peak amplitude; and (ii) acceleration of the current inactivation rate. This action was blocked by substituting Cs for K in the recording pipette and by the substance P antagonist, [ d-Arg, d-Phe, d-Trp, Leu]-substance P. As indicated by conductance-voltage relationship, the reduction in current peak amplitude as a result of substance P application was not due to a shift of the voltage dependence of the channel. Raising intracellular free calcium concentration from 2 to 200 nm reversed the reduction, induced by substance P, in current peak amplitude and disclosed an apparent desensitization towards the neuropeptide action. The treatment, however, did not reverse substance P-induced acceleration of the rate of current decay. Intracellular administration of hydrolysis-resistant guanosine triphosphate (to persistently activate GTP-binding protein) and guanosine diphosphate (to competitively inhibit GTP-binding proteins) analogues mimicked and inhibited substance P-induced reduction of K conductance, respectively. The data demonstrate a modulation of T-lymphocyte K channels by substance P and substantiate a possible role for GTP-binding proteins in this modulation. [ABSTRACT FROM AUTHOR]

Effects of tetraalkylammonium ions, having tetraalkyl chains of increasing length from ethyl to octyl, on inositol-trisphosphate (Ins P)-induced Ca release and contractile mechanics were examined in guinea-pig skinned ileal smooth muscle longitudinal strips. Although tetrahexylammonium ions (THexA) appeared to be the most potent inhibitor of Ca release among the tetraalkylammonium ions examined, an additional and more prominent effect was found, i.e., the contraction induced by Ca release showed a large sustained component in the presence of THexA. Potentiation of the contraction by THexA (above 30 μM) was also observed in skinned fibers in which the sarcoplasmic reticulum function was destroyed by treatment with A23187. The potentiating effect of THexA was the most potent by far among the tetraalkylammonium ions examined and was elicited by Ca-dependent and GTP-binding-protein-independent mechanisms. The potentiation was not due to activation of myosin light-chain kinase. The selective inhibitors of myosin light-chain kinase, protein kinase C and calmodulin reduced THexA-induced potentiation of contraction only at concentrations above 30 μM, at which non-specific effects are likely. Furthermore, relaxation induced by changing pCa from 4.5 to 8.5 was not affected by 1 mM THexA, suggesting that the potentiating effect is not mainly due to inhibition of myosin light-chain phosphatase. In conclusion, THexA sensitizes guinea-pig skinned ileal smooth muscle to Ca in a structure-selective manner. This sensitization appears not to be mediated mainly by a GTP-binding protein, by activation of myosin light-chain kinase or protein kinase C, by enhanced Ca binding to calmodulin, or by inhibition of myosin light-chain phosphatase. [ABSTRACT FROM AUTHOR]

Effects of protein kinase C (PKC) on a non-selective cation channel current ( I) were investigated using smooth-muscle cells of the rabbit portal vein. Neither bath application of the PKC activator, phorbol 12,13-dibutyrate (PDBu; 1 μM), nor the internal application of guanosine 5′-[ γ-thio]-triphosphate (GTP[ γS]; 3 μM) elicited any current at the holding potential of −60 mV. However, when GTP[ γS] (3 μM) was present in the pipette, PDBu elicited a sustained inward current, in a concentration-dependent manner, at the holding potential of −60 mV. On the other hand, an inactive phorbol ester, 4α-phorbol 12,13-didecanoate (300 nM and 1 μM) had no effect on the membrane current even when GTP[ γS] (3 μM) was in the pipette. The current amplitude induced by PDBu in the presence of GTP[ γS] in the pipette was markedly reduced following pretreatment with 10 μM staurosporine, a PKC inhibitor. Neither a reduction in the Cl concentration in the pipette nor addition of niflumic acid to the bath inhibited the inward current, and the reversal potential estimated from the current/voltage relationship was about −5 mV (physiological salt solution containing 5 mM Ba/high CSCl), which revealed that the main component of the current is I. An internal application of pertussis toxin markedly reduced the amplitude of I induced by PDBu. These results indicate that PKC activates a sustained component of I in cooperation with an activated pertussis-toxin-sensitive G protein in the rabbit portal vein. [ABSTRACT FROM AUTHOR]

