Alpha7-nicotinic acetylcholine receptors mediate an Abeta(1-42)-induced increase in the level of acetylcholinesterase in primary cortical neurones.

The beta-amyloid protein (Abeta) 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 Abeta have suggested that this increase may be due to a direct action of Abeta on AChE expression in cells adjacent to amyloid plaques. The aim of the present study was to examine the mechanism by which Abeta increases levels of AChE in primary cortical neurones. Abeta1-42 was more potent than Abeta1-40 in its ability to increase AChE in primary cortical neurones. The increase in AChE was unrelated to the toxic effects of the Abeta peptides. The effect of Abeta1-42 on AChE was blocked by inhibitors of alpha7 nicotinic acetylcholine receptors (alpha7 nAChRs) as well as by inhibitors of L- or N-type voltage-dependent calcium channels (VDCCs), whereas agonists of alpha7 nAChRs (choline, nicotine) increased the level of AChE. The results demonstrate that the effect of Abeta1-42 on AChE is due to an agonist effect of Abeta1-42 on the alpha7 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.

A novel alpha-conotoxin identified by gene sequencing is active in suppressing the vascular response to selective stimulation of sensory nerves in vivo.

We describe the identification of a conopeptide sequence in venom duct mRNA from Conus victoriae that suppresses a vascular response to pain in the rat. PCR-RACE was used to screen venom duct cDNAs for those transcripts that encode specific antagonists of vertebrate neuronal nicotinic acetylcholine receptors (nAChRs). One of these peptides, Vc1.1, was active as an antagonist of neuronal nAChRs in receptor binding and functional studies in bovine chromaffin cells. It also suppressed the vascular responses to unmyelinated sensory nerve C-fiber activation in rats. Such vascular responses are involved in pain transmission. Furthermore, its ability to suppress C-fiber function was greater than that of MVIIA, an omega-conotoxin with known analgesic activity in rats and humans. Vc1.1 has a high degree of sequence similarity to the alpha-conotoxin family of peptides and has the 4,7 loop structure characteristic of the subfamily of peptides that act on neuronal-type nAChRs. The results suggest that neuronal alpha-conotoxins should be further investigated with respect to their potential to suppress pain.

Differential involvement of conotoxin-sensitive mechanisms in neurogenic vasodilatation responses: effects of age.

During aging there is a decline in sensory nerve function that is associated with reduced neurogenic inflammation and poor wound repair. The cellular mechanism(s) responsible for this decline in function with age is not well understood. We previously reported that sensory nerves in aged rats release sensory neuropeptides preferentially in response to low-frequency (5 Hz) as compared with higher-frequency (15 Hz) antidromic electrical stimulation, and that low-frequency transcutaneous electrical nerve stimulation accelerates wound healing. The present study investigates possible mechanisms for this preferential response. Using laser Doppler techniques, we have measured changes in blood flow in the base of vacuum-induced blisters induced in the rat hind footpad of young and old animals in response to low-frequency (5 Hz) or high-frequency (15 Hz) electrical stimulation (20 V, 2 ms for 1 minute) of the sciatic nerve. The relative contributions of the sensory neuropeptides, substance P and calcitonin gene-related peptide (CGRP), and of N-type voltage-gated calcium channels to the vascular responses were assessed by using the specific receptor antagonists RP67580, which is 2-(1-imino-2-(2 methoxy phyenyl) ethyl)-7,7 diphenyl-4 perhydroisoindolone-(3aR, 7aR); CGRP(8-37); and omega-conotoxin GVIA (Conus geographus), respectively. The results showed a greater involvement of substance P at high-frequency electrical stimulation and of CGRP at low-frequency stimulation. Our finding that omega-conotoxin-sensitive N-type calcium channel function was preserved with age and was only involved in the vascular response to low-frequency electrical stimulation could explain our previous report demonstrating beneficial effects of low-frequency transcutaneous electrical nerve stimulation to wound repair in aged animals. The current results have important practical implications for improving tissue repair in the aged.

Leu(10) of alpha-conotoxin PnIB confers potency for neuronal nicotinic responses in bovine chromaffin cells.

