Biomedical subjects

J Bockaert

Publications and source records attributed to J Bockaert.

At least 235 records · Page 13Linked to original sources

PubMed1988

Immunocytochemical localization of the guanine nucleotide-binding protein Go in primary cultures of neuronal and glial cells.

We have localized the guanine nucleotide-binding protein, Go, in primary cultures of pure neuronal and glial cells prepared from different mouse brain areas. Immunoblotting experiments with selective affinity-purified polyclonal rabbit antibodies to the 39 kDa alpha subunit of Go (Go alpha) indicated that Go is distributed in both neurons and glial cells. Go alpha accounts for 0.3% of total membrane proteins in striatal neurons. High specific Go immunoreactivity was also detected in cortical neurons and cerebellar granule cells. Similarly, striatal glial cells contain large amounts of Go (0.2% of total membrane proteins), as do glial cells from cerebral cortex and colliculi. Surprisingly, Go was barely detectable in cerebellar glial cells. 32P-ADP-ribosylation of the same neuronal and glial cell membranes with pertussis toxin indicated the presence of at least 3 substrates related to Go alpha, Gi alpha (41 kDa), and a 40 kDa protein. This 40 kDa protein is the major pertussis toxin substrate in glial cells, while Go alpha is predominant in neuronal membranes. Confirming immunoblotting, no labeled band was detected at 39 kDa in cerebellar glial cells with pertussis toxin. Indirect immunofluorescence staining of cerebellar granule cells and striatal neurons with purified Go alpha antibodies was pronounced at the plasma membrane level, particularly at cell-cell contact areas, and in neurite arborization. More discrete staining was also apparent in the cytoplasm, whereas nuclei remained unstained. In striatal glial cells, specific immunolabeling was more diffused over the whole cell, and dense around the nucleus. The localization of Go suggests that this protein must perform important functions in both the neuronal and glial cells that are discussed.

Animals↗

PubMed1987

An islet activating protein-sensitive G protein is involved in dopamine inhibition of angiotensin and thyrotropin-releasing hormone-stimulated inositol phosphate production in anterior pituitary cells.

In primary culture of anterior pituitary cells, dopamine inhibited the angiotensin (AII)-stimulated inositol phosphate production by 28 +/- 2.5% (n = 14), with an EC50 of 660 +/- 228 nM (n = 8). This effect was blocked by (+)-butaclamol, a specific dopamine receptor antagonist. RU 24926, a D2 specific agonist, but not SKF 38393, a specific D1 agonist, inhibited AII-stimulated inositol phosphate production, suggesting that this dopamine effect is mediated through a dopamine receptor of the D2 subtype. Dopamine also partially inhibited (25%) inositol phosphate production stimulated by thyrotropin-releasing hormone (TRH). Our results suggest that the dopamine-mediated inhibition of hormonally stimulated inositol phosphate production is probably not mediated through the known inhibitory effects of dopamine on cAMP and Ca2+ intracellular concentrations. Although unknown, the mechanism by which dopamine inhibited the AII and TRH-stimulated inositol phosphate production implicates a GTP binding protein sensitive to the islet activating protein (IAP) since dopamine effects were blocked by this toxin. The alpha subunit of the GTP binding protein involved could be one of the three ADP-ribosylated proteins found in anterior pituitary cells in primary cultures, the alpha o (39 kDa), the alpha i (41 kDa), and an alpha subunit of 40 kDa. Indeed, we show here that this 40-kDa IAP substrate, already described in a few tissues, is present in anterior pituitary cells. The negative coupling between dopamine receptors and the AII or TRH inositol phosphate production systems, could be implicated in the dopamine inhibition of the AII- and TRH-stimulated prolactin release since such an inhibition is blocked by IAP. Our results suggest that the negative regulation of inositol phosphate production is one of the mechanisms by which dopamine controls hormonally stimulated prolactin release.

Adenosine Diphosphate Ribose↗

PubMed1987

G-proteins in Torpedo marmorata electric organ. Differential distribution in pre- and post-synaptic membranes and synaptic vesicles.

