Endothelin-B receptors activate Galpha13.

Endothelin (ET) receptors activate heterotrimeric G proteins that are members of the Gi, Gq, and Gs families but may also activate members of other families such as Galpha12/13. Galpha13 has multiple complex cellular effects that are similar to those of ET. We studied the ability of ET receptors to activate Galpha13 using an assay for G protein alpha-chain activation that is based on the fact that an activated (GTP-bound) alpha-chain is resistant to trypsinization compared with an inactive (GDP-bound) alpha-chain. Nonhydrolyzable guanine nucleotides and AlMgF protected Galpha13 from degradation by trypsin. In membranes from human embryonic kidney 293 cells that coexpress ETB receptors and alpha13, ET-3 and 5'-guanylylimidodiphosphate [Gpp(NH)p] increased the protection of alpha13 compared with Gpp(NH)p alone. The specificity of ETB receptor-alpha13 coupling was documented by showing that beta2 receptors and isoproterenol or ETA receptors and ET-1 did not activate alpha13 and that a specific antagonist for ETB receptors blocked ET-3-dependent activation of alpha13.

p115 RhoGEF, a GTPase activating protein for Galpha12 and Galpha13.

Members of the regulators of G protein signaling (RGS) family stimulate the intrinsic guanosine triphosphatase (GTPase) activity of the alpha subunits of certain heterotrimeric guanine nucleotide-binding proteins (G proteins). The guanine nucleotide exchange factor (GEF) for Rho, p115 RhoGEF, has an amino-terminal region with similarity to RGS proteins. Recombinant p115 RhoGEF and a fusion protein containing the amino terminus of p115 had specific activity as GTPase activating proteins toward the alpha subunits of the G proteins G12 and G13, but not toward members of the Gs, Gi, or Gq subfamilies of Galpha proteins. This GEF may act as an intermediary in the regulation of Rho proteins by G13 and G12.

Direct stimulation of the guanine nucleotide exchange activity of p115 RhoGEF by Galpha13.

Signaling pathways that link extracellular factors to activation of the monomeric guanosine triphosphatase (GTPase) Rho control cytoskeletal rearrangements and cell growth. Heterotrimeric guanine nucleotide-binding proteins (G proteins) participate in several of these pathways, although their mechanisms are unclear. The GTPase activities of two G protein alpha subunits, Galpha12 and Galpha13, are stimulated by the Rho guanine nucleotide exchange factor p115 RhoGEF. Activated Galpha13 bound tightly to p115 RhoGEF and stimulated its capacity to catalyze nucleotide exchange on Rho. In contrast, activated Galpha12 inhibited stimulation by Galpha13. Thus, p115 RhoGEF can directly link heterotrimeric G protein alpha subunits to regulation of Rho.

The monomeric G-proteins Rac1 and/or Cdc42 are required for the inhibition of voltage-dependent calcium current by bradykinin.

Although regulation of voltage-dependent calcium current (ICa,V) by neurotransmitters is a ubiquitous mechanism among nerve cells, the signaling pathways involved are not well understood. We have determined previously that in a neuroblastoma-glioma hybrid cell line (NG108-15), the heterotrimeric G-protein G13 mediates the inhibition of ICa,V produced by bradykinin (BK) via an unknown mechanism. Various reports indicate that G13 can couple to RhoA, Rac1, and Cdc42, which are closely related members of the Rho family of monomeric G-proteins. We have investigated their role as signaling intermediates in the pathway used by BK to inhibit ICa,V. Using immunoblot analysis and the PCR, we found evidence that RhoA, Rac1, and Cdc42 all are expressed in NG108-15 cells. Intracellularly perfused recombinant Rho-GDI (an inhibitor of guanine nucleotide exchange specific for the Rho family) attenuated the inhibition of ICa,V by BK. These findings indicate that activation of RhoA, Rac1, or Cdc42 may be required for the response to BK. To determine whether any of these monomeric G-proteins mediate the response to BK, we have intracellularly applied blocking antibodies specific for each of the candidate proteins. Only the anti-Rac1 antibody blocked the response to BK. In parallel experiments, peptides corresponding to the C-terminal regions of Rac1 and Cdc42 blocked the same response. These data indicate a novel functional contribution of Rac1 and possibly also of Cdc42 to the inhibition of ICa,V by neurotransmitters.