In mouse peritoneal macrophages, α-adrenoceptor stimulation evokes a Ca-dependent K current [ I(Adr)] [Hara et al. (1991) Pflügers Arch 419:371-379]. The roles of D- myo-inositol 1,4,5-trisphosphate (Ins P) and a GTP-binding protein (G protein) in I(Adr) were investigated with tight-seal whole-cell recordings and fura-2 fluorescence measurements. Intracellular injection of lns P (5-50 μM) evoked transient outward currents [ I(Ins P)] with or without damped oscillations in membrane currents at -40 mV. Dialysis with 0.2 mM guanosine 5′-[3-thio]triphosphate (GTP[ γS], a poorly hydrolysable GTP analogue) at -40 mV activated oscillatory outward currents or a slowly developing steady current on which such oscillations were superimposed after a delay of 10-90 s. I(Ins P) and the GTP[ γS]-induced current { I(GTP[ γS])} were accompanied by an increase in conductance. Reversal potentials of both responses closely depended on the extracellular K concentration. Fura-2 measurements revealed that I(Ins P) and I(GTP[ γS]) result from a rise in intracellular free Ca concentration ([Ca]). Removal of extracellular Ca did not abolish I(Ins P) and I(GTP[ γS]). Both were blocked by bath-applied charybdotoxin. Intracellular D- myo-inositol 1,3,4,5-tetrakisphosphate (Ins P, 50 μM) did not evoke any responses, whereas D- myo-inositol 2,4,5-trisphosphate [Ins P(2,4,5), 20 μM] elicited an outward current at -40 mV. I(Ins P) was completely blocked by prior dialysis with the Ins P receptor antagonist heparin (5 mg/ml). Inclusion of guanosine 5′-[2-thio] diphosphate (GDP[ βS], 2 mM) or heparin (5 mg/ml) together with GTP[ γS] in the patch pipette solution completely blocked I(GTP[ γS]). These results indicate that intracellular injection of Ins P or GTP[ γS] mimic I(Adr). Furthermore, intracellular dialysis with heparin (3 mg/ ml) or GDP[ βS] (2 mM) greatly accelerated a run-down of I(Adr). On the other hand, I(Adr) was markedly prolonged in a cell dialysed with GTP[ γS] (0.2 mM). Therefore, it is concluded that I(Adr) results from stimulation of α-adrenoceptor and Ins P formation via a G protein. [ABSTRACT FROM AUTHOR]

In the heart, the guanosine 5′-triphosphate (GTP)-binding protein G is activated by hormone binding to β-adrenergic receptors and stimulates the intracellular cyclic adenosine 3′,5′-monophosphate (cAMP) pathway that leads to phosphorylation of L-type Ca channels by the cAMP-dependent protein kinase A [28]. Additionally, G can modulate cardiac Ca channels directly in cell-free systems [57]. In order to examine the question of whether these pathways could be separated functionally and whether they act independently or synergistically on L-type Ca channels in intact cells, the whole-cell Ca current ( I) and the respective current density were measured in guinea-pig ventricular myocytes at 0 mV. The following results were obtained. First, typically, the I density increased from 12 to 40 μA/cm following application of 1 μM isoproterenol (ISP) to myocytes bathed in solutions containing 1.8 mM CaCl. However, 1 μM ISP enhanced I only from 9 to 17 μA/cm after inhibition of the protein kinase A by dialysis of 0.5 mM Rp-cAMPS (the Rp-isomer of adenosine 3′,5′-monophosphorothioate) in the presence of 0.5 mM GTP. Withdrawal of GTP from the dialysate attenuated the effects of ISP on I. Thus, Rpc-AMPS unmasks a GTP-dependent component of the β-adrenergic stimulation of I, which probably reflects the direct stimulation of Ca channels by G under block of cAMP-dependent phosphorylation. Second, in cells under dialysis with 100 or 200 μM cAMP, bath application of 20-40 μM 3-isobutyl-1-methylxanthine (IBMX) enhanced the I density to about 41 μA/cm indicating saturation of the cAMP pathway. Under this condition, 1 μM ISP was without significant effect on I. This result may suggests that direct G stimulation is rather ineffective on Ca channels after maximal cAMP-dependent phosphorylation. Alternatively, maximal stimulation of the cAMP pathway may also interfere with the activation of the G pathway in intact myocytes. Third, simultaneous application of 1 μM ISP and 40 μM IBMX enhanced I up to densities of around 75 μA/cm during cell dialysis with 100 μM cAMP, an effect much stronger than that exerted by IBMX alone under similar conditions. Since it seems likely that G is activated more quickly, than the cAMP pathway during application of the ISP/IBMX mixture, the latter result suggests that a direct effect of G may act to prime L-type Ca channels for cAMP-dependent phosphorylation during β-adrenergic stimulation of cardiac myocytes. [ABSTRACT FROM AUTHOR]

