Biomedical subjects

I A Hendry

Publications and source records attributed to I A Hendry.

At least 19 recordsLinked to original sources

PubMed1997

In sympathetic but not sensory neurones, phosphoinositide-3 kinase is important for NGF-dependent survival and the retrograde transport of 125I-betaNGF.

The way in which the same ligands and receptors have different functional effects in different cell types must depend on subtle differences in the second messenger cascades. Sensory and sympathetic neurones both retrogradely transport nerve growth factor (NGF) and depend on NGF for their developmental survival. NGF binding to the high affinity tyrosine kinase (TrkA) receptors initiates second messenger signalling cascades, one of which includes the activation of phosphoinositide-3 kinase (PI3-kinase). We demonstrate that 100-fold higher concentrations of the PI3-kinase inhibitor, Wortmannin, are required to inhibit the survival effects and retrograde axonal transport of NGF in sensory neurones than in sympathetic neurones. Similarly, although less potently than Wortmannin, the PI3-kinase inhibitor LY294002 required a 10-fold higher concentration to inhibit the survival effects of NGF in sensory than in sympathetic neurones. Inhibitors of other second messengers, including staurosporine, pertussis and cholera toxins, failed to have an effect on the transport of the NGF receptor complex in both cell types. Also, Wortmannin did not affect the structural integrity of the sympathetic nerve terminals. As PI3-kinase is present in both neuronal populations, this suggests that the Wortmannin sensitive isoform of PI3-kinase (p110) is essential in sympathetic neurones both for survival and for NGF-TrkA receptor complex trafficking. As sensory neurones also depend on NGF for their developmental survival and endocytose and retrogradely transport the NGF-TrkA receptor complex, this population of neurones may either recruit a different isoform of PI3-kinase or utilize PI3-kinase independent signalling pathways for these cellular functions.

1-Phosphatidylinositol 4-Kinase↗

PubMed1997

Identifying the G protein, Gz alpha, and its associated proteins in nervous tissue using mass spectrometry and microsequencing techniques.

The signaling pathway associated with pertussis and cholera toxin sensitive G proteins have been extensively investigated. In contrast, the function and associated signal transduction cascade for the pertussis toxin insensitive G protein, Gz alpha have remained elusive. Therefore, the aim of this study was to identify the signal transduction pathway associated with Gz alpha by using the protein identification techniques of matrix assisted laser desorption ionization-time of flight mass spectroscopy and N-terminal Edman sequencing. We have chosen this technique to identify proteins that Gz alpha associates with and to gain insights into the potential role this G protein plays in cells. As Gz alpha is predominantly localized in neuronal tissues, homogenates of whole brain tissue were used. Gz alpha and its associated proteins were immunoprecipitated from brain tissue and identified. The immunoprecipitation of four proteins (140, 46, 41 and 36 kDa) was shown to be inhibited in the presence of the Gz alpha peptide. These proteins were subsequently identified as phospholipase C (PLC)-gamma, beta or gamma-actin, Gz alpha and G beta, the beta subunit of heterotrimeric G proteins, respectively. These results suggest that Gz alpha exists in a protein complex with the actin cytoskeleton and an important intracellular signalling enzyme, PLC-gamma. These methods are powerful techniques for determining protein-protein interactions, and provide the first step to the identification of signalling proteins that Gz alpha associates with. However further experimentation will be required to determine the biological relevance of these protein interactions.

Amino Acid Sequence↗

PubMed1996

Signal transduction from membrane to nucleus: the special case for neurons.

Neurons have a unique problem with signal transduction from the membrane in the region of their terminals back to the cell body and nucleus. This distance may be several meters in some nerves in some species, so there is a requirement for some mechanism to stabilize the signal. This review examines two complementary mechanisms for this signal transduction, either by the retrograde axonal transport of the neurotrophic factor together with its receptor, or the transport of a stable activated second messenger molecule. Extrapolation of studies on the fibroblast signal transduction pathway, where it has been shown that G1 can translocate from the membrane to the nucleus, has led to the demonstration of the retrograde axonal transport of several putative signaling molecules. The alpha subunits of both G1 and Gz are retrogradely transported and Gz alpha or possibly the intact heterotrimeric Gz subsequently accumulates in dorsal root ganglia nuclei. Thus Gz1 Gi1 and potentially other G-proteins and distinct signaling molecules may provide additional signal transduction pathways to that of the neurotrophins from terminal to nucleus.

