Dose-dependent effects of nicotine on proliferation and differentiation of human bone marrow stromal cells and the antagonistic action of vitamin C.

A range of biological and molecular effects caused by nicotine are considered to effect bone metabolism. Vitamin C functions as a biological antioxidant. This study was to evaluate the in vitro effects of nicotine on human bone marrow stromal cells and whether Vitamin C supplementation show the antagonism action to high concentration nicotine. We used CCK-8, alkaline phosphatase (ALP) activity assay, Von Kossa staining, real-time polymerase chain reaction and Western Blot to evaluate the proliferation and osteogenic differentiation. The results indicated that the proliferation of BMSCs increased at the concentration of 50, 100 ng/ml, got inhibited at 1,000 ng/ml. When Vitamin C was added, the OD for proliferation increased. For ALP staining, we found that BMSCs treated with 50 and 100 ng/ml nicotine showed a higher activity compared with the control, and decreased at the 1,000 ng/ml. Bone morphogenetic protein-2 (BMP-2) expression and the calcium depositions decreased at 100 and 1,000 ng/ml nicotine, while the addition of Vitamin C reversed the down regulation. By real-time PCR, we detected that the mRNA expression of collagen type I (COL-I) and ALP were also increased in 50 and 100 ng/ml nicotine groups (P < 0.05), while reduced at 1,000 ng/ml (P < 0.05). When it came to osteocalcin (OCN), the changes were similar. Taken all together, it is found that nicotine has a two-phase effect on human BMSCs, showing that low level of nicotine could promote the proliferation and osteogenic differentiation while the high level display the opposite effect. Vitamin C could antagonize the inhibitory effect of higher concentration of nicotine partly.

Acute behavioural responses to nicotine and nicotine withdrawal syndrome are modified in GABA(B1) knockout mice.

Nicotine is the main active component of tobacco, and has both acute and chronic pharmacological effects that can contribute to its abuse potential in humans. The aim of the present study was to evaluate a possible role of GABA(B) receptors in acute and chronic responses to nicotine administration, by comparing GABA(B1) knockout mice and their wild-type littermates. In wild-type mice, acute nicotine administration (0.5, 1, 3 and 6 mg/kg, sc) dose-dependently decreased locomotor activity, and induced antinociceptive responses in the tail-immersion and hot-plate tests. In GABA(B1) knockout mice, the hypolocomotive effect was observed only with the highest dose of nicotine, and the antinociceptive responses in both tests were significantly reduced in GABA(B1) knockout mice compared to their wild-type littermate. Additionally, nicotine elicited anxiolytic- (0.05 mg/kg) and anxiogenic-like (0.8 mg/kg) responses in the elevated plus-maze test in wild-type mice, while selectively the anxiolytic-like effect was abolished in GABA(B1) knockout mice. We further investigated nicotine withdrawal in mice chronically treated with nicotine (25 mg/kg/day, sc). Mecamylamine (1 mg/kg, sc) precipitated several somatic signs of nicotine withdrawal in wild-type mice. However, signs of nicotine withdrawal were missing in GABA(B1) knockout mice. Finally, there was a decreased immunoreactivity of Fos-positive nuclei in the bed nucleus of the stria terminalis, basolateral amygdaloid nucleus and hippocampal dentate gyrus in abstinent wild-type but not in GABA(B1) knockout mice. These results reveal an interaction between the GABA(B) system and the neurochemical systems through which nicotine exerts its acute and long-term effects.

The serotonin 2C receptor agonist Ro-60-0175 attenuates effects of nicotine in the five-choice serial reaction time task and in drug discrimination.