The nature of the G-proteins present in the pre- and post-synaptic plasma membranes and in the synaptic vesicles of cholinergic nerve terminals purified from the Torpedo electric organ was investigated. In pre- and post-synaptic plasma membranes, Bordetella pertussis toxin, known to catalyze the ADP-ribosylation of the alpha-subunit of several G-proteins, labels two substrates at 41 and 39 kDa. The 39 kDa subunit detected by ADP-ribosylation in the synaptic plasma membrane fractions was immunologically similar to the Go alpha-subunit purified from calf brain. In contrast to bovine chromaffin cell granules, no G-protein could be detected in Torpedo synaptic vesicles either by ADP-ribosylation or by immunoblotting.

Adrenal Medulla↗

PubMed1987

Opioid receptors inhibit the adenylate cyclase in guinea pig cochleas.

The effects of mu- and delta-preferring agonists on adenylate cyclase activity have been investigated in vitro in homogenates of guinea pig cochleas. Morphine, Leu-enkephalin, D-Ala2, N-methyl-Phe4, Gly-ol5-enkephalin (DAGO) and D-Ser2-Leu-enkephalin-Thr (DSLET) each inhibited the synthesis of cyclic AMP. This effect was reversed by naloxone which had a greater affinity in blocking the effect of the mu-preferring agonists (morphine, DAGO) than in blocking the effect of the delta-preferring agonists (Leu-enkephalin, DSLET). Finally, no additive effects were observed when various combinations of two agonists were used. These results indicate that opioid receptors exist in the guinea pig cochlea and that they are negatively linked to adenylate cyclase. The different affinities shown by naloxone to reverse the inhibition induced by the mu- and delta-preferring agonists suggest that morphine and DAGO act through mu-receptors, whereas Leu-enkephalin and DSLET act through delta-receptors. Since no additive effects have been found when combining two different agonists, it can be hypothesized that the mu- and delta-receptors are coupled to the same pool of adenylate cyclase. It may be proposed from these findings that in vivo enkephalins inhibit the synthesis of cyclic AMP via mu- and delta-receptors. However, whether this effect occurs at a presynaptic level (within opioid-containing olivocochlear varicosities) or at the postsynaptic level (within dendrites of the primary auditory neurons) remains to be determined.

Adenylyl Cyclases↗

PubMed1987

Characterization of subtypes of excitatory amino acid receptors involved in the stimulation of inositol phosphate synthesis in rat brain synaptoneurosomes.

The action of excitatory amino acids (EAA) on inositol phosphates (IPs) synthesis was examined in forebrain synaptoneurosomes of Long Evans rats (6-9 days old). Glutamate (GLU) (EC50: 23 microM) and quisqualate (QA) (EC50: 0.12 microM) enhanced IPs turnover. N-methyl-D-aspartate (NMDA) and kainate (KA) were less potent. The EAA-elicited IPs response was not blocked by tetrodotoxin (2 microM) or by the absence of Ca2+. This suggests that the activation of EAA receptors stimulates directly the phosphodiesterase responsible for phosphoinositide breakdown. The three main agonists (QA, KA and NMDA) tested in pairs, induced additive responses on IPs accumulation. In synaptoneurosomes prepared from adult rat, the relative responses to QA and GLU were dramatically reduced, whereas those to KA and NMDA remained unchanged. We concluded that GLU stimulates IPs formation mainly via a QA-like receptor subtype (AA2). This stimulation is transient and could play a key role during synaptogenesis. GLU also enhanced IPs accumulation via other receptor subtypes (probably of the NMDA- or AA1-like class).

Aging↗

PubMed1987

Heavy isotope labeling study of the turnover of forskolin-stimulated adenylate cyclase in BC3H1 cell line.