Animals↗

PubMed1995

Retrograde axonal transport of the alpha subunit of the GTP-binding protein Gz to the nucleus of sensory neurons.

Nerve cells are exquisitely sensitive to target tissue derived factors and the discovery that nerve growth factor could be retrogradely transported in axons suggested that the physical translocation of proteins along the axon could be a mechanism to convey this signal. This message is not due to the neurotrophic factor itself but rather due to second messengers generated by interaction with receptors. We have previously demonstrated the retrograde axonal transport of the alpha subunits of two putative second messenger molecules Gi and Gz. We have investigated more thoroughly the transport of the alpha subunit of Gz (Gz alpha) and in order to be more certain that the immunoreactivity seen is due to Gz alpha, we have made antibodies to peptides from both the N- and C-terminal regions of Gz alpha, which recognise the same 41 kDa band on Western blots of brain and sciatic nerve extracts. This band is eliminated when the antibodies are previously incubated with the specific peptide to which they were made. Using these antibodies for immunohistochemical localisation for Gz alpha, we now report that the GTP-binding protein Gz, is not only retrogradely transported in axons but that it translocates to the neuronal nucleus. Furthermore, the levels seen in the nuclear compartment decline after axotomy or ligation of the mice under ether anaesthetic, suggesting it is the retrogradely transported Gz alpha that is accumulating in the nucleus after activation at the nerve terminal.

Amino Acid Sequence↗

PubMed1995

Retrograde axonal transport of signal transduction proteins in rat sciatic nerve.

Neurons require a mechanism to transmit stable signals over the large distance from the nerve growth cone or terminal to the cell body, in order that information from the target tissue can be relayed to the cell body where it is required. Nerve growth factor (NGF), a target-derived neurotrophic factor, is thought to signal over this distance by receptor mediated internalization of NGF, followed by retrograde axonal transport of the NGF-receptor complex. In this paper we show, by immunohistochemistry of rat sciatic nerve, accumulation of phosphotyrosine immunoreactivity only on the distal side of a nerve crush, suggesting axonal transport of tyrosine kinases and/or tyrosine phosphorylated proteins primarily in a retrograde direction. Furthermore, we also show retrograde axonal transport of phosphoinositide 3-kinase, ERK, MEK and MEK kinase, of which all but MEK kinase are known to be activated downstream of tyrosine receptor kinase activation. The retrograde transport of these proteins suggests that they may be involved in transmission of signals along the axon, relaying neurotrophic factor receptor activation at the nerve terminal to the nerve cell body.

Animals↗

PubMed1995

Acidic and basic fibroblast growth factor bind with differing affinity to the same heparan sulfate proteoglycan on BALB/c 3T3 cells: implications for potentiation of growth factor action by heparin.

Heparan sulfate proteoglycans on the cell surface act as low affinity binding sites for acidic and basic fibroblast growth factor (FGF) [Moscatelli (1987): J Cell Physiol 131:123-130] and play an important role in the interaction of FGF with the FGF receptor (FGFR). In this study, several aspects of the interaction of FGFs with cell surface heparan sulfate proteoglycans were examined. Reciprocal cross blocking studies demonstrated that acidic FGF (aFGF) and basic FGF (bFGF) bind to identical or closely associated heparan sulfate motifs on BALB/c 3T3 cell surface heparan sulfate proteoglycans. However, the binding affinity of the two growth factors for these heparan sulfate proteoglycans differs considerably, competition binding data indicating that aFGF has a 4.7-fold lower affinity than bFGF for 3T3 heparan sulfate proteoglycan. Subsequent studies of dissociation kinetics demonstrated that bFGF dissociates from the FGFR at least 10-fold slower than aFGF, whereas, following removal of cell surface heparan sulfate proteoglycans by heparinase treatment, the dissociation rate of both FGFs is similar and rapid. These results support the concept that cell surface heparan sulfate proteoglycans stabilize the interaction of FGF with FGFR, possibly by the formation of a ternary complex.