Regulation of eukaryotic phosphatidylinositol-specific phospholipase C and phospholipase D.

This review focuses on two phospholipase activities involved in eukaryotic signal transduction. The action of the phosphatidylinositol-specific phospholipase C enzymes produces two well-characterized second messengers, inositol 1,4,5-trisphosphate and diacylglycerol. This discussion emphasizes recent advances in elucidation of the mechanisms of regulation and catalysis of the various isoforms of these enzymes. These are especially related to structural information now available for a phospholipase C delta isozyme. Phospholipase D hydrolyzes phospholipids to produce phosphatidic acid and the respective head group. A perspective of selected past studies is related to emerging molecular characterization of purified and cloned phospholipases D. Evidence for various stimulatory agents (two small G protein families, protein kinase C, and phosphoinositides) suggests complex regulatory mechanisms, and some studies suggest a role for this enzyme activity in intracellular membrane traffic.

Three distinct G protein pathways mediate inhibition of neuronal calcium current by bradykinin.

1. In NG108-15 cells dialyzed with 10 mM ethylene glycolbis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or bis (o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA), bradykinin (BK) selectively inhibited the N-type calcium current. This effect of BK was blocked by an antibody directed against the G protein G13. Thus under these conditions G13 mediates the inhibition of voltage-dependent calcium current (ICa, V) by BK. In contrast, activation of K+ currents by BK is mediated by Gq/11. BK also couples to Gi2. 2. We now examine the involvement of G proteins in the inhibition of ICa, V by BK when NG108-15 cells are dialyzed with 1 mM BAPTA. Under these conditions, BK inhibited both the N- and L-type, but not the T-type, calcium currents. Intracellular application of anti-G13 antibody did not suppress the response to BK. Applications of either anti-Gq/11 antibody or pertussis toxin (PTX, to block Gi2) were similarly ineffective. Even combined application of anti-Gq/11 and -G13 antibodies, or PTX together with either antibody, did not block inhibition of ICa, V by BK. However, the combination of both antibodies with PTX blocked the response to BK in low BAPTA. In conclusion, both Gq/11 and a PTX-sensitive G protein (presumably Gi2), together with G13, are involved in the inhibition of ICa, V by BK. 3. Gq/11 inhibited only the L-type calcium current, whereas the PTX-sensitive G protein inhibited both the N- and L-type calcium currents. 4. The BAPTA dependence of the Gq/11 and PTX-sensitive inhibitions may reflect a Ca2+ requirement of the pathway(s) acting on the L current and/or a direct suppressive effect of BAPTA.

Gq/11 and PLC-beta 1 mediate the substance P-induced inhibition of an inward rectifier K+ channel in brain neurons.

1. Substance P (SP) induces a slow neuronal excitation in cholinergic neurons from the nucleus basalis by suppressing an inwardly rectifying K+ current (Kir). We have determined which G protein alpha-subunit mediates this SP effect. 2. After intracellularly injecting antibody against each alpha-subunit of G proteins (Gq alpha/11 alpha, G12 alpha, and G13 alpha) with an Eppendorf microinjector, we examined, by using the whole cell patch-clamp and the ON-cell mode of single-channel recording, the effect of SP on Kir in cultured neurons of the nucleus basalis. The effect of SP on Kir was substantially reduced in neurons injected with antibodies to Gq alpha/11 alpha but not with antibodies to G12 alpha or G13 alpha. 3. The effects of antibodies against three isozymes of phospholipase C (PLC-beta 1, PLC-beta 2, and PLC-beta 3) were tested. The SP-induced suppression of Kir was reduced by antibody against PLC-beta 1 but not by antibodies against PLC-beta 2 or PLC-beta 3. 4. We conclude that the SP-induced inhibition of Kir in nucleus basalis neurons is mediated by Gq/11 and PLC-beta 1.