We have used the method of heavy isotope labeling to study the metabolic turnover of adenylate cyclase in a nonfusing muscle cell line, the BC3H1 cells. These cells contains an adenylate cyclase coupled to beta-adrenergic receptors and highly stimulated by forskolin, a potent activator of the enzyme. After transfer of the cells from normal medium to heavy medium (a medium containing heavy labeled amino acids, 2H, 13C, 15N), heavy isotope-labeled adenylate cyclase molecules progressively replace pre-existing light molecules. In sucrose gradient differential sedimentation, after a 5-day switch in heavy medium, the enzyme exhibited a higher mass (s = 8.40 +/- 0.03 S, n = 13) compared to the control enzyme (s = 7.40 +/- 0.04 S, n = 36). Indeed, the increase in the sedimentation coefficient of the heavy molecules was due to the synthesis of new molecules of adenylate cyclase labeled with heavy isotope amino acids since in the presence of cycloheximide, an inhibitor of protein synthesis, no change in the sedimentation pattern of the forskolin-stimulated adenylate cyclase occurred. After incorporation of heavy isotope amino acids in the adenylate cyclase molecules, the kinetics parameters of the enzyme (i.e. Km for ATP and EC50 for Mn2+ or Mg2+) did not change. However, adenylate cyclase from cells incubated with heavy medium exhibits an activity about 2-fold lower than control (cells in light medium). After switching the cells to the heavy medium, the decrease of the activity of the enzyme occurred during the first 24 h and thereafter remained at a steady state for at least 4 days. In contrast, 24 h after the switch, the sedimentation coefficient of forskolin-stimulated adenylate cyclase was progressively shifted to a higher value indicating that the heavy isotope-labeled enzyme replaced the pre-existing light form of the molecule. These observations show that the rapid decrease in adenylate cyclase activity and the synthesis of heavy adenylate cyclase molecules are two separate events. The relative amounts of heavy and light components of forskolin-stimulated adenylate cyclase obtained in sucrose gradient differential sedimentation were determined as a function of time beginning 24 h after the transfer into the heavy medium. The decrease of the pre-existing light form could be represented by simple first order kinetics with a half-time of 40 h. This result suggests that the metabolic renewal of forskolin-stimulated adenylate cyclase is comparable to that of most plasma membrane proteins.

Adenylyl Cyclases↗

PubMed1987

Presence of three pertussis toxin substrates and Go alpha immunoreactivity in both plasma and granule membranes of chromaffin cells.

GTP-binding proteins have been proposed to be involved in some secretory processes. Bordetella pertussis toxin is known to catalyze ADP-ribosylation of several GTP-binding proteins. In this paper, the subcellular localization of B. pertussis toxin substrates has been explored in chromaffin cells of bovine adrenal medulla. With appropriate gel electrophoresis conditions, three ADP-ribosylated substrates of 39, 40 and 41 kDa were detectable in both plasma and granule membranes. The more intense labelling occurred on the 40 kDa component, while the 41 kDa species exhibited electrophoretic mobility similar to that of Gi alpha. Significant immunoreactivity with anti-Go alpha antibodies was detected at the level of the 39 kDa faster component. The association of G-proteins with granule and plasma membranes suggests the involvement of these proteins in the exocytotic process or in its regulation.

Adenosine Diphosphate Ribose↗

PubMed1987

Chloride transport blockers inhibit the chloride-dependent glutamate binding to rat brain membranes.

The effects of a series of chloride transport blockers (ethacrynate, furosemide, torasemide, 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene and diphenylcarboxylate) on Cl(-)-dependent L-[3H]glutamate (Glu) binding were tested in rat brain membranes, Cl-transport blockers inhibit the Ca2+/Cl(-)-induced increase in L-[3H]Glu binding, some of them without affecting the Ca2+/Cl(-)-independent L-[3H]Glu binding. Increasing the medium osmolarity by augmenting the sucrose concentration also inhibited the Ca2+/Cl(-)-induced increase in L-[3H]Glu binding. The effects of both sucrose and Cl-transport blockers were not additive, suggesting that they acted on the same type of mechanism. We recently suggested that L-[3H]Glu binding to brain membranes corresponds to Glu uptake in membrane vesicles. Therefore we propose that the Cl-transport blockers inhibit a Cl(-)-dependent Glu accumulation into these vesicles.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1987

Multiple Cl(-)-independent binding sites for the excitatory amino acids: glutamate, aspartate and cysteine sulfinate in rat brain membranes.

