Bruce G. Livett

Alpha-conotoxin Vc1.1 alleviates neuropathic pain and accelerates functional recovery of injured neurones.

This paper demonstrates the capacity of the neuronal nicotinic acetylcholine receptor (nAChR) antagonist alpha-conotoxin Vc1.1 to inhibit pain responses in vivo. Vc1.1 suppressed pain behaviors when tested in two models of peripheral neuropathy of the rat sciatic nerve, the chronic constriction injury (CCI) and partial nerve ligation (PNL) models. Mechanical hyperalgesia was assessed using an Ugo Basile Analgesymeter. Vc1.1 was administered by intramuscular bolus injection near the site of injury at doses of 0.036 microg, 0.36 microg and 3.6 microg in CCI rats and at a dose of 0.36 microg in PNL rats. Vc1.1 was also administered contralaterally in CCI rats at doses of 0.36 microg and 3.6 microg. Treatment started after the development of hyperalgesia and continued for 7 days. Vc1.1 significantly attenuated mechanical hyperalgesia in both CCI and PNL rats for up to a week following cessation of treatment. Vc1.1 also accelerated functional recovery of injured neurones. A blister was raised over the footpad innervated by the peripheral terminals of the injured nerve. The ability of these terminals to mount an inflammatory vascular response upon perfusion of the blister base with substance P provided a measure of functional recovery. This study shows that alpha-conotoxin Vc1.1, a neuronal nAChR antagonist, suppressed mechanical pain responses associated with peripheral neuropathy in rats in vivo and accelerated functional recovery of the injured neurones. A role for neuronal nAChRs in the analgesic activity of Vc1.1 is proposed.

Drugs from the sea: conopeptides as potential therapeutics.

Marine cone snails from the genus Conus are estimated to consist of up to 700 species. These predatory molluscs have devised an efficient venom apparatus that allows them to successfully capture polychaete worms, other molluscs or in some cases fish as their primary food sources. The toxic venom used by the cone shells contains up to 50 different peptides that selectively inhibit the function of ion channels involved in the transmission of nerve signals in animals. Each of the 700 Conus species contains a unique set of peptides in their venom. Across the genus Conus, the conotoxins represent an extensive array of ion channel blockers each showing a high degree of selectivity for particular types of channels. We have undertaken a study of the conotoxins from Australian species of Conus that have the capacity to inhibit specifically the nicotinic acetylcholine receptors in higher animals. These conotoxins have been identified by mass spectroscopy and their peptide sequences in some cases deduced by the application of modern molecular biology to the RNA extracted from venom ducts. The molecular biological approach has proven more powerful than earlier protein/peptide based technique tor the detection of novel conotoxins [1,2]. Novel conotoxins detected in this way have been further screened for their abilities to modify the responses of tissues to pain stimuli as a first step in describing their potential as lead compounds for novel drugs. This review describes the progress made by several research groups to characterise the properties of conopeptides and to use them as drug leads for the development of novel therapeutics for the treatment of a range of neurological conditions.

α7‐Nicotinic acetylcholine receptors mediate an Aβ1−42‐induced increase in the level of acetylcholinesterase in primary cortical neurones

The β‐amyloid protein (Aβ) is the major protein component of amyloid plaques found in the Alzheimer brain. Although there is a loss of acetylcholinesterase (AChE) from both cholinergic and non‐cholinergic neurones in the brain of Alzheimer patients, the level of AChE is increased around amyloid plaques. Previous studies using P19 cells in culture and transgenic mice which overexpress human Aβ have suggested that this increase may be due to a direct action of Aβ on AChE expression in cells adjacent to amyloid plaques. The aim of the present study was to examine the mechanism by which Aβ increases levels of AChE in primary cortical neurones. Aβ1−42 was more potent than Aβ1−40 in its ability to increase AChE in primary cortical neurones. The increase in AChE was unrelated to the toxic effects of the Aβ peptides. The effect of Aβ1−42 on AChE was blocked by inhibitors of α7 nicotinic acetylcholine receptors (α7 nAChRs) as well as by inhibitors of L‐ or N‐type voltage‐dependent calcium channels (VDCCs), whereas agonists of α7 nAChRs (choline, nicotine) increased the level of AChE. The results demonstrate that the effect of Aβ1−42 on AChE is due to an agonist effect of Aβ1−42 on the α7 nAChR.

Drugs From the Sea: Conotoxins as Drug Leads for Neuropathic Pain and Other Neurological Conditions

The oceans are a source of a large group of structurally unique natural products that are mainly found in invertebrates such as sponges, tunicates, bryozoans, and molluscs. It is interesting to note that the majority of marine compounds currently in clinical trials or under preclinical evaluation are produced by these species rather than as secondary metabolites by marine algae. Through the combined efforts of marine natural products chemists and pharmacologists a number of promising compounds have been identified that are either already at advanced stages of clinical trials such as the new anti-cancer drug marine alkaloid ecteinascidin 743, or have been selected as promising candidates for extended preclinical evaluation. This is the case for conotoxins, (Table 1) where a number of conopeptides are currently being developed as analgesics for the treatment of neuropathic pain.