Induction of inducible nitric-oxide synthase by the heterotrimeric G protein Galpha13.

While the functions of several G protein alpha subunits such as alpha(s( and alpha(q) are relatively well understood, the action of others such as alpha13 remain largely undefined. Because of recent interest in regulation of nitric-oxide synthase (NOS) by G protein-coupled signaling systems and findings that receptors for two proinflammatory substances, thrombin and thromboxane couple to alpha13, we studied the effect of alpha13 on NOS activity in a renal epithelial cell line. We found that stable overexpression of alpha13 or its GTPase-deficient mutant, alpha13Q226L, in a continuous renal epithelial cell line (MCT) increased NOS activity. The increased NOS activity was due to increased expression of the macrophage-inducible form of NOS (iNOS). iNOS protein and activity were not increased in similar cells expressing an activated alpha(s) (alpha(s)Q227L) or were minimally increased in cells expressing activated alpha(i1) (alpha-i1Q204L) and alpha(q) (alpha(q)Q209L), members of the three other G protein alpha chain families. Transient co-expression of alpha13 or alpha13Q226L increased the activity of an iNOS promoter-CAT construct demonstrating that alpha13 increases iNOS expression through transcription. Consequently, alpha13 induces iNOS through a novel mechanism that is distinct from that of other G protein alpha chains and that may mediate the actions of G protein-dependent proinflammatory agents.

Regulation of phospholipase D by protein kinase C is synergistic with ADP-ribosylation factor and independent of protein kinase activity.

Phospholipase D (PLD) which was partially purified from membranes of porcine brain could be stimulated by multiple cytosolic components; these included ADP-ribosylation factor (Arf) and RhoA, which required guanine nucleotides for activity, and an unidentified factor which activated the enzyme in a nucleotide-independent manner (Singer, W. D., Brown, H. A., Bokoch, G. M., and Sternweis, P. C. (1995) J. Biol. Chem. 270, 14944-14950). Here, we report purification of the latter factor, its identification as the alpha isoform of protein kinase C (PKCalpha), and characterization of its regulation of PLD activity. Stimulation of PLD by purified PKCalpha or recombinant PKCalpha (rPKCalpha) occurred in the absence of any nucleotide and required activators such as Ca2+ or phorbol ester. This action was synergistic with stimulation of PLD evoked by either Arf or RhoA. Dephosphorylation of rPKC alpha with protein phosphatase 1 or 2A resulted in a loss of its kinase activity, but had little effect on its ability to stimulate PLD either alone or in conjunction with Arf. Staurosporine inhibited the kinase activity of PKCalpha without affecting activation of PLD. Finally, gel filtration of PKCalpha that had been cleaved with trypsin demonstrated that stimulatory activity for PLD coeluted with the regulatory domain of the enzyme. These data indicate that PKC may regulate signaling events through direct molecular interaction with downstream effectors as well as through its well characterized catalytic modification of proteins by phosphorylation.

Resolved phospholipase D activity is modulated by cytosolic factors other than Arf.

Phospholipase D, which has been extracted from porcine brain membranes and chromatographically enriched 100-fold, was activated better by impure preparations of Arf than by purified or recombinant Arf. Examination of brain cytosol with this enriched preparation of PLD activity revealed at least three stimulatory components. One of these is Arf or the first cytoplasmic factor. A second peak of PLD-stimulating activity (cytoplasmic factor II, CFII) was resolved from Arf by anion exchange and gel filtration. This CFII can be further separated into multiple activities by chromatography with heparin-agarose. The activities were differentiated by their stimulatory properties as measured in the absence or presence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) alone and in the presence of added Arf and GTP gamma S. While all of the CFII pools stimulated PLD activity to some degree and showed synergistic activation when administered in conjunction with Arf, they could be classified into two groups with distinct behavior. When used together, pools from the two respective groups showed synergistic activation of PLD. The first set of pools contained the RhoA monomeric G protein. Recombinant RhoA was used to show that it could indeed activate this enriched PLD activity and act synergistically with Arf proteins. A related monomeric G protein, Cdc42, was also effective. The second set of CFII pools were devoid of RhoA and, in contrast to the first group, demonstrated significant stimulating activity in the absence of guanine nucleotides. These data indicate that the PLD activity from brain can be modulated by several cytosolic factors and that Arf-sensitive PLD may represent a complex activity that can be regulated in an interactive fashion by a variety of cellular signaling events.