As we have recently reported that Cl(-)-dependent glutamate (GLU) binding reflects GLU accumulation into membrane vesicles, the characteristics, kinetics and pharmacological specificities of L-[3H]glutamate (L-[3H]GLU) binding to crude rat brain synaptic membranes, were investigated in Cl(-)-free medium. L-[3H]GLU binding was systematically compared to that of L-[3H]cysteine sulfinate (L-[3H]CSA) and L-[3H]ASP), two other putative excitatory amino acids. A high affinity site was determined for each of these radioactive ligands (L-[3H]GLU: Kd = 0.14 microM, Bm = 3.4 pmol/mg protein; L-[3H]CSA: Kd = 0.07 microM, Bm = 2.2 pmol/mg protein; L-[3H]ASP: Kd = 5.8 microM, Bm = 31.2 pmol/mg protein). The pharmacological specificity of these Cl(-)-independent binding sites indicate the existence of at least 3 distinct high affinity sites, all different from the Cl(-)-dependent GLU binding 'site': one having a similar affinity for GLU and CSA, a second one preferring CSA, and a third one preferring ASP. Among the large quantity of structural analogs of the neuroexcitatory amino acids tested, only endogenous compounds (GLU, ASP and CSA) (except hydroxylamine-o-sulfate) were able to interact efficiently. No inhibition by classical agonists and antagonists (such as N-methyl-D-aspartate, quisqualate, kainate, 2-amino-4-phosphonobutyrate, or 2-amino-5-phosphonovalerate) was found. In addition to their high specificity, these Cl(-)-independent sites possess most other biochemical characteristics of receptor proteins.

Animals↗

PubMed1987

Multiple voltage-sensitive calcium channels are probably involved in endogenous GABA release from striatal neurones differentiated in primary culture.

Calcium-dependent release of neurotransmitters is thought to be due to Ca2+ entry into nerve terminals, but the identities of the various voltage-sensitive Ca2+ channels (VSCC) involved in this process remain obscure. To elucidate the types of VSCCs involved in the release process, we studied the effects of various organic Ca2+ channel antagonists and agonists on the release of endogenous gamma-aminobutyric acid (GABA) from mouse striatal neurones differentiated in primary culture. Diltiazem, verapamil and methoxyverapamil (D 600) inhibited K+-evoked (30 mM) GABA release at very high concentrations (greater than 1 microM). The dihydropyridine (DHP) nifedipine, at low concentrations (0.01-1.00 microM), was able to inhibit part of the K+-evoked GABA release (25.6 +/- 7.3% inhibition at 1 microM). This is in agreement with the high affinity of nifedipine for DHP binding sites. The DHPs, BAY K 8644 (EC50 = 41 +/- 15 nM) and CGP 28.392, which possess agonist properties at VSCCs, increased the 15 mM K+-evoked GABA release. The release evoked by the combination of K+ (15 mM) and BAY K 8644 (up to 10 microM) remained smaller than the release elicited by 30 mM K+. The effect of BAY K 8644 (1 microM) was inhibited by nifedipine (IC50 0.55 +/- 0.05 microM). When Na+ ions were replaced by choline, basal and K+-evoked GABA release was significantly increased. Even in the absence of external Na+, nifedipine (1 microM) was not able to totally block the K+ effect. Moreover amiloride, a drug known to inhibit Na+/Ca2+ exchange, and tetrodotoxin (TTX), did not modify the 30 mM K+ response.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1987

Piperazine derivatives including the putative anxiolytic drugs, buspirone and ipsapirone, are agonists at 5-HT1A receptors negatively coupled with adenylate cyclase in hippocampal neurons.

