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Clostridial neurotoxins potently and specifically inhibit neurotransmitter release in defined cell types. Here we report that a catalytically active derivative (termed LH(N)/A) of the type A neurotoxin from Clostridium botulinum has been coupled to a lectin obtained from Erythrina cristagalli to form a novel conjugate. This conjugate exhibits an in vitro selectivity for nociceptive afferents compared with the anatomically adjacent spinal neurons, as assessed using in vitro primary neuronal culture systems to measure inhibition of release of neurotransmitters. Chemical conjugates prepared between E. cristagalli lectin and either natively sourced LH(N)/A or recombinant LH(N)/A purified from Escherichia coli are assessed, and equivalence of the recombinant material are demonstrated. Furthermore, the dependence of inhibition of neurotransmitter release on the cleavage of SNAP-25 is demonstrated through the use of an endopeptidase-deficient LH(N)/A conjugate variant. The duration of action of inhibition of neurotransmitter released by the conjugate in vitro is assessed and is comparable with that observed with Clostridium botulinum neurotoxin. Finally, in vivo electrophysiology shows that these in vitro actions have biological relevance in that sensory transmission from nociceptive afferents through the spinal cord is significantly attenuated. These data demonstrate that the potent endopeptidase activity of clostridial neurotoxins can be selectively retargeted to cells of interest and that inhibition of release of neurotransmitters from a neuronal population of therapeutic relevance to the treatment of pain can be achieved.

We have described, in undifferentiated SH-SY5Y human neuroblastoma cells, the relative potency of Clostridium botulinum neurotoxin (BoNT) serotypes A-F Sensitivity of stimulated [3H]-noradrenaline ([3H]-NA) release to the toxins had a rank order of potency of: C > D > A > B > F after 3 days exposure. The difference between the most potent (BoNT/C: IC50 0.54 nM) and the least (BoNT/F: IC50 > 300 nM) was approximately 1,000-fold. Though fluid phase endocytosis may have been the mechanism of entry for low potency toxins the far higher potency of BoNT/C would suggest receptor-driven entry. Potency was not a reflection of the dependence of the release mechanism on a particular SNARE since the substrate specificities were mixed throughout the potency order. This indicated that the toxins differed in their efficiency of binding/endocytosis or enzymatic activity inside the cell. The serotypes that cleaved vesicle-associated membrane protein (VAMP) isoforms (BoNT/B, D and F) did not fully inhibit [3H]-NA release. Cleavage of the appropriate substrate proteins was observed for all serotypes. SNAP-25 cleavage by BoNT/A was shown to be a dose-dependent and correlated closely with reduction of release, supporting proteolysis as the mechanism by which toxin inhibited secretion. Comparison of the SH-SY5Y cell line sensitivity to BoNT/A with glycine releasing rat primary spinal cord neuron cultures, revealed a massive difference in potency; the primary cultures being approximately 200,000-fold more sensitive. The demonstration, using BoNTs, of the crucial role of SNAP-25, VAMP and syntaxin in SH-SY5Y cells suggests the use of this neuroblastoma as a model in the study of these proteins in neurotransmitter release.

Clostridium botulinum neurotoxins (BoNT) are zinc dependent endopeptidases which, once internalised into the neuronal cytosol, block neurotransmission by proteolysis of membrane-associated proteins putatively involved in synaptic vesicle docking and fusion with the plasma membrane. Although many studies have used a variety of cellular systems to study the neurotoxins, most require relatively large amounts of toxin or permeabilisation to internalise the neurotoxin. We present here a primary culture of embryonic rat dorsal root ganglia (DRG) neurons that exhibits calcium-dependent substance P secretion when depolarised with elevated extracellular potassium and is naturally BoNT sensitive. The DRG neurons showed a different IC50 for each of the toxins tested with a 1000 fold difference between the most and least potent neurotoxins (0.05, 0.3, 30 and approximately 60 nM for A, C, F and B, respectively). BoNT/A cleavage of SNAP-25 was seen as early as 2 h, but substance P secretion was not significantly inhibited until 4 h intoxication and the effects of BoNT/A were observed for as long as 15 days. This primary neuronal culture system represents a new and sensitive cellular model for the in vitro study of the botulinum neurotoxins.