G alpha q and G alpha 13 regulate NHE-1 and intracellular calcium in epithelial cells.

To understand the mechanisms by which G protein-coupled signaling systems regulate NHE-1 in epithelial cells, we expressed G alpha q and G alpha 13 in a renal epithelial cell line. We studied two signaling systems that have been implicated in NHE-1 regulation [intracellular Ca (Cai) and phospholipase C activity] and measured NHE-1 activity, mRNA, and antigen. Expression of alpha qWT and alpha qQ209L (a GTPase-deficient mutant) increased basal Cai and altered the kinetics of the bradykinin-induced Cai signal. The initial bradykinin-induced spike in Cai was prolonged and the plateau was higher in cells expressing alpha qWT and alpha qQ209L than in control cells. Cells expressing alpha 13WT also had a higher basal Cai and plateau after stimulation by bradykinin, but Ca release from intracellular stores was similar to that in control cells. Expression of all three alpha-chains increased NHE-1 activity, antigen, and mRNA. The alpha qQ209L had the greatest effect increasing activity by approximately twofold. The alpha 13WT increased NHE-1 activity by approximately 1.5-fold, and alpha qWT increased activity 1.2-fold. These studies demonstrate that alpha q and alpha 13 alter regulation of Cai but by different mechanisms. The Ca signal or another signal generated by alpha q and alpha 13 regulate(s) NHE-1 at the levels of activity, antigen, and mRNA.

Polymerization of Antarctic fish tubulins at low temperatures: role of carboxy-terminal domains.

We have proposed previously that the efficient polymerization of tubulins from Antarctic fishes at low, physiological temperatures (-1.8 to +2 degrees C) may result in part from adaptations (e.g., reductions in acidic residues) located in their carboxy-terminal (C-terminal) tails [Detrich & Overton (1986) J. Biol. Chem. 261, 10922-10930]. To test this hypothesis, we have examined the polymerization of Antarctic fish neural tubulins modified at their C termini by proteolysis or by neutralization of carboxyl groups. Addition of subtilisin to low concentrations of Notothenia coriiceps tubulin induced a biphasic assembly reaction: stage I corresponded to the C-terminal cleavage of beta chains to produce alpha beta s dimers, and stage II coincided with the slower, C-terminal cleavage of alpha chains to yield alpha s beta s. Both stage I and stage II polymers consisted of protofilament sheets and microtubules with attached sheets. The critical concentration for assembly of the stage II polymer was at least 10-fold lower than that of untreated tubulin. Neutralization of Glu and Asp carboxyls in Gobionotothen gibberifrons microtubules by the carbodiimide-catalyzed incorporation of glycine ethyl ester (GEE) moieties produced a tubulin, modified largely in its C termini, that assembled more readily than did control tubulin. When 12 GEE groups were incorporated per dimer, the critical concentrations for assembly of modified tubulin at 5-10 degrees C were 2-3-fold smaller than those for the unmodified protein. Comparably modified bovine tubulin (10 GEE/dimer) assembled at 37 degrees C with a critical concentration 2.6-fold lower than that for the unmodified tubulin.(ABSTRACT TRUNCATED AT 250 WORDS)

The G protein G13 mediates inhibition of voltage-dependent calcium current by bradykinin.

In neuroblastoma-glioma hybrid cells, bradykinin has dual modulatory effects on ion channels: it activates a K+ current as well as inhibits the voltage-dependent Ca2+ current (ICa,V). Both of these actions are mediated by pertussis toxin-insensitive G proteins. Antibodies raised against the homologous Gq and G11 proteins suppress only the activation of the K+ current; this suggested that at least two distinct G protein pathways transduce diverse effects of this transmitter. Here, we show that the inhibition of ICa,V by bradykinin is suppressed selectively by intracellular application of antibodies specific for G13. This novel G protein may play a general role in the inhibition of ICa,V by pathways resistant to pertussis toxin.