Two putative anxiolytic drugs [ipsapirone (TVXQ 7821) and buspirone], structurally unrelated to benzodiazepines, have negligible ataxic and sedative side effects. These drugs are piperazine analogs which interact at 5-HT1 binding sites. It is demonstrated here that these drugs and two other piperazine derivatives, trifluoromethylphenylpiperazine (TFMPP) and m-chlorophenylpiperazine (mCPP), are agonists at 5-HT1A receptors, a subclass of the 5-HT1 receptor, mediating inhibition of forskolin (100 microM) stimulated adenylate cyclase in particulate fractions of guinea pig hippocampus as well as inhibition of the formation of cyclic AMP promoted by vasoactive intestinal polypeptide (0.1 microM) plus forskolin (1 microM) in mouse hippocampal neurons in primary culture. This study demonstrates that these piperazine based drugs act in both brain homogenate preparations and in intact neurons in a similar manner. The biochemical models described here may aid in the development of even more active drugs in this class.

Adenylyl Cyclases↗

PubMed1987

Multiple coupling of neurohormone receptors with cyclic AMP and inositol phosphate production in anterior pituitary cells.

Regulation of adenohypophyseal hormone secretions has been shown to involve cyclic AMP production, modulation of phosphatidyl inositol diphosphate breakdown and Ca2+ mobilization. Various neurohormone receptors are positively or negatively coupled to adenylate cyclase activity in anterior pituitary cells. The effects of these neurohormones on adenylate cyclase activity are consistent with the effect on hormone secretions, suggesting that modulation of the enzyme activity is actually involved in the regulation of adenohypophyseal secretions. Thus DA inhibits, whereas VIP stimulates adenylate cyclase activity of the same cell type, which, according to the effect of these neurohormones on prolactin secretion, appear to be lactotrophs. On the other hand, SRIF inhibits, whereas GRF stimulates the adenylate cyclase activity of another cell type, namely somatotrophs, whereas CRF appears to act on a third cell type, corticotrophs. Peripheral hormones have been shown to modulate the sensitivity of anterior pituitary cells to these neurohormones. Estradiol long-term treatment has an anti-dopaminergic effect on prolactin secretion. The steroid also suppresses the dopamine inhibition of adenylate cyclase. This effect appears selective to the DA inhibition, since AII inhibition of the enzyme is only partially reduced, whereas the somatostatin inhibition is markedly increased. Peripheral hormones seem to affect the sensitivity of adenohypophyseal cells not only by modulating the number of receptors for a given neurohormone but also by interfering with the coupling mechanisms of these receptors. AII and DA inhibit the adenylate cyclase activity of lactotroph cells. The prolactin stimulation induced by angiotensin is not consistent with the effect of the peptide on adenylate cyclase.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylate Cyclase Toxin↗

PubMed1987

GTP binding proteins: a key role in cellular communication.

One of the major steps in the understanding of the hormonal and sensory transduction mechanisms in eukaryotic cells has been the discovery of a family of GTP binding proteins which couple receptors to specific cellular effectors. The absolute requirement of GTP for hormonal stimulation of adenylate cyclase was the initial observation which led to the purification of the protein involved: Gs. Gs couples stimulatory receptors to adenylate cyclase. It is a heterotrimer composed of an alpha chain (45 or 52 kDa), a beta chain (35-36 kDa) and a gamma chain (8 kDa). Several other G proteins of known functions have been purified: Gi, which couples inhibitory receptors to adenylate cyclase, and transducin which couples photoexcited rhodopsin to cyclic GMP phosphodiesterase. Some G proteins of uncertain function have also been purified: Go, a G protein mainly localized in nervous tissues and Gp, a G protein isolated from placenta and platelets. All these G proteins have a common design. Like Gs they all consist of 3 chains: alpha, beta and gamma. The beta chains are nearly identical, whereas the gamma chains are more variable. The alpha chains are different, but share common domains (especially at the level of the GTP binding site). These domains of homologies are also similar to those of other GTP binding proteins, such as the product of the ras gene (p21) and the initiation or elongation factors. alpha Chains are also ADP ribosylated by bacterial toxins. Gs and transducin are targets for cholera toxin, whereas Gi, Go and transducin are targets for pertussis toxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1987

Go, a major brain GTP binding protein in search of a function: purification, immunological and biochemical characteristics.