RATIONALE: There is evidence that serotonin(2C) (5-HT(2C)) receptors can modulate some behavioural effects of nicotine, but the generality of this action is not known. OBJECTIVE: To analyse the influence of the 5-HT(2C) agonist Ro-60-0175 on responses to nicotine in the five-choice serial reaction time task (5-CSRTT) and on its discriminative stimulus effect; these procedures constitute models for attention-enhancing and subjective effects of nicotine, respectively. MATERIALS AND METHODS: In the 5-CSRTT, rats were trained to obtain food reinforcers by detecting light stimuli and then challenged with Ro-60-0175 (0.3-0.8 mg/kg) and nicotine (0.2 mg/kg). For drug discrimination studies, rats were trained to discriminate nicotine (0.2 mg/kg) from saline in a two-lever procedure using a tandem schedule of food reinforcement. RESULTS: In the 5-CSRTT, nicotine positively influenced most response indices, confirming previous results. Ro-60-0175 increased response latencies and omission errors and reduced anticipatory responding but had little effect on response accuracy; importantly, it counteracted the effects of nicotine on response speed and omission errors. Pentobarbitone (10-14 mg/kg) also slowed performance of the 5-CSRTT but did not weaken the nicotine-induced enhancement of performance. In the drug discrimination procedure, Ro-60-0175 was not generalised with nicotine but shifted the nicotine dose-response curve to the right in a dose-related manner. CONCLUSIONS: The data suggest that selective occupancy of 5-HT(2C) receptors can attenuate some effects of nicotine in the 5-CSRTT and weaken the nicotine discriminative stimulus; these effects cannot be explained by a sedative action of Ro-60-0175.

Nicotine suppression of gustatory responses of neurons in the nucleus of the solitary tract.

This study investigated effects of nicotine applied to the tongue surface on responses of gustatory neurons in the nucleus of the solitary tract (NTS) in rats. In pentobarbital-anesthetized rats, single-unit recordings were made from NTS units responsive to one or more tastants (sucrose, NaCl, citric acid, monosodium glutamate, quinine). Application of nicotine (0.87, 8.7, or 600 mM) excited gustatory NTS units and significantly attenuated NTS unit responses to their preferred tastant in a dose-dependent manner. The depressant effect of nicotine was equivalent regardless of which tastant best excited the NTS unit. Nicotinic excitation of NTS units and depression of their tastant-evoked responses were both significantly attenuated by the nicotinic antagonist mecamylamine, which itself did not excite NTS units. In rats with bilateral trigeminal ganglionectomy, nicotine still excited nearly all NTS units but no longer depressed tastant-evoked responses. Nicotine did not elicit plasma extravasation when applied to the tongue. The results indicate that nicotine directly excites NTS units by gustatory nerves and inhibits their tastant-evoked responses by a nicotinic acetylcholine receptor-mediated excitation of trigeminal afferents that inhibit NTS units centrally.

Chronic caffeine exposure in rats blocks a subsequent nicotine-conditioned taste avoidance in a one-bottle, but not a two-bottle test.

Two experiments were conducted in order to investigate nicotine-conditioned taste avoidance (CTA) following chronic preexposure to caffeine. Rats were given daily intraperitoneal injections of caffeine anhydrous (0, 10, or 30 mg/kg) for 10 or 30 days. Training of the nicotine-CTA began after the last day of caffeine preexposure. On five separate occasions access to a saccharin solution was followed immediately by an injection of 1.2 mg/kg nicotine hydrogen tartrate salt or saline. Nicotine-CTA readily developed in saline-preexposed controls. That is, paired rats drank less saccharin solution than unpaired rats after repeated saccharin-nicotine pairings. A similar pattern of nicotine-CTA was found for rats preexposed to 30 mg/kg caffeine for 10 days. Following 10 days of preexposure to 10 mg/kg caffeine, however, CTA did not develop under standard testing conditions. Thirty days of caffeine preexposure did not affect the development of a nicotine-CTA even though the anorexic effects of caffeine were evident after exposure to 30 mg/kg for this duration. Thus, caffeine exposure appears to weaken acquisition or expression of the conditioned avoidance properties of nicotine. This effect is sensitive to the dose of caffeine and duration of preexposure. Importantly, the pattern of nicotine-CTA does not appear to be due to nonspecific effects of caffeine.