Two alpha-conotoxins PnIA and PnIB (previously reported as being "mollusc specific") which differ in only two amino acid residues (AN versus LS at residues 10 and 11, respectively), show markedly different inhibition of the neuronal nicotinic acetylcholine receptor response in bovine chromaffin cells, a mammalian preparation. Whereas alpha-conotoxin PnIB completely inhibits the nicotine-evoked catecholamine release at 10 microM, with IC(50) = 0.7 microM, alpha-conotoxin PnIA is some 30-40 times less potent. Two peptide analogues, [A10L]PnIA and [N11S]PnIA were synthesized to investigate the extent to which each residue contributes to activity. [A10L]PnIA (IC(50) = 2.0 microM) completely inhibits catecholamine release at 10 microM whereas [N11S]PnIA shows little inhibition. In contrast, none of the peptides inhibit muscle-type nicotinic responses in the rat hemi-diaphragm preparation. We conclude that the enhanced potency of alpha-conotoxin PnIB over alpha-conotoxin PnIA in the neuronal-type nicotinic response is principally determined by the larger, more hydrophobic leucine residue at position 10 in alpha-conotoxin PnIB.

Alpha-conotoxin ImI inhibits the alpha-bungarotoxin-resistant nicotinic response in bovine adrenal chromaffin cells.

The activity of alpha-conotoxin (alpha-CTX) ImI, from the vermivorous marine snail Conus imperialis, has been studied on mammalian nicotinic receptors on bovine chromaffin cells and at the rat neuromuscular junction. Synthetic alpha-CTX ImI was a potent inhibitor of the neuronal nicotinic response in bovine adrenal chromaffin cells (IC50 = 2.5 microM, log IC50 = 0.4 +/- 0.07), showing competitive inhibition of nicotine-evoked catecholamine secretion. Alpha-CTX ImI also inhibited nicotine-evoked 45Ca2+ uptake but not 45Ca2+ uptake stimulated by 56 mM K+. In contrast, alpha-CTX ImI had no effect at the neuromuscular junction over the concentration range 1-20 microM. Bovine chromaffin cells are known to contain the alpha3beta4, alpha7, and (possibly) alpha3beta4alpha5 subtypes. However, the secretory response of bovine chromaffin cells is not inhibited by alpha-bungarotoxin, indicating that alpha7 nicotinic receptors are not involved. We propose that alpha-CTX Iml interacts selectively with the functional (alpha3beta4 or alpha3beta4alpha5) nicotinic acetylcholine receptor to inhibit the neuronal-type nicotinic response in bovine chromaffin cells.

alpha-conotoxin EpI, a novel sulfated peptide from Conus episcopatus that selectively targets neuronal nicotinic acetylcholine receptors.

We have isolated and characterized alpha-conotoxin EpI, a novel sulfated peptide from the venom of the molluscivorous snail, Conus episcopatus. The peptide was classified as an alpha-conotoxin based on sequence, disulfide connectivity, and pharmacological target. EpI has homology to sequences of previously described alpha-conotoxins, particularly PnIA, PnIB, and ImI. However, EpI differs from previously reported conotoxins in that it has a sulfotyrosine residue, identified by amino acid analysis and mass spectrometry. Native EpI was shown to coelute with synthetic EpI. The peptide sequence is consistent with most, but not all, recognized criteria for predicting tyrosine sulfation sites in proteins and peptides. The activities of synthetic EpI and its unsulfated analogue [Tyr15]EpI were similar. Both peptides caused competitive inhibition of nicotine action on bovine adrenal chromaffin cells (neuronal nicotinic ACh receptors) but had no effect on the rat phrenic nerve-diaphragm (muscle nicotinic ACh receptors). Both EpI and [Tyr15]EpI partly inhibited acetylcholine-evoked currents in isolated parasympathetic neurons of rat intracardiac ganglia. These results indicate that EpI and [Tyr15]EpI selectively inhibit alpha3beta2 and alpha3 beta4 nicotinic acetylcholine receptors.

Substance P and epibatidine-evoked catecholamine release from fractionated chromaffin cells.

Bovine chromaffin cells were separated by density gradient centrifugation into subfractions enriched with either > 90% adrenaline- or 70-80% noradrenaline-producing cells. Concentrations of epibatidine (an alkaloid with nicotinic receptor activity) as low as 10 nM released adrenaline and noradrenaline from both fractions of cells maintained as monolayer cultures. The maximal effect was evoked by 30 nM epibatidine and was comparable to that evoked by 10 microM nicotine. The catecholamine release from the noradrenaline fraction was 30-40% higher than from the adrenaline fraction. Initial exposure to 50 nM epibatidine reduced release induced by a second exposure to the drug. There was cross-desensitization between epibatidine and nicotine. Substance P inhibited the epibatidine-evoked catecholamine release from both fractions by up to 85% (IC50 = 3-5 microM). The release of noradrenaline was inhibited more than that of adrenaline. In addition, substance P protected the chromaffin cells against desensitization of the nicotinic receptor by epibatidine. The C-terminal heptapeptide sequence of substance P was 10 x less active, two N-terminal sequences did not modulate the catecholamine release.

