Biomedical subjects

I A Hendry

Publications and source records attributed to I A Hendry.

At least 19 recordsLinked to original sources

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↗

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

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↗

PubMed1992

Binding and retrograde transport of leukemia inhibitory factor by the sensory nervous system.

Leukemia inhibitory factor (LIF), a peptide growth factor with multiple activities, has recently been shown to support the generation and survival of sensory neurons in cultures of mouse neural crest and dorsal root ganglia (DRG). We have conducted binding experiments with 125I-LIF on cultures of DRG to determine the receptor distribution for LIF on these cells and found that at least 60% of the sensory neurons in the cultures bound 125I-LIF, all of which could be eliminated by the addition of unlabeled LIF. The other cells in the culture, which morphologically appeared to be Schwann cells, did not bind appreciable quantities of 125I-LIF. In order to investigate whether LIF is retrogradely transported to sensory neurons in vivo, 125I-LIF was injected into the footpads and gastrocnemius muscles of newborn and adult mice, following sciatic nerve ligation. Radioactivity accumulated in the distal portion of the sciatic nerve, indicating retrograde transport of LIF. Subsequent experiments on mice with unligated sciatic nerves showed that 125I-LIF is specifically transported into the sensory neurons of the DRG. There was no apparent transport of 125I-LIF into motor neurons in the spinal cord. These experiments demonstrate that LIF can specifically bind to and be transported by sensory neurons and further support the idea that LIF acts as a target-derived neurotrophic factor, analogous to NGF.

Animals↗

PubMed1991

Retrograde axonal transport of the GTP-binding protein Gi alpha: a potential neurotrophic intra-axonal messenger.

When a neuronal target is to provide information to the nucleus of the neurone innervating it, it faces the problem of getting its message up the long length of axon separating the cell body from the site of receptor activation at the terminal. The retrograde axonal transport of the neurotrophic molecule, nerve growth factor (NGF), provided one possible mechanism for this information transfer in the sympathetic nervous system. However, some neurotrophic molecules are not retrogradely transported, indicating the message is carried back by a different mechanism. In this paper, we examined such a novel mechanism mediated by the retrograde axonal transport of the alpha subunit of the second messenger protein, Gi. It is proposed that some non-transported neurotrophic molecules may produce a stable second messenger that is itself transported to the nucleus to convey the target derived information for survival.

Animals↗

PubMed1991

Class 1 heparin binding growth factor promotes the differentiation but not the survival of ciliary neurones in vivo.

Molecules isolated from target tissues of various neuronal populations have been shown to enable the survival of those innervating neurones. A molecule derived from bovine heart which can support the survival of parasympathetic neurones of the chick embryo ciliary ganglion in vitro has been sequenced and identified as a member of the class 1 heparin binding growth factors (HBGF-1). When injected into developing chick embryos during the period of naturally occurring cell death in the ciliary ganglion, HBGF-1 failed to rescue those neurones which normally die. There was, however, a premature increase in the activity of choline acetyltransferase within the ganglion and a shift to the right in the size distribution of the neurones. We conclude that HBGF-1 may promote the differentiation of some subpopulation of neurones of the ciliary ganglion of the chick embryo but does not act in vivo as a survival molecule.

Amino Acid Sequence↗

PubMed1991

Retinoic acid potentiates the neurotrophic but not the mitogenic action of the class 1 heparin-binding growth factor (HBGF-1).

Retinoic acid promotes the neuronal survival properties of the class 1 heparin-binding growth factor (HBGF-1) on 8-day-old chick ciliary and sensory neurones. It has little or no effect on the survival promoting properties of class 2 heparin-binding growth factor (HBGF-2) on these cell types. Nerve growth factor (NGF) promoted survival of sympathetic and sensory neurones, and was unaffected at any concentration of retinoic acid. The mitogenic effect of both HBGF-1 and HBGF-2 was unaltered at any concentration of retinoic acid. It is suggested that naturally occurring gradients of retinoic acid, for example, as occur in the developing limb bud, could have a role in the development of normal innervation patterns through selective interaction with neurotrophic molecules.

Animals↗

PubMed1991

Co-activation of insulin-like growth factor-I receptors and protein kinase C results in parasympathetic neuronal survival.

We have studied the interaction between several growth factors to promote parasympathetic neuronal survival. Neither insulin nor insulin-like growth factor-I (IGF-I) had any effect on the survival of embryonic day 8 chick ciliary neurons in culture. Similarly, the protein kinase C activator phorbol dibutyrate (PdBu) had only a minor survival-promoting activity. In combination with PdBu, however, IGF-I or insulin, at concentrations sufficient to act through the IGF-I receptor, were highly synergistic. In a similar fashion, acidic fibroblast growth factor (aFGF)-induced neuronal survival was greatly enhanced by PdBu, as well as by insulin or IGF-I. When added alone, aFGF-induced cell survival required the presence of 1% serum. However, addition of aFGF, IGF-I, or insulin with PdBu under serum-free conditions replaced the serum requirement. That is, these agonist combinations could apparently induce the second messenger requirement for ciliary neuronal survival. Therefore, IGF-I must now be included in the list of candidate molecules responsible for directing parasympathetic nerve formation. The synergy between agonists observed in these experiments highlights the possibility that combinations of growth factors, rather than sole molecules, may dictate parasympathetic nervous system development in vivo.

Animals↗

PubMed1991

Intraspinal release of substance P and calcitonin gene-related peptide during opiate dependence and withdrawal.

The antibody microprobe technique was used to study the release of immunoreactive substance P and immunoreactive calcitonin gene-related peptide within the lower lumbar spinal cord of anaesthetized spinalized cats pretreated twice daily for 3.5 days with increasing doses of morphine hydrochloride (2-20 mg/kg, i.p.). Both peptides were released in the region of the substantia gelatinosa during noxious cutaneous thermal stimulation or high-intensity electrical stimulation of a hind limb nerve. Intravenous administration of naloxone increased the nociceptive excitation of lumbar dorsal horn neurons, but did not alter the evoked release of immunoreactive substance P or immunoreactive calcitonin gene-related peptide in the superficial gray matter dorsal to these neurons. In addition, the release of both peptides was not significantly different to that detected under similar experimental conditions in opioid-naive cats. The results suggest that alterations in neuropeptide release from the central terminals of nociceptive primary afferent neurons do not occur during states of opiate dependence and withdrawal, and thus do not contribute to the characteristic signs of these phenomena in dependent animals.

Animals↗