Nitric oxide mediates a therapeutic effect of nicotine in ulcerative colitis.

BACKGROUND: Ulcerative colitis is a condition of nonsmokers in which nicotine is of therapeutic benefit. AIMS: To examine the in vitro effect of nicotine on colonic smooth muscle activity and the role of nitric oxide (NO) as a mediator. METHODS: Nicotine, 1-10 microM, was administered to strips of circular muscle from the distal sigmoid colon of 9 patients with active ulcerative colitis and 18 with colorectal cancer. The effect of electrical field stimulation (EFS) was examined before nicotine was added. Finally L-NAME, a NO synthetase inhibitor, was added before nicotine was administered again. RESULTS: Muscle strips developed similar spontaneous resting tone. In response to EFS, ulcerative colitis tissue developed lower tensions than the controls. Nicotine significantly reduced the resting tone and peak tension after EFS, with a greater effect in controls. With L-NAME, peak tensions were increased more in ulcerative colitis than controls, and nicotine produced a much smaller reduction. CONCLUSIONS: Nicotine reduces circular muscle activity, predominantly through the release of nitric oxide-this appears to be 'up-regulated' in active ulcerative colitis. These findings may explain some of the therapeutic benefit from nicotine (and smoking) in ulcerative colitis and may account for the colonic motor dysfunction in active disease.

Nicotine-induced excitation of locus coeruleus neurons is blocked by elevated levels of endogenous kynurenic acid.

The present electrophysiological study shows that manipulation with endogenous brain kynurenic acid (KYNA) is able to affect the response of central noradrenergic neurons to nicotine. Previous studies have shown that systemically administered nicotine in low doses is associated with a marked, but short-lasting increase in the firing rate of rat noradrenergic neurons in the locus coeruleus (LC). This action of nicotine is of peripheral origin and finally mediated via a release of glutamate within the LC. KYNA is an endogenous glutamate receptor antagonist, which shows an uneven distribution in human brain. Previous studies have shown that a potent inhibitor of kynurenine 3-hydroxylase, PNU 156561A, is able to dose-dependently increase the levels of KYNA in brain. Anesthetized rats were given PNU 156561A in a dose that caused a 5-fold increase in brain KYNA levels after 3-6 hours (40 mg/kg, i.v. ). This treatment was found to abolish the increase in firing rate of LC neurons induced by nicotine (25-200 microg/kg, i.v.). The results of the present study show that an increased concentration of endogenous brain KYNA is able to inhibit the activation of central noradrenergic neurons by nicotine. In addition, our results highlight the role of endogenous KYNA in brain as a potentially important modulator of brain glutamatergic responses.

Inhibitory effect of propofol on sympathetic neurotransmission results in changes of plasma neuropeptide Y in rats.

1. The effects of propofol on sympathetic neurotransmission and changes of plasma level of neuropeptide Y-like immunoreactivity (NPY-ir) were investigated in rats. 2. Intraperitoneal injection of propofol into rats lowered the systemic blood pressure and plasma NPY-ir in a dose-dependent manner. 3. Decrease of plasma NPY-ir induced by propofol was not modified in adrenalectomized rats. In the activation of adrenergic neurotransmission by a ganglionic nicotinic agonist, elevation of plasma NPY-ir was also reduced by propofol indicating the direct effect on peripheral adrenergic nerve terminals. 4. Plasma level of NPY-ir reversed in parallel with the recovery of anaesthesia induced by propofol. After an intracerebroventricular injection of propofol into the rats, both the lowering of plasma NPY-ir and the induction of anaesthesia were observed. Thus, a central nervous system effect of propofol can also be considered in its effect on plasma NPY-ir. 5. The data suggest that propofol has the ability to lower plasma NPY-ir in rats through an inhibition of adrenergic neurotransmission via central nervous pathway and/or peripheral nerve terminal blockade.