The GTP-binding proteins involved in signal transduction now constitute a large family of so called 'G proteins'. Among them, Gs and Gi mediate the stimulation and inhibition of adenyl cyclase, respectively. Recently, another G protein (Go) abundant in brain was purified, but its function is still unknown. Like other G proteins, Go is a heterotrimer (alpha, beta, gamma) and the beta-gamma subunits seem to be identical to those of Gs and Gi. The alpha subunit of Go (Go-alpha) has a molecular weight of 39 kDa lower than those of Gi (41 kDa) or Gs (45-52 kDa). A positive immunoreativity with antibodies against Go-alpha was found in peripheral nervous tissues, adrenal medulla, heart, adenohypophysis and adipocytes. Go ressembles Gi in its ability to be ADP-ribosylated by pertussis toxin, and sequence analysis reveals a 68% homology between their alpha subunits. The GTPase activity of Go is several times higher than that of Gi. The affinity of the beta-gamma entity is about 3 times higher for Gi than for Go. In reconstitution studies, Go does not mimic the inhibitory effect of Gi on adenyl cyclase-stimulated by Gs. On the contrary, Go is as efficient as Gi in reconstituting the functional coupling with the muscarinic, alpha 2-adrenergic and chemotactic agent f-Met-Leu-Phe (fMLP), receptors. Recent studies seem to rule out Go as the coupling G protein of phospholipase C, the enzyme involved in phosphatidyl inositol trisphosphate hydrolysis. However, Go remains a putative candidate for transduction mechanisms coupled to a potassium channel or to a voltage-dependent calcium channel.

Animals↗

PubMed1987

Metabolism of adrenergic receptors and adenylate cyclase.

The alpha 1 and beta-adrenergic receptor metabolism was studied at cell confluency in BC3H1 and C6 glioma cells. After their irreversible blockade with phenoxybenzamine and a bromoacetyl derivative of pindolol (Br-AAM-pindolol) respectively the receptor reappearance allows to determine a half life of 23 hours for the alpha 1-adrenergic receptor in BC3H1 and a quasi absence of beta-adrenergic receptor metabolism in C6 glioma cells at confluency. In contrast, beta-adrenergic receptor is rapidly synthesized during cell division. This metabolic stability of beta-adrenergic receptor at confluency was also observed in BC3H1 cells using the heavy isotope labeling of the beta-adrenergic receptor (half life of 8 days). This stability was also confirmed by the observation that at confluency in C6 glioma cells, beta adrenergic receptors reappeared at the cell surface after a complete down-regulation. In parallel with the study of the half life of adrenergic receptors, we determined in BC3H1 the half life of the forskolin stimulated catalytic unit of the adenylate cyclase using heavy isotope labeling method. In heavy amino-acid medium the apparent sedimentation coefficients of the adenylate cyclase increased from 7.4 +/- 0.04S (n = 36) to 8.4 +/- 0.03S (n = 13). This increase was due to the synthesis of new heavy molecule since it was blocked by cycloheximide. The analysis of the kinetic of synthesis of heavy molecules allowed to calculate a half life of 36 hours. The comparison between the half life of several regulatory membrane proteins in BC3H1 indicate that each of them has a specific metabolism.

Adenylyl Cyclases↗

PubMed1987

Dual action of excitatory amino acids on the metabolism of inositol phosphates in striatal neurons.

Glutamate is able to stimulate inositol phosphate (IP) formation in striatal neurons in primary culture, mainly via an excitatory amino acid receptor of the quisqualate subtype. In the present study we show that carbachol (Carb)-(a cholinergic agonist), but not neurotensin or norepinephrine-induced IP production could be reduced by 40% when measured in the presence of Glu. The inhibition of the Carb response by Glu was dose dependent and reproduced by N-methyl-D-aspartate (NMDA). Quisqualate elicited an additive response with Carb. 2-Amino-5-phosphonovalerate (APV) completely reversed the NMDA-induced inhibition. APV had no significant effect on Glu- or kainate-induced inhibition. Therefore, striatal neurons contain at least three different excitatory amino acid receptors: a quisqualate receptor triggering the stimulation of IP metabolism, and an NMDA and a kainate receptor, both able to decrease the Carb-induced IP formation.