1. Cultures of endothelial cells derived from the microvasculature of human frontal lobe have been investigated for phospholipase C (PLC) responses to histamine, endothelins and purinoceptor agonists. 2. Using cells prelabelled with [3H]-inositol and measuring total [3H]-inositol (poly)phosphates, histamine acting at H1 receptors stimulated a substantial response with an EC50 of about 10 microM. 3. Endothelin-1 also gave a clear stimulation of phosphoinositide-specific phospholipase C. Both concentration-response curves and binding curves showed effective responses and binding in the rank order of endothelin-1 > sarafotoxin S6b > endothelin-3, suggesting an ETA receptor. 4. Assay of total [3H]-inositol (poly)phosphates showed no response to the purinoceptor agonists, 2-methylthioadenosine 5'-trisphosphate (2MeSATP), adenosine 5'-O-(3-thiotrisphosphate) (ATP gamma S) or beta,gamma-methylene ATP. Both ATP and UTP gave a small PLC response. 5. Similarly, when formation of [32P]-phosphatidic acid from cells prelabelled with 32Pi was used as an index of both PLC and phospholipase D, a small response to ATP and UTP was seen but there was no response to the other purinoceptor agonists tested. 6. Study by mass assay of stimulation by ATP of inositol (1,4,5) trisphosphate accumulation revealed a transient response in the first few seconds, a decline to basal, followed by a small sustained response. 7. These results show that human brain endothelial cells in culture are responsive to histamine and endothelins in a manner which may regulate brain capillary permeability. Purines exert a lesser influence.

1. We have investigated whether the 'atypical' P2-purinoceptor previously described on adrenal microvasculature endothelial cells is a nucleotide receptor (responds to pyrimidines and purines) and is linked to phospholipase D as well as phospholipase C. 2. Cultured bovine adrenal medullary endothelial (BAME) cells responded to the pyrimidine UTP, as well as the purines. The total [3H]-inositol phosphate responses were with a rank order of UTPATP- = adenosine 5'-O-(3-thio-triphosphate) (ATP gamma S)2MeSATP. The selective P2x agonist beta, gamma-methylene ATP was inactive. 3. Construction of dose-response curves to ATP, ATP gamma S and UTP in the presence and absence of additional agonists showed that responses to ATP gamma S and UTP were not additive, nor were those to UTP and ATP. This suggests that purines and pyrmidines acted via a common nucleotide receptor. 4. 32P-labelled BAME cells, in the presence of butanol, produced [32P]-phosphatidylbutanol (PBut) when stimulated with ATP gamma S or the protein kinase C activator, tetradecanoyl phorbol acetate (TPA). 5. Cells labelled with [3H]-palmitate and stimulated in the presence of butanol generated [3H]-PBut with the same order of agonist potencies seen for inositol phosphate responses. 6. The protein kinase C inhibitor, Ro 31-8220, abolished TPA and agonist stimulation of [3H]-PBut production. 7. These observations, and our related studies on bovine aortic endothelial cells, provide the first demonstration of a phospholipase C linked nucleotide receptor on vascular endothelial cells. It is concluded that BAME cells express a nucleotide receptor linked to phospholipase C and phospholipase D, but that activation of phospholipase D is probably down-stream of phospholipase C.

Clostridial neurotoxins potently and specifically inhibit neurotransmitter release in defined cell types. Previously reported data have demonstrated that the catalytically active LH(N) endopeptidase fragment of botulinum neurotoxin type A (termed LH(N)/A) can be retargeted to a range of cell types in vitro to lead to inhibition of secretion of a range of transmitters. Here, we report the synthesis of endopeptidase conjugates with in vitro selectivity for nociceptive afferents compared to spinal neurons. Chemical conjugates prepared between Erythrina cristagalli lectin and LH(N)/A are assessed in vitro and in in vivo models of pain. Chemical conjugates prepared between E. cristagalli lectin and either natively sourced LH(N)/A, or recombinant LH(N)/A purified from Escherichia coli are assessed, and equivalence of the recombinant material is demonstrated. The duration of action of inhibition of neurotransmitter release by the conjugate in vitro is also assessed and is comparable to that observed with Clostridium botulinum neurotoxin. Selectivity of targeting and therapeutic potential have been confirmed by in vivo electrophysiology studies. Furthermore, the analgesic properties of the conjugate have been assessed in in vivo models of pain and extended duration effects observed. These data provide proof of principle for the concept of retargeted clostridial endopeptidases as novel analgesics.