Nicotinic antagonist-produced frequency-dependent changes in acetylcholine release from rat motor nerve terminals.

1. The frequency (0.5-150 Hz) and calcium dependence (0.5-2.0 mM) of the effects of the nicotinic antagonist tubocurarine (0.2 microM) on acetylcholine (ACh) liberation from motor nerve terminals has been examined using binomial analysis of quantal transmitter release. 2. At an extracellular calcium ion concentration ([Ca2+]o) of 2.0 mM, tubocurarine produced a decrease in the endplate current (EPC) quantal content of approximately 30% at high frequencies of motor nerve stimulation (50-150 Hz). In contrast, at low frequencies of stimulation (0.5-1.0 Hz), tubocurarine enhanced the EPC quantal content by approximately 20%. 3. The enhancement of EPC quantal content produced by tubocurarine at low frequencies of motor nerve stimulation was [Ca2+]o dependent, being abolished when [Ca2+]o was lowered from 2.0 to 0.5 mM. In contrast, the decrease in quantal content produced by tubocurarine at high frequencies of motor nerve stimulation was independent of [Ca2+]o, being approximately 30% at all calcium ion concentrations studied. 4. In direct contrast to tubocurarine, the nicotinic antagonist vecuronium (1.0 microM) produced no increase in EPC quantal content at low frequencies of nerve stimulation. However, at high frequencies of nerve stimulation it decreased EPC quantal content to a similar extent to 0.2 microM tubocurarine. The frequency-dependent decrease in EPC quantal content produced by 1.0 microM vecuronium in 2.0 mM [Ca2+]o was very similar to that seen with 0.2 microM tubocurarine in 0.5 mM [Ca2+]o. 5. Binomial analysis revealed that all the changes in EPC quantal content associated with both nicotinic antagonists were due to changes in the size of the pool of quanta in the nerve terminal available for immediate release with no effect on the probability of release of an individual quantum. 6. The results are interpreted in terms of two separately identifiable prejunctional actions of the nicotinic antagonists, both involving an action at nicotinic ACh receptors situated on the motor nerve terminal. Thus, at high frequencies of motor nerve stimulation tubocurarine and vecuronium produce a [Ca2+]o-independent decrease in ACh release, probably through an inhibitory action on a positive-feedback prejunctional nicotinic autoreceptor closely related to the muscle-type nicotinic ACh autoreceptor. However, at low frequencies of motor nerve stimulation we suggest that tubocurarine, but not vecuronium, produces a [Ca2+]o-dependent increase in ACh release through an action at a negative-feedback prejunctional neuronal-type nicotinic ACh autoreceptor.

Potentiation by tonic A2a-adenosine receptor activation of CGRP-facilitated [3H]-ACh release from rat motor nerve endings.

1. The effect of calcitonin gene-related peptide (CGRP) on [3H]-acetylcholine ([3H]-ACh) release from motor nerve endings and its interaction with presynaptic facilitatory A2a-adenosine and nicotinic acetylcholine receptors was studied on rat phrenic nerve-hemidiaphragm preparations loaded with [3H]-choline. 2. CGRP (100-400 nM) increased electrically evoked [3H]-ACh release from phrenic nerve endings in a concentration-dependent manner. 3. The magnitude of CGRP excitation increased with the increase of the stimulation pulse duration from 40 microseconds to 1 ms, keeping the frequency, the amplitude and the train length constants. With 1 ms pulses, the evoked [3H]-ACh release was more intense than with 40 microseconds pulse duration. 4. Both the nicotinic acetylcholine receptor agonist, 1,1-dimethyl-4-phenylpiperazinium, and the A2a adenosine receptor agonist, CGS 21680C, increased evoked [3H]-ACh release, but only CGS 21680C potentiated the facilitatory effect of CGRP. This potentiation was prevented by the A2a adenosine receptor antagonist, PD 115,199. 5. Adenosine deaminase prevented the excitatory effect of CGRP (400 nM) on [3H]-ACh release. This effect was reversed by the non-hydrolysable A2a-adenosine receptor agonist, CGS 21680C. 6. The nicotinic antagonist, tubocurarine, did not significantly change, whereas the A2-adenosine receptor antagonist, PD 115,199, blocked the CGRP facilitation. The A1-adenosine receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine, potentiated the CGRP excitatory effect. 7. The results suggest that the facilitatory effect of CGRP on evoked [3H]-ACh release from rat phrenic motor nerve endings depends on the presence of endogenous adenosine which tonically activates A2a-adenosine receptors. Since both CGRP and A2a-adenosine receptors are positively coupled to the adenylate cyclase/cyclic AMP system, cooperation between these receptors might occur at the second messenger transduction system level.