Effects of opioid peptides and morphine on histamine‐induced catecholamine secretion from cultured, bovine adrenal chromaffin cells

1 The effect of opioid peptides and morphine on histamine‐induced catecholamine secretion has been studied in monolayer cultures of dispersed, bovine adrenal chromaffin cells. 2 Histamine‐induced a dose‐dependent secretion of both adrenaline and noradrenaline with a threshold dose of approximately 5 nM, an EC50 of 150 nM and maximal secretion at 10 μM. 3 Catecholamine secretion induced by 1 μM histamine was completely dependent on extracellular calcium, was inhibited in a dose‐dependent manner by mepyramine (1 nM‐1 μM), and was unaffected by cimetidine (10 μM) and hexamethonium (0.1 mM). 4 Dynorphin‐1–13 (1 nM‐20μM), metorphamide (0.1 nM‐10μM), morphine (1 nM − 0.1 mM) and diprenorphine (1 nM − 0.1 mM) each had no effect on adrenaline or noradrenaline secretion induced by 1 μM histamine. 5 The characteristics of histamine‐induced catecholamine secretion from bovine adrenal chromaffin cells were similar to those reported previously for cat and rat adrenal medulla being calcium‐dependent and mediated by H1 histamine‐receptors. The results with opioid peptides and morphine suggest that endogenous adrenal opioid peptides do not act on the opioid binding sites found on adrenal medullary chromaffin cells to modify their secretory response to histamine.

Chromogranin A: Secretion of Processed Products from the Stimulated Retrogradely Perfused Bovine Adrenal Gland

Chromogranin A (CGA) is a member of a family of highly acidic proteins co‐stored and co‐secreted with adrenaline and noradrenaline in the adrenal medulla. A number of biologically active fragments of CGA (CGAFs) have been characterized including a group of small N‐terminal fragments collectively named vasostatins due to their vascular inhibitory activity. In the present study, the release of CGAFs, including CGA N‐terminal fragments, from the isolated, retrogradely perfused bovine adrenal gland, has been studied under basal conditions and during nerve stimulation and perfusion with acetylcholine. The CGAFs were characterized by SDS‐PAGE followed by immunoblotting with antisera to specific sequences within the CGA molecule. Many different CGAFs were released during stimulation of the glands. Antisera to CGA1–40 and CGA44–76 detected a 7 kD protein whose release was increased during stimulation. This component co‐migrated with synthetic CGA1–76, was not immunoreactive to antisera to CGA79–113 or CGA124–143, and was seen whether or not the serine protease inhibitor aprotinin was present in the perfusion medium. The release of an ∼ 18 kD component, which stained with antisera to CGA1–40, CGA44–76 and CGA79–113, but not to chromostatin (CGA124–143), was also increased during stimulation. Components of 22 kD and larger were detected with antisera to chromostatin, but not with antisera to CGA1–40, CGA44–78 and CGA79–113. Two of these components of 22 to 24 kD were enhanced during nerve stimulation in the presence of aprotinin. The results indicate that processed Chromogranin A fragments are secreted from the bovine adrenal medulla during stimulation of chromaffin cells. The major fragments secreted appear to be the N‐terminal fragments of CGA, CGA1–76 and CGA1–113, which would arise as a result of processing of CGA at the first and second pairs of basic amino acids. A number of larger CGAFs, possibly containing the chromostatin sequence CGA124–143 at their N‐terminal, and components similar in size to intact CGA and to proteoglycan forms of CGA, are also secreted from the perfused bovine adrenal gland during stimulation.

SUBSTANCE P-CONTAINING SENSORY NEURONS IN THE RAT DORSAL ROOT GANGLIA INNERVATE THE ADRENAL MEDULLA

The adrenal medulla is innervated by both cholinergic and substance P (SP)-containing fibres via the splanchnic nerve. SP has been shown to modulate catecholamine (CA) secretion in isolated chromaffin cells and in the perfused rat adrenal gland, however, the origin of SP-containing fibres is not known. In the present study, we have combined the techniques of SP immunohistochemistry and retrograde tracing with Fast blue injected into the left adrenal medulla of the rat in order to study whether SP-containing sensory neurons in the dorsal root ganglia innervate the adrenal medulla. The results showed that there were on average 281 +/- 31 SP-like immunoreactive cells in each left dorsal root ganglion, T3-T13 (range, 234 +/- 19 in T4 to 372 +/- 43 in T13, n = 8). The average total number of Fast blue-labelled cells (T3-T13) in 8 experiments was 172 +/- 26, distributed normally about a peak at T8 (33.8 +/- 6.3 cells) and T9 (33.3 +/- 6.8 cells) with the least at T3 (1.5 +/- 0.8) and T13 (5.2 +/- 2.0). No Fast blue-labelled cells were found in the right DRG. In the left DRG, the average number of cells exhibiting both SP and Fast blue labelled cells were distributed from T7 to T9. These results demonstrate that SP-containing sensory neurons in the dorsal root ganglia provide an ipsilateral innervation of the adrenal medulla in rats.