3T3 Cells↗

PubMed1995

Evidence that carboxyl-reduced heparin fails to potentiate acidic fibroblast growth factor activity due to an inability to interact with cell surface heparin receptors.

Recently we reported that carboxyl-reduced heparin (CR-heparin), despite binding acidic fibroblast growth factor (aFGF) as effectively as native heparin, was much less potent at augmenting aFGF-induced mitogenesis. This paper describes experiments which examined this phenomenon in more detail in the hope that it would shed light on the mechanism by which heparin potentiates aFGF activity. Initial studies confirmed that heparin, with 60% of its carboxyl groups reduced, although binding aFGF with the same affinity as native heparin (Kd 35 +/- 5 nM), was a poor potentiator of aFGF-induced mitogenic activity. Proteolysis protection experiments also revealed that CR-heparin was as effective as native heparin at protecting aFGF from proteolytic degradation. In contrast, CR-heparin was considerably less effective than native heparin at enhancing the binding of aFGF to the fibroblast growth factor receptor (FGFR) on 3T3 cells. Furthermore, CR-heparin only bound to a subset (approximately 1/3) of heparin receptors on 3T3 cells. Based on these data, it is proposed that CR-heparin is less efficient than heparin at facilitating the formation of a quaternary complex among aFGF, the FGFR, and cell surface heparin receptors.

3T3 Cells↗

PubMed1995

Developmental signalling.

1. In investigating the communication paths between target tissue and neurons we have been led to propose two classes of neurotrophic factors. One comprises the factors which transport themselves, the other factors relying on the transport of a second messenger. The former may have labile second messenger systems necessitating the translocation of agonist and receptor from the nerve terminal to the cell body and the latter must possess a stable second messenger system that itself is sufficiently robust to survive the transport to the cell body. 2. One such class of stable messengers may be the GTP-binding protein family and it has been shown that the alpha subunits of both Gi alpha and Gz alpha can be retrogradely transported in the mouse sciatic nerve. 3. Examination of the cell bodies in the dorsal root ganglia revealed that Gz alpha accumulated in the nucleus of cells with intact axons but that 24 h after axonal ligation this immunoreactivity decreased. 4. It is suggested that Gz is activated at the nerve terminal and it, or at least its alpha subunit, undergoes retrograde transport to the cell body where it accumulates in the nucleus.

Animals↗

PubMed1995

Interaction of the GTP-binding protein Gi2 with a protein kinase A-like kinase in mouse fibroblasts.

We have previously shown that the GTP-binding protein, Gi2 of mouse Balb/c3T3 cells is linked to a serine kinase which phosphorylates the alpha-subunit of Gi itself. In this report we show that Gi is coupled to a second protein kinase. This kinase does not phosphorylate G but phosphorylates another protein bound non-covalently to G. Phosphorylation of the Gi-linked protein induces its release from Gi. Kinase activity is slightly enhanced by GTPyS, suggesting that this kinase may be physiologically regulated by Gi. In an attempt to identify the kinase we have examined the effect of peptide substrates and inhibitors on kinase activity. We found that the protein kinase A inhibitory peptide, PK1 5-24, inhibited the kinase activity, but at concentrations above those usually required to block protein kinase A. The protein kinase A substrate peptide, kemptide, acted as a substrate of the kinase, and was an inhibitor of the phosphorylation of the Gi-linked protein. However, a protein kinase A, catalytic subunit antibody failed to react with any proteins linked to Gi., A protein kinase C inhibitory peptide had no effect on phosphorylation of the Gi-linked protein. Thus, the identity of this kinase has not been resolved, but it may form part of the signalling system of activated Gi in fibroblasts.

Animals↗

PubMed1994

Retrograde axonal transport of the alpha-subunit of the GTP-binding protein GZ in mouse sciatic nerve: a potential pathway for signal transduction in neurons.