Catecholamine release from fractionated chromaffin cells.

Bovine chromaffin cells were separated by density gradient centrifugation into subfractions. After centrifugation on a self-generating Percoll gradient (42.75% isotonic Percoll, 30,000 x g for 22 min at 20 degrees C), the chromaffin cells were found in two clearly distinguishable peaks. The peak with the lower density contained most of the noradrenaline-producing cells (approximately 80%), whereas the adrenaline-producing cells were equally distributed between the two peaks. After collection of suitable fractions from the gradient, cell cultures were obtained, which were enriched with either > 90% adrenaline- or approximately 65% noradrenaline-producing cells. When stimulated by nicotine or carbachol, the dose-response curves of both cell fractions yielded similar EC50s for the release of adrenaline and noradrenaline. On the other hand, the cells of the less dense fraction released 30% more catecholamines (adrenaline as well as noradrenaline) than the cells of the more dense fraction. It is suggested that there are subpopulations among the adrenaline- and noradrenaline-producing cells with differences in receptor-effector coupling.

Anatoxin-a is a potent agonist of the nicotinic acetylcholine receptor of bovine adrenal chromaffin cells.

(+)-Anatoxin-a is a neurotoxic alkaloid produced by the cyanobacterium Anabaena flos-aquae. In this study synthetic (+/-)-anatoxin-a was tested on isolated bovine adrenal chromaffin cells to determine its ability to evoke secretion of endogenous catecholamines through neuronal-type nicotinic receptor activation. Anatoxin-a was found to act as a potent agonist of the secretory response of chromaffin cells with an EC50 of 1-2 microM, compared with an EC50 of 4-5 microM for nicotine. The cells responded to anatoxin-a and nicotine with bell-shaped concentration-response curves consistent with desensitisation at concentrations of anatoxin-a greater than 5 microM and of nicotine greater than 20 microM. The secretion of catecholamines stimulated by anatoxin-a was completely inhibited in a non-competitive manner by the nicotinic antagonist mecamylamine with an IC50 of 0.4-0.5 microM. In the presence of depolarising concentrations of K+ (15 or 50 mM), anatoxin-a increased the secretion of catecholamines in a concentration-dependent manner up to the same maximum as that achieved by anatoxin-a alone. It is concluded that anatoxin-a acts as a potent and selective nicotinic agonist, capable of evoking secretion of endogenous catecholamines from chromaffin cells via their neuronal-type nicotinic receptor.

Expression of dimeric and tetrameric acetylcholinesterase isoforms on the surface of cultured bovine adrenal chromaffin cells.

Acetylcholinesterase is a highly polymorphic enzyme, which can be anchored to the cell surface through several different mechanisms. Dimeric (G2) acetylcholinesterase isoforms are attached by a glycosyl-phosphatidyl-inositol (GPI) linkage, whereas tetrameric (G4) forms are linked through a 20 kilodalton hydrophobic subunit. Although cells of haemopoietic origin contain large amounts of G2 GPI-linked acetylcholinesterase, most tissues express only trace amounts of this isoform. We examined the expression of acetylcholinesterase isoforms in cultured bovine adrenal medullary chromaffin cells. Two major isoforms (G2 and G4) were identified on the cell surface. The G2 isoform, which accounted for approximately half the cell-surface enzyme activity, was linked to the membrane through a GPI anchor. After treatment with diisopropylfluorophosphate to completely inhibit cellular acetylcholinesterase, the G4 isoform was found to be resynthesised and transported to the cell surface more rapidly than the G2 isoform. As the addition of GPI anchors is known to be a very rapid step, this finding suggested that the G2 and G4 isoforms might be transported to the cell surface by two different mechanisms. This conclusion was supported by results from subcellular fractionation experiments. The ratio of G4/G2 membrane-bound acetylcholinesterase varied between different subcellular fractions. The membrane-bound G2 isoform was greatly enriched in a high-speed "microsomal" fraction. G4 acetylcholinesterase is known to be actively secreted by chromaffin cells in culture. Although the G4 isoform was present on the cell surface, most of the secreted enzyme was derived from an intracellular pool. Thus, it is unlikely that the cell-surface G4 isoform contributes significantly to the pool of secreted enzyme. Instead, the expression of two different membrane-bound isoforms may provide a means by which chromaffin cells can target the enzyme to different locations on the cell surface.