Capsaicin, the pungent component of "hot" chili peppers, selectively activates a distinct population of primary sensory neurons responsive to noxious stimuli. Many of these fibres express neuropeptides including the tachykinin, substance P. Using cultured dorsal root ganglion neurons, we found that capsaicin (10 microM) stimulated a 2-fold increase in release of substance P in the absence of extracellular Ca(2+). Elevated potassium (75 mM) was unable to induce release under these conditions. The introduction of Ca(2+) enhanced capsaicin-induced release and brought about a robust response to potassium. Preincubation of cells with botulinum neurotoxin A (100 nM) completely blocked potassium-induced release but the capsaicin response, in the absence of Ca(2+), was unaffected. However, toxin treatment dramatically reduced capsaicin-stimulated release in the presence of Ca(2+). It is concluded that capsaicin induces release of substance P from dorsal root ganglion neurons via two mechanisms, one requiring extracellular Ca(2+) and the intact synaptosomal-associated protein 25 kDa (SNAP-25), and the other independent of extracellular Ca(2+) and not involving SNAP-25.

Nerve growth factor (NGF) receptor binding, internalisation and transportation of NGF has been identified as a potential route of delivery for other molecules. A derivative of Clostridium botulinum neurotoxin type A (LHN) that retains catalytic activity but has significantly reduced cell-binding capability has been prepared and chemically coupled to NGF. Intact clostridial neurotoxins potently inhibit neurotransmitter release at the neuromuscular junction by proteolysis of specific components of the vesicle docking/fusion complex. Here we report that the NGF-LHN/A conjugate, when applied to PC12 cells, significantly inhibited neurotransmitter release and cleaved the type A toxin substrate. This work represents the successful use of NGF as a targeting moiety for the delivery of a neurotoxin fragment.

Clostridial neurotoxins potently and specifically inhibit neurotransmitter release in defined cell types by a mechanism that involves cleavage of specific components of the vesicle docking/fusion complex, the SNARE complex. A derivative of the type A neurotoxin from Clostridium botulinum (termed LH N /A) that retains catalytic activity can be prepared by proteolysis. The LH N /A, however, lacks the putative native binding domain (H C ) of the neurotoxin and is thus unable to bind to neurons and effect inhibition of neurotransmitter release. Here we report the chemical conjugation of LH N /A to an alternative cell-binding ligand, wheat germ agglutinin (WGA). When applied to a variety of cell lines, including those that are ordinarily resistant to the effects of neurotoxin, WGA-LH N /A conjugate potently inhibits secretory responses in those cells. Inhibition of release is demonstrated to be ligand mediated and dose dependent and to occur via a mechanism involving endopeptidase-dependent cleavage of the natural botulinum neurotoxin type A substrate. These data confirm that the function of the H C domain of C. botulinum neurotoxin type A is limited to binding to cell surface moieties. The data also demonstrate that the endopeptidase and translocation functions of the neurotoxin are effective in a range of cell types, including those of nonneuronal origin. These observations lead to the conclusion that a clostridial endopeptidase conjugate that can be used to investigate SNARE-mediated processes in a variety of cells has been successfully generated.

In adherent SH-SY5Y human neuroblastoma cells cultured for 14 days to promote uptake and release of [3H]-noradrenaline, ionomycin induced a biphasic elevation of the intracellular [Ca2+] ([Ca2+]i). This consisted of a rapid transient elevation followed by a marked, persistent secondary elevation. Further study indicated that the peak [Ca2+]i elevation was dependent upon intracellular Ca2+ whilst the secondary elevation was dependent upon extracellular Ca2+. This profile of response and dependence upon intracellular and extracellular sources of Ca2+ was similar to that evoked by the muscarinic agonist, methacholine but was independent of inositol 1,4,5-trisphosphate generation. Ionomycin also stimulated the release of [3H]-noradrenaline from preloaded cells. Both intracellular and extracellular sources of Ca2+ were needed for the full response and synergised to effect release. Thus, in adherent SH-SY5Y cells, ionomycin elevates [Ca2+]i in a complex way in a manner partly analogous to the elevation of [Ca2+]i by agonists of phosphoinositidase C-linked receptors. Furthermore the effects of [Ca2+]i elevation on [3H]-noradrenaline release by these two processes are similar. Such functional consequences may, however, differ under circumstances where the profile and source of Ca2+ for ionomycin-mediated changes differs to that of receptor agonists.