2-Amino-5-phosphonovalerate↗

PubMed1987

The 5-hydroxytryptamine (5-HT2) receptor stimulates inositol phosphate formation in intact and broken WRK1 cells: determination of occupancy-response relationships for 5-HT agonists.

5-Hydroxytryptamine (5-HT) stimulates the accumulation of inositol-trisphosphate in WRK1 cells, a cell line originating from a rat mammary tumor. 5-HT acts via a single receptor type for which it has an affinity constant estimated to be 1.27 microM. A series of agonists known to act at 5-HT2 receptors are partial agonists in this system and have a rank order of relative intrinsic efficacies corresponding to that seen in other systems possessing 5-HT2 receptors. There is an essentially linear occupancy-response relationship for 5-HT and other agonists indicating the absence of a strong amplification mechanism between receptor activation and inositol phosphate formation. The selective blockade of the 5-HT response by nanomolar concentrations of 5-HT2 selective antagonists but not by drugs acting at other 5-HT receptor subtypes suggest that the receptor in WRK1 cells is of the 5-HT2 type. Additionally, we demonstrate that in WRK1 membranes 5-HT acts via the 5-HT2 receptor to elicit a GTP dependent increase in the production of inositol-bisphosphate and inositol-trisphosphate. These properties of the WRK1 cell line indicate that it is a useful model with which to study the nature of 5-HT receptor coupling to the putative second messenger(s), the inositol phosphates.

5-Methoxytryptamine↗

PubMed1987

Immunological localization of the GTP-binding protein Go in different tissues of vertebrates and invertebrates.

Bovine brain contains two GTP-binding proteins, Gi and Go, which are substrates for ADP ribosylation by pertussis toxin. The Gi protein mediates hormone and GTP inhibition of adenylate cyclase, but the function and the precise tissue distribution of Go are unknown. To immunologically probe the localization of Go, we have purified the Go alpha and G beta, gamma subunits of Go and have raised antibodies against them. The polyclonal anti-Go alpha antibodies obtained were very selective for Go alpha compared to Gi alpha or Gs alpha. The positive Go alpha and G beta, gamma immunoreactivities were investigated in different tissues of vertebrates and invertebrates on immunoblots after gel electrophoresis of the crude membranes. The anti-G beta, gamma antibodies recognized a 35-36-kDa protein in brain of vertebrates such as mammals (rat), avians (pigeon), amphibians (frog), fish (trout), and reptiles (turtle) but not in the invertebrates such as molluscs (snail) and insects (locust). With the anti-Go alpha antibodies a high level of immunoreactivity was detected at molecular weights of 39,000-40,000 in the brain of invertebrates as well as in the central nervous system of vertebrates. Moreover, ADP ribosylation with pertussis toxin occurred in the nervous system of invertebrates. These results suggest that the GTP-binding proteins of invertebrates either are devoid of G beta, gamma subunit or, more probably, possess immunologically different G beta, gamma subunits when compared to those of vertebrates. In the vertebrates, Go alpha immunoreactivity was also present in the peripheral nervous system in areas such as the superior cervical ganglia and sciatic nerve. When examined with the anti-Go alpha antibodies, the neuro-and adenohypophysis exhibited a similar immunoreactivity which was about 6 times lower than in brain. Our antibodies also recognized a 40-kDa protein in human adipocytes but at a concentration 17 times lower than that recognized in brain. Taken together, these data show that the Go alpha subunit is well conserved through evolution and, furthermore, confirm that Go alpha is not strictly limited to the nervous system. This suggests that the protein Go ensures a function required for neuronal activity but also present in some other non-nervous tissues.

Adenosine Diphosphate Ribose↗