Purification and characterization of the alpha subunit of G13.

Specific antisera were produced to peptides representing the carboxyl terminus of alpha 13, a recently identified alpha subunit of the heterotrimeric guanine nucleotide-binding proteins (G proteins). Immunodetection with the antisera indicated that the 43-kDa protein is expressed ubiquitously at low levels (0.005-0.05% of membrane protein) in tissues and cultured cells. A combination of conventional and immunoaffinity chromatographic techniques was used to purify small quantities of alpha 13 from bovine brain. Quantities of protein sufficient for biochemical analysis could be produced by concurrent expression of alpha 13 with G protein beta 2 and gamma 2 subunits using a baculovirus system. The rate of dissociation of GDP from recombinant alpha 13 (r alpha 13) is slow (0.01-0.02 min-1 at 30 degrees C), and relatively high concentrations of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) are required to observe nucleotide binding. This binding was reduced significantly in the presence of 20 mM Mg2+. Rates of hydrolysis of GTP by alpha 13 were limited by nucleotide exchange; attempts to measure the intrinsic rate of hydrolysis indicate that it is greater than 0.2 min-1. Stoichiometric concentrations of beta gamma subunits inhibited binding of GTP gamma S to and hydrolysis of GTP by alpha 13. By reconstitution, the purified alpha 13 did not affect the activity of several known effector enzymes. The availability of purified r alpha 13 and knowledge of its biochemical properties will allow further characterization of its interactions with receptors and effectors.

Cellular uptake and tubulin binding properties of four Vinca alkaloids.

The in vitro effects of four Vinca alkaloids, vinblastine (VLB), vincristine (VCR), vindesine (VDS) and vinepidine (VPD), on B16 melanoma proliferation, binding to bovine brain tubulin and B16 melanoma cell extracts, and uptake by the B16 cells were compared. The relative binding affinities to bovine brain tubulin were VPD greater than VCR congruent to VDS greater than VLB with the Ka for VPD being about 4-fold higher than that for VLB. On the other hand, the relative effects on B16 cell proliferation were exactly the opposite. Differences were found in the degree of concentration of the four alkaloids by the cells: 100-fold for VLB, 50-fold for VCR and VDS, and 20-fold for VPD. At the extracellular concentrations of drugs which inhibit proliferation by 50%, the intracellular concentration would still be far less than the tubulin concentration. Thus, it is likely that all of the Vinca alkaloids would be bound to tubulin and difference in uptake rather than Ka values is the major factor in determining the relative effectiveness of the drugs. L cells showed 50% the sensitivity of B16 melanoma cells toward VLB and 30% the sensitivity toward VPD. The L cells also concentrated these drugs to a lesser extent than did the B16 cells.

The incidence of seizures during tricyclic antidepressant drug treatment in a brain-injured population.

Tricyclic antidepressants (TCAs) have been associated with the occurrence of seizures both with overdoses and with therapeutic doses. Seizures with therapeutic doses of TCA have been reported in patients both with and without previous histories of seizures. The incidence of seizures possibly precipitated by TCAs was examined retrospectively in a population of 68 severely brain-injured patients, all of whom were at high risk for the development of seizures. Seizure histories, anticonvulsant use, comedication use, and other pertinent data were recorded before, during, and after TCA use. We conclude that 14 patients (19%) developed seizures largely caused by TCAs. Other possible contributing factors, clinical outcomes, and some recommendations are discussed.

Binding of vinblastine to stabilized microtubules.