We have utilized antibodies against the alpha subunit of GZ in fluorescence immunohistochemistry to determine whether this GTP-binding protein can translocate along nerves by intra-axonal transport. After ligation of the mouse sciatic nerve we found an increase in GZ-like immunoreactivity on the proximal and distal side with time, suggesting that the alpha subunit undergoes orthograde axonal transport and also returns to the cell body by retrograde axonal transport in the sciatic nerve. Unlike the retrograde transport of Gi alpha, shown in a previous study to be present in most sciatic axons, GZ alpha only accumulated in a subpopulation of axons, suggesting that different G-proteins could convey information specific to neuronal subtypes. These results support our proposal that GZ may play a second messenger role in communicating information from the terminals back to cell bodies. Gi alpha and GZ alpha may be representative of relatively stable signalling molecules by which the signal from some neurotrophic molecules can be translocated from the neuron periphery to the cell body without the need for the retrograde transport of the neurotrophic factor itself.

Amino Acid Sequence↗

PubMed1993

Investigation of the ability of several naturally occurring and synthetic polyanions to bind to and potentiate the biological activity of acidic fibroblast growth factor.

The ability of several animal, plant, and bacterial derived polyanions (PAs) as well as synthetic PAs to compete with heparin for the binding of acidic fibroblast growth factor (aFGF) was correlated with their ability to potentiate the mitogenic and neurotrophic actions of this factor. Dextran sulphate, kappa-carrageenan, pentosan sulphate, polyanethole sulfonate, heparin, and fucoidin competed for the heparin binding site on aFGF at relatively low concentrations (< 50 micrograms/ml). lambda-carrageenan, iota-carrageenan, and polyvinyl sulphate exhibited lower affinity for aFGF, whereas hyaluronic acid, dermatan sulphate, chondroitin-6-sulphate, chondroitin-4-sulphate, and uncharged dextran displayed very low or no demonstrable affinity. Potentiation of the mitogenic action of aFGF for Balb/c 3T3 fibroblasts tended to be in general agreement with the aFGF binding affinity of the PAs. However, polyanethole sulfonate, the carrageenans, polyvinyl sulphate, fucoidin, and pentosan sulphate exerted a mitogenic action on the 3T3 cells that was independent of, and in addition to, the ability of these GAGs to potentiate the action of aFGF. The ability to potentiate the neurotrophic action of aFGF for E8 chick ciliary neurons was a general property of those PA with low or no activity in the mitogen assay. Thus hyaluronic acid, dermatan sulphate, chondroitin-4-sulphate, chondroitin-6-sulphate, and even unchanged dextran all potentiated aFGF induced neuronal survival. The differential effects of these PA in potentiating the biological activities of aFGF are discussed in relation to their ability to compete for the heparin-binding site of aFGF.

3T3 Cells↗

PubMed1993

Growth factor second messenger systems: oncogenes and the heterotrimeric GTP-binding protein connection.

We feel that there is now compelling evidence that the GTP-binding proteins play more than just a coordinating role in the actions of both tyrosine kinase and nontyrosine kinase receptor signal transduction. These similarities appear to represent just a small component of the convergence in the signaling pathways for structurally dissimilar receptor subsets. Future years will see further understanding of the intricacies of these G-protein-proto-oncogene interactions, and the extension into the potential role in growth factor action played by the expanding number of known members of this G-protein family.

Animals↗

PubMed1993

Gi alpha and Gi beta are part of a signalling complex in Balb/c3T3 cells: phosphorylation of Gi beta in growth-factor-activated fibroblasts.