Beta A4(25-35) modulates substance P effect on rat skin microvasculature in aged rats: pharmacological manipulation using SEC-receptor ligands.

The primary constituent of the senile plaque core in Alzheimer's disease (AD) is the beta-amyloid protein (beta A4). A discrete 11 amino acid fragment of the beta A4, beta A4(25-35), has been implicated in mediating in vitro neurotoxicity and an inflammatory response surrounding senile plaques in AD via interaction with the Serpin Enzyme Complex (SEC) receptor. Substance P (SP), a neuropeptide of the tachykinin family and a major mediator of neurogenic inflammation, shows sequence homology to beta A4(25-35) and has been shown to protect against the neurotoxicity of beta-amyloid. SP also competes with beta A4(25-35) for binding to the SEC-receptor. SP neurons have also been found to be depleted in AD. Using a blister model of inflammation in the rat hind footpad, we have examined the effect of beta A4(25-35) and its interaction with SP in rat skin microvasculature and determined age-related changes to these phenomena. In addition, pharmacological manipulation of these responses using SEC-receptor ligands (peptide 105Y and 105C) was also undertaken. Because of the evidence for co-existence and co-release of SP and calcitonin gene-related peptide (CGRP) from the peripheral terminals of sensory nerves, it was of interest to examine the interaction of CGRP with beta A4(25-35) on rat skin microvasculature. beta A4(25-35) (10 microM) was perfused over the base of a blister raised on the hind footpad of anaesthetised young and old rats. This was followed by perfusion of SP (1 microM) or CGRP (1 microM) after Ringer's solution. Relative blood flow was monitored using a Laser-Doppler Flowmeter.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional studies with substance P analogues: effects of N-terminal, C-terminal, and C-terminus-extended analogues of substance P on nicotine-induced secretion and desensitization in cultured bovine adrenal chromaffin cells.

Substance P (SP) and SP analogues, including C-terminal, N-terminal, and C-terminus-extended analogues, have been investigated for their ability to modulate nicotine-induced secretion from bovine adrenal chromaffin cells in culture. Secretion was monitored by measuring the release of endogenous catecholamines by electrochemical detection following separation on HPLC and the release of endogenous ATP with an on-line luciferin-luciferase bioluminescence technique. SP is known to have the following two effects on nicotine-induced secretion of catecholamines (see Livett and Zhou, 1991): inhibition of the nicotinic response and protection against nicotinic desensitization. Secretion induced by 10(-5) M nicotine was inhibited 70-80% by SP, SP-methyl ester, and the C-terminus-extended analogue SP-Tyr12-NH2, 65% by (Ala3)SP-NH2, 45% by the C-terminal analogue SP(4-11), and 20 and 5% by the N-terminal analogues SP(1-7) and SP(1-5), respectively, when these peptides were present at 3 x 10(-5) M concentrations. The order of potency was SP = SP-methyl ester = SP-Tyr12-NH2 > (Ala3)SP-NH2 > SP(4-11) > SP(1-7) > SP(1-5). SP, SP-methyl ester, and (Ala3)SP-NH2 protected against nicotinic desensitization by 40-55%, and SP(4-11) protected by 20% (all at 3 x 10(-5) M). In contrast, the N-terminal analogues SP(1-7) and SP(1-5) and the C-terminus-extended analogue SP-Tyr12-NH2 at 3 x 10(-5) M did not protect against nicotinic desensitization. Cyclo-SP(3-9), Ac-SP(3-9)-NH2, SP(3-9), and SP(3-6) had neither inhibitory nor facilitatory effects on secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Noncholinergic control of adrenal catecholamine secretion.

It has been known for over 70 years that adrenal catecholamine secretion can be modulated or elicited by noncholinergic neurotransmitters and hormones. However, our understanding of the cellular mechanisms by which these agents produce their effects and the physiological conditions under which they act are not well characterised. Here we briefly review the mechanisms by which one such agent (the neuropeptide substance P) modulates the cholinergic secretory response of adrenal chromaffin cells, and another agent (angiotensin II) elicits catecholamine secretion independently of the cholinergic innervation.

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 approximately 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, CGA44-76 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.(ABSTRACT TRUNCATED AT 250 WORDS)

An amyloid peptide, beta A4 25-35, mimics the function of substance P on modulation of nicotine-evoked secretion and desensitization in cultured bovine adrenal chromaffin cells.