A high density of nerve fibers containing calcitonin-gene-related peptide (CGRP) is present in the atria. Recently CGRP was reported to open ATP-sensitive K channels in arterial smooth muscle cells. This study examines whether CGRP activates a similar K channel in cardiac cells. In voltage-clamped whole cells loaded with GTP and ATP, CGRP reversibly evoked an inwardly rectifying K current. To identify the K channel that gives rise to this current, three types of K channel (resting, ATP-sensitive and acetylcholine-activated) were examined. CGRP failed to activate or inhibit the ATP-sensitive or the resting K channel. However, CGRP (0.1-1 μM) caused activation of single channels with kinetics similar to that of the muscarinic K channel (35-40 pS conductance and approx. 1 ms mean open time in symmetrical 140 mM K). In excised, inside-out (CGRP in pipette) or in outside-out (GTP in pipette) patches, the K current was activated by perfusion with GTP or CGRP, respectively, suggesting that CGRP activated the muscarinic K channel via GTP-binding protein. Treatment with pertussis toxin inhibited the activation of the K channel, suggesting that CGRP receptor may be coupled to a G or a G type of GTP-binding protein. Together with previous findings, these results suggest that CGRP modulates several types of ion channels to produce its cellular effects. [ABSTRACT FROM AUTHOR]

Inhibitory modulation of the high-voltage-activated (HVA) Ca-channel current by 2-chloroadenosine (2CA) was studied in chick sensory neurons using the whole-cell clamp method. 2CA reduced the ωCTX-sensitive HVA-current (Aosaki and Kasai 1989) in a dose-dependent manner with a K of 0.8 μM. The inhibition by 2CA was also voltage-dependent, being maximal at hyperpolarized potentials, and completely removed at potentials more positive than 30 mV. This voltage-dependence of 2CA action was also evident as a progressive increase in Ca-channel current magnitude during a depolarization which could be described by a single exponential function and which became faster at larger depolarizations. The concentration of 2CA affected the steady-state reduction in Ca-channel current, but did not alter the time-course of current increase during depolarization. The voltage-dependent effect of 2CA was mimicked by intracellular application of GTP-γS, but not by phorbol ester, arachidonic acid or nitroprusside. These results are consistent with model in which 2CA activates a G-protein, which then unmasks an additional activation gate on the Ca-channel. [ABSTRACT FROM AUTHOR]

Effects of intracellular Mg in the activation of a muscarinic K channel were examined in single atrial cells, using patch-recording techniques. In 'cell-attached' patch recordings, acetylcholine (ACh) or adenosine (Ado), present in the pipette, activated a specific population of K channels. In 'inside-out' patches, openings of the K channel by ACh or Ado diminished and did not resume until Mg was added to the perfusate which contained GTP or GTP-γS, a non-hydrolyzable GTP analogue. Channel openings caused by GTP faded by removing Mg, while GTP-γS-induced openings persisted steadily even when both Mg and GTP-γS were removed. In contrast to the case of GTP-induced channel openings, the GTP-γS-induced openings were not inhibited by the A protomer of pertussi toxin with NAD. From these observations, we concluded: 1) Intracellular Mg is essential for GTP to activate the GTP-binding protein. 2) Deactivation of the N protein may be caused by hydrolysis of GTP to GDP. This process may not require Mg. 3) During the activation by GTP analogues, the N protein may be dissociated into its subunits. [ABSTRACT FROM AUTHOR]

Carbamylcholine and GTP act synergistically in stimulating the production of [H]inositol-1-phosphate by digitonized tumoral islet cells (RINm5F line) prelabeled with myo-[2-H(N)]inositol. The response to these two agents is similar to that evoked by GTPγS. These findings suggest that a GTP-binding regulatory protein couples the occupancy of muscarinic receptors to activation of phospholipase C in pancreatic islet cells. [ABSTRACT FROM AUTHOR]