Stimulation of division of Balb/c3T3 cells by epidermal growth factor (EGF) and/or insulin is inhibited by pertussis toxin. The G-protein involvement in this response includes the growth factor receptor-induced translocation of the alpha-subunit of Gi (Gi alpha) to the nucleus, where Gi alpha binds specifically to chromatin of dividing cells. This paper reports the first data of studies on the mode of interaction of tyrosine kinase growth factor receptors with Gi alpha, and the mechanism by which Gi affects cell proliferation. When Gi alpha was immunoprecipitated from Triton X-100 extracts of Balb/c3T3 cells, several other proteins were co-precipitated. The major proteins, of 110,000, 60,000 and 36,000 M(r), were not directly recognized by the Gi alpha antibody, showing that Gi alpha was in a complex with these proteins. The 36,000 M(r) protein was recognized by G beta-common antiserum, so confirming its identity as Gi beta. The 36,000 M(r) protein was phosphorylated in cells activated for 20 h with platelet-derived growth factor, epidermal growth factor and insulin, but not after 3 min or 1 h of stimulation. Both Gi alpha and G beta-common antibodies precipitated the phosphorylated 36,000 protein. Gi beta phosphorylation was similarly observed in response to activation by EGF alone for 20 h, but to a lesser extent. Phosphotyrosine antibodies also precipitated a 36,000 M(r) phosphorylated protein from growth factor-activated cells, suggesting that Gi beta may be phosphorylated on tyrosine. Therefore, Gi beta phosphorylation appears to represent a late event after activation of cells by tyrosine kinase growth factor receptors. We are currently examining the role of this event in signal transduction, particularly in relation to control of nuclear responses.

3T3 Cells↗

PubMed1993

Age-related changes in tyrosine hydroxylase and choline acetyltransferase in sympathetic ganglia of a rat strain with reduced sympathetic neuron numbers.

In Wistar rats, a subpopulation of sympathetic ganglionic neurons dies during ageing, but in the GH strain, these same neurons die during the period of perinatal maturation. We have compared tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT) in superior cervical ganglia of GH and control rats at different ages. Ganglionic TH rose to near adult levels between postnatal weeks 1 and 2. No significant differences in TH values were seen between GH and control ganglia at any age, indicating that reduced neuron numbers are compensated for by increased cellular activity. Ganglionic ChAT rose initially in parallel with TH and then more slowly over postnatal weeks 3-4, reaching adult levels that were about 20% lower in GH than in normal ganglia. During ageing, TH remained constant but ChAT continued to rise slowly in GH ganglia, whereas ChAT in normal ganglia fell by about 10%. Both the strain difference in ChAT during development and the fall in ChAT during ageing in normal animals parallel the differences in ganglion cell numbers seen under these circumstances.

Aging↗

PubMed1993

Involvement of pertussis toxin-sensitive and -insensitive mechanisms in alpha-adrenoceptor modulation of noradrenaline release from rat sympathetic neurones in tissue culture.

1. Sympathetic neurones derived from superior cervical ganglia of neonatal rats and maintained in tissue culture were used to investigate the modulation of neurotransmitter release by presynaptic receptors. Three week old cultures of neurones were loaded with [3H]-noradrenaline to label endogenous neurotransmitter stores. Release of noradrenaline was evoked by depolarization with raised extracellular K+ in the presence of desipramine and corticosterone to prevent uptake of released catecholamine. 2. Potassium (55 mmol l-1) depolarization for 30 s caused more than a four fold increase in 3H overflow from basal levels but this increase was reduced by up to 40% in the presence of exogenous noradrenaline (1 mumol l-1). The inhibition by noradrenaline of depolarization-evoked overflow was blocked by the alpha 1/alpha 2-adrenoceptor antagonist, phentolamine. Phentolamine alone did not increase K(+)-evoked 3H overflow. 3. The alpha 2-adrenoceptor antagonist, yohimbine, produced a concentration-dependent block of the inhibition by noradrenaline of K(+)-evoked overflow, while the alpha 1-adrenoceptor antagonist, prazosin, was without effect at concentrations up to 0.1 mumol l-1. 4. The beta-adrenoceptor antagonist, propranolol, neither reduced K(+)-evoked overflow nor increased the degree of inhibition caused by the addition of 1 mumol l-1 noradrenaline. 5. The alpha 2-adrenoceptor agonist, clonidine (1 mumol l-1) was less effective than noradrenaline at inhibiting K(+)-evoked overflow, while the alpha 1-adrenoceptor agonist, phenylephrine (1 mumol l-1) had no significant effect. 6. The L-channel calcium blocker, nicardipine (1 mumol l-1) significantly inhibited 3H overflow evoked by K+. In the presence of L-channel block, however, noradrenaline still inhibited residual evoked overflow.7. In the presence or absence of nicardipine, pertussis toxin pretreatment (1 nmol 1-1) reduced, but did not prevent, the effect of noradrenaline (1 micromol 1-1). Pertussis toxin alone caused a significant enhancement of K+-evoked 3H overflow.8. The data indicate that on postganglionic neurones of cultured rat sympathetic ganglia there are alpha 2-adrenoceptors that modulate neurotransmitter release, but no functional beta-adrenoceptors that mediate an enhancement of transmitter release. The data suggest further that in this preparation the mechanism of alpha2-adrenoceptor modulation may involve pertussis toxin sensitive and insensitive G-proteins and effects on calcium channels other than L-type.