Memory modulation with peripherally acting cholinergic drugs.

Physostigmine (PHYSO), in doses as low as 0.003 mg/kg IP, antagonized scopolamine (SCOP, 3 mg/kg) induced amnesia of step-through passive avoidance in mice. The peripherally acting acetylcholinesterase (AChE) inhibitor neostigmine (NEO) was also found to reliably, though less strongly, antagonize the SCOP induced amnesia at a dose of 0.03 mg/kg. The NEO antagonism of the SCOP amnesia could be reversed with SCOP (0.3, 1, and 3 mg/kg) and mecamylamine (MECA, 1, 3, and 10 mg/kg), muscarinic and nicotinic antagonists, respectively, which are active both peripherally and centrally, as well as with M-SCOP (0.3 and 1 mg/kg) and hexamethonium (HEX, 1 and 3 mg/kg), muscarinic and nicotinic antagonists, respectively, which are active only in the periphery. In contrast to the ability of these four compounds to attenuate the SCOP amnesia, only the centrally acting compounds SCOP (3 mg/kg) and MECA (10 mg/kg) induced an amnesia when administered alone. These findings suggest that the induction of amnesia of passive avoidance involves central cholinergic systems, whereas the NEO, and possibly PHYSO, reversal of the SCOP induced amnesia is mediated peripherally by both muscarinic and nicotinic receptors. It is hypothesized that the release of adrenal catecholamines, the influence of which on memory processes is well known, and secondarily glucose, may be responsible for the NEO antagonism of the SCOP amnesia.

1,3-Di(2-tolyl)guanidine blocks nicotinic response in guinea pig myenteric neurons.

Ditolylguanidine (DTG) is a ligand which binds with high affinity to neuronal sigma receptors. Activation of sigma receptors inhibits the release of acetylcholine (ACh) from guinea pig ileum myenteric plexus preparations. A study was therefore undertaken to investigate the action of sigma receptor ligands on single neurons. Nicotinic responses to locally applied ACh onto single neurons of the guinea pig ileum myenteric plexus were studied using intracellular recording techniques. DTG and (+)-SKF10047 (N-allylnormetazocine) produced a concentration-dependent suppression of the depolarization of enteric neurons evoked by ionophoresis of ACh. The EC50 values for DTG and (+)-SKF10047 were 4.7 and 3.8 microM, respectively, and were similar to that for hexamethonium (3.2 microM). The inhibition of the ACh-depolarization was not mediated at sigma receptors because (-)SKF10047 and Bridge-DPG (2-imino-1,3H-dibenzo[d,f]-[1,3]-diazepine), which are inactive at sigma receptors, were as potent as DTG and (+)-SKF10047. DTG and hexamethonium (each at 1 microM) were more effective blockers of ACh-induced inward currents at a holding potential of -100 mV than at -40 mV. This voltage dependence is consistent with a channel blocking mechanism. DTG (10 microM) did not affect the depolarization (mediated by 5-HT3 receptors) induced by pressure application of 5-HT onto single neurons. DTG and Bridge-DPG inhibited contractures of the longitudinal muscle-myenteric plexus preparation elicited by dimethylphenylpiperazinium noncompetitively (EC50 values were 8.0 and 12.3 microM, respectively) whereas DTG but not Bridge-DPG inhibited 5-HT-induced contractions of the longitudinal muscle-myenteric plexus noncompetitively.(ABSTRACT TRUNCATED AT 250 WORDS)

Frequency dependent inhibition of the nicotinic transmission by serotonin in vesical pelvic ganglia of the rabbit.