Addition of 2-200 microM vinblastine to microtubules at steady state in vitro causes the microtubules to depolymerize, with the formation of protofilament spirals and other aggregated forms of microtubule protein. The presence of such spirals and protein aggregates, which are difficult to separate from microtubules, has complicated attempts to measure the binding of vinblastine to microtubules. We have found that stabilizing bovine brain microtubules in vitro with dimethyl sulfoxide, taxol, or a combination of dimethyl sulfoxide and taxol prevents or greatly retards the formation of protofilamentous spirals, thus permitting us to measure the binding of vinblastine to intact microtubules. Reciprocal plots of binding data indicate the presence of 1.4-1.7 vinblastine binding sites/mol of tubulin in the microtubule, with a Ka of approximately 3-4 x 10(3) M-1. The Ka value obtained is within 1 order of magnitude of the apparent intrinsic binding constant for the binding of vinblastine to tubulin dimers. The results support the idea that depolymerization of microtubules by intermediate and high concentrations of vinblastine occurs by stoichiometric binding of vinblastine to tubulin along the microtubule surface.

Carbamazepine replacement of phenytoin, phenobarbital and primidone in a rehabilitation setting: effects on seizure control.

Most patients who receive anticonvulsants after traumatic brain injury are treated with the sedative anticonvulsants phenytoin and/or phenobarbital, or perhaps primidone. However, there is considerable evidence demonstrating that these medications have a deleterious effect on cognitive function. Thus, in a rehabilitation setting, alternatives should be sought. Carbamazepine has been found to be relatively free of such effects, and would be an optimum alternative if seizure control were comparable. We have studied the effects of withdrawing phenytoin, phenobarbital and primidone, and using carbamazepine as the primary anticonvulsant in 27 patients at the Greenery Rehabilitation and Skilled Nursing Center for whom ongoing anticonvulsant treatment was considered to be necessary due to previous seizures or a high risk of the occurrence of seizure. We compared a 3 month baseline period (just prior to carbamazepine introduction or sedative anticonvulsant tapering), to a 3 month post-withdrawal period immediately following sedative anticonvulsant withdrawal, when carbamazepine was the sole anticonvulsant. In 20 out of 21 patients in whom carbamazepine replaced sedative anticonvulsants seizure control was essentially similar or somewhat improved. In only one patient did the substitution with carbamazepine result in a loss of seizure control. Six patients were initially receiving carbamazepine in combination with phenytoin and/or phenobarbital. The removal of phenytoin and phenobarbital, leaving carbamazepine as sole therapy, resulted in improved seizure control in three patients and no change in the other three. In the light of carbamazepine's reportedly less detrimental effects on cognitive function and behaviour in other patient populations, it should perhaps be considered as a first line anticonvulsant, especially for patients in rehabilitation settings.

Effect of solution variables on the binding of vinblastine to tubulin.

Vinblastine binding to tublin was measured in different buffers using tubulin prepared by two different methods and three different binding assay methods. In 100 mM 1,4-piperazinediethanesulfonic acid (Pipes) buffer containing 1 mM MgSO4 and 1 mM ethylene glycol bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), the data appeared to be consistent with one site with a Ka value of 3.4 X 10(6) M-1 and another site with a Ka value of 2.8 X 10(5) M-1. However, in buffers of lower ionic strengths and without Mg2+ the Ka values were lower. The lowest value (2 X 10(4) M-1) was obtained in 10 mM phosphate buffer, in which only one site was evident under the conditions used. Neither the binding assay used nor the method for tubulin preparation affected the Ka value. Using HPLC, aggregation induced by vinblastine was evident in buffers which gave the largest Ka values. Tubulin aggregation in the presence of vinblastine was also confirmed by analytical ultracentrifugation. The results support the proposal of Na and Timasheff [Biochemistry 25, 6214 (1986)] that the apparent Ka value is influenced by the degree of aggregation induced by vinblastine and that the intrinsic binding constant to the dimer is represented by the lowest value, about 2 X 10(4) M-1.

Effect of pyridoxine deficiency in the neuronally mature rat.

Pyridoxine deficiency was induced in neuronally mature rats by combination of dietary deprivation and ingestion of 4-deoxypyridoxine. Overt signs of deficiency were evident. At this time the activity of glutamic acid decarboxylase as also the content of serotonin decreased in several areas of the brain. Catecholamine contents were unchanged from controls. Light microscopic observations indicated no difference in CNS myelin between the deficient and control animals. There was no difference between deficient and control groups in their auditory evoked responses.