The role of sensory fibres in the rat splanchnic nerve in the regulation of adrenal medullary secretion during stress.

We have studied the involvement of sensory nerves containing substance P (SP) in the modulation of stress‐induced catecholamine (CA) secretion from the sympathetic nervous system and adrenal medulla. Adrenaline and noradrenaline (NA) levels were measured in blood samples withdrawn from the inferior vena cava (i.v.c.) at 5 or 15 min intervals for periods of up to 60 min, in adult rats during stress induced by insulin or cold. Insulin stress caused a biphasic elevation of plasma CA. Previous studies from our laboratory have shown that the first phase lasting 30 min is neurogenic, and the second phase from 30 to 60 min is non‐neurogenic in mechanism. In control adult rats (with normal levels of SP in their splanchnic nerve), insulin stress caused a slow and progressive secretion of adrenaline into the circulation for the first 30 min (neurogenic phase). In the period 30‐60 min (non‐neurogenic phase) plasma adrenaline and NA levels rose at a much higher rate. In capsaicin‐pre‐treated rats (in which SP levels in the splanchnic nerve were depleted by 68%) insulin stress produced a steady increase in plasma adrenaline levels for up to 5 min similar to that in insulin‐stressed control animals; however, by 10 min the plasma adrenaline levels had fallen to basal and remained low up to 30 min. From 30 to 60 min, plasma adrenaline and NA levels rose steeply as seen with control animals. We conclude that capsaicin pre‐treatment affected the neurogenic phase but did not affect the non‐neurogenic phase. Cold stress increased the plasma adrenaline levels by a neurogenic mechanism over 30 min in control rats. In contrast, in capsaicin‐pre‐treated, cold‐stressed rats, plasma adrenaline did not increase significantly. Plasma NA levels were also significantly lowered in capsaicin‐pre‐treated, cold‐stressed rats during the neurogenic phase but NA increases were not dependent on an intact adrenal innervation. The results using both insulin stress and cold stress suggest that capsaicin‐sensitive (sensory) nerve fibres in the adrenal medulla and in sympathetic ganglia are capable of modifying the secretory responses of these tissues to stress. Results from our previous in vitro work are compatible with the view that SP may be the neuromodulator released from such sensory nerves to produce these effects. This suggests that the previously reported ability of SP to modulate nicotinic receptor function in vitro by either inhibiting the nicotinic response or protecting against nicotinic desensitization may be more than a mere pharmacological curiosity.(ABSTRACT TRUNCATED AT 400 WORDS)

Induction of phenylethanolamine N-methyltransferase mRNA expression by glucocorticoids in cultured bovine adrenal chromaffin cells

The effects of glucocorticoids on the expression of phenylethanolamine N-methyltransferase (PNMT) mRNA and proenkephalin A (ProEnk A) mRNA in cultures of bovine adrenal chromaffin cells were examined. The expression of PNMT mRNA (approx. 1.1 kilobases) was induced in the presence of glucocorticoids. This induction was of high potency with an EC50 in the range of 1-10 nM for dexamethasone, and was blocked by high concentrations of the glucocorticoid antagonist RU-38486. Cortisol, prednisolone and Reichstein substance S (11-deoxy-17-hydroxycorticosterone) were all effective in stimulating PNMT mRNA expression while cortisone, progesterone and beta-estradiol were without effect. These results indicate that the effects are mediated by specific glucocorticoid receptor activation and exhibited a strict structural requirement for the ability of glucocorticoids to induce PNMT mRNA expression. By contrast, glucocorticoids had no significant effect on the expression of ProEnk A mRNA. In summary, this study provides evidence that glucocorticoids act to regulate PNMT (but not ProEnk A) at the transcriptional level. This differential effect of glucocorticoids suggests that different mechanisms govern the expression of mRNAs required for synthesis of the co-stored secretory components, the enkephalins and adrenaline within the chromaffin cells of the adrenal medulla.

Differences between the mechanisms of adrenaline and noradrenaline secretion from isolated, bovine, adrenal chromaffin cells

Cultured, bovine, adrenal medullary chromaffin cells have been used to study the secretion of adrenaline (A) and noradrenaline (NA) in response to two consecutive stimulation periods. Cells were first exposed to nicotine, then rapidly washed before being exposed to elevated K+ levels. Both A and NA cell types secreted their catecholamine in response to nicotine; however, NA cells secreted 3 times more of their catecholamine stores as did A cells. The NA cells were desensitized to stimulation by K+ if they had previously been exposed to nicotine. A cells, on the other hand, were up to twice as responsive to K+ stimulation after having been stimulated with nicotine, in spite of substantial depletion of their catecholamine stores. This sensitization of A cells to K+ was neither due to carryover of nicotine from the first stimulation period, nor to accumulation of calcium in the cells during the nicotinic stimulation. The results suggest that stimulus-secretion coupling or the exocytotic machinery in A and NA chromaffin cells is different. These differences may contribute to the ratio A:NA secreted from the adrenal medulla during stress.