Intracellular recordings were made from neurons in rabbit vesical pelvic ganglia (VPG), in vitro. Increasing the frequency of preganglionic-nerve stimulations from 0.1-1 Hz to 10-20 Hz facilitated fast excitatory postsynaptic potentials (EPSPs), resulting in a generation of action potentials. Acetylcholine-induced response was not altered during the facilitation of the fast EPSP. Serotonin blocked action potentials elicited by preganglionic-nerve stimulations at 0.1 Hz, while it caused no blockade at 10 Hz. Serotonin may potentiate the feature of high-pass filter in transmission of VPG. Immunohistochemical study demonstrated serotonin-like varicose terminals in rabbit VPG.

Neurotransmission in the frog retina: possible physiological and histological correlations.

In the frog retina, extracellular recordings of transient ganglion cells have shown that the inhibitory surround of the receptive field of these cells was mediated by gamma-aminobutyric acid and acetylcholine (through the nicotinic receptors). Histoautoradiographic and immunocytochemical studies for the two respectively have shown that these neurotransmitters can act through horizontal and amacrine cells. The separation of the ON and OFF channels mediated by glutamate at the bipolar cell level may also be obtained by glycine and/or acetylcholine (through muscarinic receptors). Respective histoautoradiographic and immunocytochemical studies indicate that these neurotransmitters act at the amacrine cell level. These data are consistent with the functional separation of spatial and temporal organization of retinal information, with horizontal cells especially responsible for the spatial organization of the ganglion cell responses and amacrine cells involved in both spatial and temporal organization of the responses.

Train-of-four as an index of neuromuscular block in cats: changes induced by atropine.

1. The present study reevaluates the effect of atropine on the rate of recovery from tetanic fade caused by intraarterial administration of neostigmine or antinicotinic agents in cat anterior tibial muscle preparations submitted to a train-of-four (TOF) pattern of nerve stimulation. The study also compares the sensitivity of the TOF and tetanic responses as indices of residual nondepolarizing block. 2. Neostigmine, hexamethonium and d-tubocurarine all produced short-lived TOF fade. Both single and TOF twitches were increased by neostigmine and depressed by the antinicotinic agents. 3. Prior administration of atropine reduced the TOF fade induced by the antinicotinic drugs but potentiated that caused by anticholinesterase drugs. 4. These results indicate that TOF fade is not the most sensitive index for studying neuromuscular blockade when drugs other than neuromuscular blockers are also present.

Antagonism by propranolol of the ganglion stimulant action of 5-hydroxytryptamine.

The effects of racemic propranolol and its constituent isomers were studied on ganglionic stimulation produced in situ by close arterial injection of 5-HT and DMPP to the superior cervical ganglion. Ganglion stimulation was recorded in terms of the resultant contraction of the nictitating membrane. d,l-Propranolol caused a biphasic antagonism of the ganglion stimulant effect of 5-HT. At low doses, 0.5-10 mug, the antagonism was surmountable by increasing the amount of 5-HT. The l-isomer (0.2-4 mug) but not d-propranolol also caused antagonism. At higher doses, 0.1-5 mg, both d,l- and d-propranolol caused a second type of blockade of 5-HT which was not surmountable and resembled that seen with procaine. The ganglion stimulant effects of DMPP and acetylcholine were only antagonised by the higher doses of d- and d,l-propranolol. d,l-Propranolol did not reduce the direct stimulation by 5-HT on the muscle of the nictitating membrane.