Adrenergic alpha-1 Receptor Antagonists↗

PubMed1992

Neonatal nerve growth factor treatment alters the preganglionic innervation pattern of rat superior cervical ganglion.

We treated rat pups with nerve growth factor (10 micrograms/animal/day s.c.) over postnatal days 1-7. Subsequent adult neuron numbers and tyrosine hydroxylase content in superior cervical ganglion were normal, but preganglionic inputs, as gauged from ganglionic choline acetyltransferase, were reduced. In parallel, intraganglionic axon terminals containing calcitonin gene-related peptide, but not those containing substance P, were increased in number. We postulate that neonatal nerve growth factor stimulates sprouting of ingrowing axons that have entered the ganglion soon after birth and that this represses subsequent establishment of cholinergic preganglionic synapses.

Aging↗

PubMed1992

Ability of different chemically modified heparins to potentiate the biological activity of heparin-binding growth factor 1: lack of correlation with growth factor binding.

A range of chemically modified heparins was examined for their ability to bind heparin-binding growth factor 1 (HBGF-1; acidic fibroblast growth factor) and potentiate the in vitro mitogenic and neurotrophic activity of HBGF-1. It was found that carboxyl-reduced heparin bound HBGF-1 as effectively as the native heparin molecule. Totally desulfated heparin and N-desulfated heparin lack HBGF-1-binding capacity, and substitution of the exposed amino group with acetyl or acetoacetyl groups only partially restored binding capacity, indicating that N-sulfates only play a limited role in growth factor binding. However, the failure of totally desulfated, N-resulfated heparin to interact with HBGF-1 demonstrated that N-sulfates alone are insufficient and ester sulfates are absolutely essential for HBGF-1 binding. In contrast, the ability of the modified heparins to potentiate the mitogenic activity of HBGF-1 correlated only to a limited extent with their affinity for HBGF-1. Thus, the carboxyl-reduced molecule which displayed similar affinity for HBGF-1 as native heparin was consistently less potent in augmenting mitogenesis. Similarly, the N-acetylated and the N-acetoacetylated species, which had much lower affinity for HBGF-1 than the carboxyl-reduced molecule, conferred similar biological activity to HBGF-1 whereas N-desulfated heparin, which was unable to bind growth factor, potentiated the mitogenic activity of HBGF-1 for both 3T3 and HUVE cells. In contrast, the neurotrophic activity of HBGF-1 was potentiated by modified heparin species which failed to bind HBGF-1 and were without activity in the mitogenic assays.(ABSTRACT TRUNCATED AT 250 WORDS)

3T3 Cells↗

PubMed1992

Retrograde factors in peripheral nerves.

The relationship between the neuron and its target is explored and the possible mechanisms for achieving correct connections are analysed. The most plausible mechanism is the presence of a retrograde intra-axonal message from the target to the neuronal cell body. The molecular form of the message and the mechanisms to achieve this signal transduction are discussed and it is proposed that there are two types of neurotrophic factors. One has a short-acting second messenger, itself incapable of surviving for the time required for transport to the cell body and thus requiring the transport of the message-generating complex to the cell body. The other has a long-lasting second messenger complex which is well able to survive the transport to the cell body so that there is no need for the transport of the neurotrophic factor itself. Thus all neurotrophic factors do not themselves require retrograde axonal transport and such non-transportable factors may generate intricate messages due to associations of signal transduction molecules via binding sites such as phosphorylated tyrosines and the src homology domain 2.

Animals↗