The human neuroblastoma cell line SH-SY5Y, maintained at confluence for 14 days, released [ 3 H]-noradrenaline ([ 3 H]NA) when stimulated with either the muscarinic receptor agonist methacholine or bradykinin. The major fraction of release was rapid, occurring in

The mechanism whereby endothelin stimulates motogenesis of vascular smooth muscle cells is not understood. Here we show that endothelin-1 stimulates phospholipase D by a protein kinase C and tyrosine kinase dependent mechanism, and present evidence that implicate the phosphatidic acid formed by phospholipase D in the mitogenic response.

1. We have examined the phospholipase C responses in bovine aortic endothelial cells to purines (ATP, ADP and analogues) and the pyrimidine, uridine triphosphate (UTP). 2. The cells responded to purines in a manner consistent with the presence of P2y purinoceptors; both 2-methylthioadenosine 5'-triphosphate (2MeSATP) and adenosine 5'-0-(2-thiodiphosphate) (ADP beta S) were potent agonists (EC50 0.41 microM and 0.85 microM respectively) while beta, gamma-methylene ATP at 300 microM was not. 3. The cells also responded to UTP. The maximal response to UTP was less than that for either 2MeSATP and ADP beta S while adenosine 5'-0-(3-thiotriphosphate) (ATP gamma S) gave the largest maximal response. 4. The concentration-effect curve to UTP was additive in the presence of either 2MeSATP or ADP beta S. However, the concentration-effect curves to ATP gamma S reached the same maximum in the presence or absence of UTP. 5. Suramin, at concentrations between 10 microM and 100 microM was a competitive antagonist for the response to ADP beta S and 2MeSATP but not the response to UTP. 6. The results show that there are two separate, co-existing, receptor populations: P2y-purinoceptors (responding to purines) and nucleotide receptors (responding to both purines and pyrimidines). We conclude that purines such as ATP/ADP may regulate aortic endothelial cells by interacting with two phospholipase C-linked receptors.

In this study we used the bovine thoracic aorta endothelial cell line AG 4762 and primary bovine aortic endothelial cells to investigate the formation of phosphatidic acid (PA) in response to activation of P 2 -purinergic receptors. 2-Methylthio ATP (2MeSATP) stimulated the formation of [ 32 P]-PA in bovine aortic endothelial cells labelled with 32 P i , for 2.5 hr. A comparison of the response to other ATP analogues suggests that this was mediated via a P 2y -purinergic receptor. Using various agonists at 30 μM there was a correlation between the formation of [ 32 P]PA and of total inositol phosphates in the presence of lithium. The 2MeSATP-stimulated accumulation of [ 32 P]PA showed an initial high rate, followed by a more sustained slower rate. The initial response was independent of extracellular calcium while the later response was dependent on calcium influx. The protein kinase C stimulator phorbol myristate acetate (PMA) produced only a very small enhancement of [ 32 P]PA accumulation compared to 2MeSATP. The 2MeSATP stimulation of both inositol phosphates and [ 32 P]PA was almost eliminated by the presence of PMA. Using cells prelabelled with [ 3 H]methylcholine 2MeSATP produced only a small non-significant enhancement of [ 3 H]choline formation; PMA by contrast formed a much larger amount of 3 H]choline. There was no evidence of a change in [ 3 H]phosphocholine. The dissociation between phospholipase D (PLD) activation and [ 32 P]PA accumulation and the correlation between stimulation of [ 32 P]PA accumulation and phospholipase C (PLC) activation all suggest that, using this protocol for labelling cells, the principle route of the stimulation of formation of [ 32 P]PA is via the activation of PLC followed by metabolism of diacylglycerol (DAG) by DAG kinase. These results show that activation of P 2y -purinergic receptors on aortic endothelial cells leads to the formation of phosphatidic acid and that both PLD and PLC pathways are likely to contribute to this response.

Endothelin-1 (ET-1) is known to stimulate phospholipase C (PLC) activity in SK-N-MC human neuroblastoma/epithelioma cells: here we show that phospholipase D(PLD) is also stimulated. The generation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3 ) by ET-1-stimulated PLC was attenuated by protein kinase C (PKC) activation and enhanced by PKC inhibition. An enhancement of ET-1-stimulated Ins(1,4,5)P 3 accumulation was also seen when the product of PLD activity was either diverted into phosphatidyl butanol in the presence of butanol, or phosphatidate phosphohydrolase (PPH) activity was inhibited by dl -propranolol. We conclude that there is an inhibitory, PKC-mediated, feedback loop in these cells which is dependent, in part, on the activation of PKC by product(s) of the PLD/PPH pathway. This provides a novel role for agonist-stimulated PLD activation.