The amyloid protein (beta A4) is found in the CNS of patients with Alzheimer's disease; however, the pathogenic role of this protein is not known. In the present study, a peptide fragment of beta A4 (beta A4 25-35; Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-NH2), which contains the conserved C-terminal sequence of substance P (X-Gly-Leu-Met-NH2), and the neuropeptide substance P (SP) were examined for their ability to modulate nicotine-evoked secretion from cultured bovine adrenal chromaffin cells. Secretion of the released endogenous catecholamines was monitored by electrochemical detection after separation by HPLC. Secretion induced by 10(-5) M nicotine was inhibited by SP and beta A4 25-35. The IC50 of SP and beta A4 25-35 was 3 x 10(-6) and 3 x 10(-5) M, respectively. SP and beta A4 25-35 both protected against nicotine receptor desensitization. However, beta A4 25-35 was approximately 10-fold less effective than SP in its protective effect. The present work shows that beta A4 25-35 can mimic the modulatory actions of SP on the nicotinic response of cultured bovine chromaffin cells, i.e., inhibition of the nicotinic response and protection against nicotinic desensitization. These modulatory actions may be associated with changes in nicotinic receptor levels reported to occur in Alzheimer's disease.

Regulation of acetylcholinesterase secretion from perfused bovine adrenal gland and isolated bovine chromaffin cells.

The secretion of acetylcholinesterase (AChE) was studied in an isolated perfused bovine adrenal gland preparation and in cultured bovine adrenal medullary chromaffin cells. Electrical field stimulation (10 Hz) of splanchnic nerve terminals in the isolated perfused gland resulted in a two-fold increase in AChE secretion from the gland. Perfusion with the cholinergic receptor antagonists mecamylamine (5 microM) and atropine (1 microM) inhibited 70% of the stimulated secretion of AChE, demonstrating that most of the stimulated secretion was derived from chromaffin cells. The effect of nicotine stimulation on the secretion of AChE from isolated bovine chromaffin cells was compared with that produced by other compounds (histamine, angiotensin II) which are known to stimulate secretion of catecholamines. Incubation with nicotine (1-25 microM) stimulated the secretion of catecholamines and AChE. Histamine (1 nM-10 microM) and angiotensin II (10 pM-10 microM) did not stimulate AChE secretion. Time-course studies of AChE resynthesis after irreversible inhibition with the esterase inhibitor diisopropylfluorophosphate (DFP) demonstrated that AChE is stored within chromaffin cells for at least 11 h before being secreted. AChE secretion was inhibited within 2-3 h by 10 micrograms/ml brefeldin A (BFA), a compound known to block protein translocation from the endoplasmic reticulum (ER) to the Golgi apparatus (GA). The results suggest that AChE may reside for 8-9 h within the lumen of the ER before being actively secreted by processing through the GA.

Identification of multiple tachykinins in bovine adrenal medulla using an improved chromatographic procedure.

Comparison of data based on the reverse-phase HPLC with two ion-pairing reagents, trifluoroacetic acid (TFA) and heptafluorobutyric acid (HFBA), together with the use of two antibodies, has allowed us to identify the various tachykinins in the bovine adrenal medulla. The results show that substance P-like, neurokinin B-like, and neurokinin A-like (including its extended forms, neuropeptide K and neuropeptide gamma) immunoreactivity are present in the bovine adrenal medulla. The concentration of SP-like immunoreactivity in the adrenal medulla was found to be substantially higher than that of NKA-like and NKB-like immunoreactivity. The strategy described here, using radioimmunoassay combined with HPLC employing TFA and HFBA as the ion-pairing reagents, should be useful for the identification of tachykinins and other peptides in the central and peripheral nervous system.

Chromatographic evidence for the presence of multiple tachykinins in the bovine adrenal medulla.

Among the mammalian tachykinins, substance P (SP) has been shown to be the most potent at modulating the response due to nicotinic acetylcholine receptor stimulation of bovine adrenal chromaffin cells. SP-like immunoreactivity has been detected in nerve terminals innervating the adrenal medulla; however, little is known of the presence of other tachykinins in this tissue. In this study, reverse-phase HPLC was used to fractionate peptides in bovine adrenal medullary extracts, and the fractions were analyzed by radioimmunoassay using antisera to SP or neurokinin A (NKA). The results show that both NKA- and SP-like immunoreactivities are present in the adrenal medulla. The presence of neurokinin B is also indicated. The presence of multiple tachykinins in this tissue raises questions as to their functions in the adrenal medulla.