Nicotine Accumulation Research Articles

Nicotine and related alkaloids accumulation in constitutive salicylic acid producing tobacco plants

Large variations in nicotine and related alkaloids accumulations were found in the leaves of constitutive salicylic acid producing (CSA)-tobacco and non transgenic tobacco plants. Statistical analysis showed that there was no significant correlation between salicylic acid (SA) levels and alkaloid accumulation in CSA-tobacco plants. The accumulation of nicotine after wounding and tobacco mosaic virus (TMV)-inoculation of leaves were measured as well. The accumulation of nicotine in the leaves of CSA-tobacco was suppressed after TMV-inoculation, whereas wounding enhanced nicotine accumulation. There was no effect of TMV-inoculation on nicotine accumulation in non-transgenic tobacco but wounding also increased nicotine accumulation.

Involvement of Specific Transport System of Renal Basolateral Membranes in Distribution of Nicotine in Rats

We measured the nicotine concentrations in tissues after a bolus i.v. administration of [(3)H]nicotine to rats to characterize the distribution profile of nicotine. The kidney showed the greatest distribution of nicotine compared to other tissues including liver, lung, heart, brain, and intestine. We also performed an HPLC assay for the determination of nicotine and its major metabolite, cotinine, and found that cotinine was negligible in the distribution of almost all tissues, except for the kidney and lung. In the kidney, cotinine was detected at a lower level than nicotine, while cotinine tended to be distributed in the lung compared to nicotine. [(3)H]Nicotine was accumulated in renal slices in a concentration dependent fashion, suggesting that the nicotine uptake in the renal tubules could be mediated by a specific transport system. Unlabeled nicotine, cotinine, and quinidine showed potent inhibitory effects on [(3)H]nicotine uptake by renal slices. In contrast, tetraethylammonium (TEA), cimetidine, and N(1)-methylnicotinamide (NMN), which were substrates of renal organic cation transporters, had no effects on the uptake. These findings suggested that a specific transporter was involved in nicotine transport at the basolateral membranes of rat renal tubules, which could mediate the high accumulation of nicotine from blood into the kidney.

Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata. IV. Insect-Induced ethylene reduces jasmonate-induced nicotine accumulation by regulating putrescine N-methyltransferase transcripts.

Attack by the specialist herbivore, Manduca sexta, on its native host Nicotiana attenuata Torr. ex Wats. produces a dramatic ethylene release, a jasmonate burst, and a suppression of the nicotine accumulation that results from careful simulations of the herbivore's damage. Methyl-jasmonate (MeJA) treatment induces nicotine biosynthesis. However, this induction can be suppressed by ethylene as pretreatment of plants with 1-methylcyclopropene (1-MCP), a competitive inhibitor of ethylene receptors, restores the full MeJA-induced nicotine response in herbivore attacked plants (J. Kahl, D.H. Siemens, R.J. Aerts, R. Gäbler, F. Kühnemann, C.A. Preston, I.T. Baldwin [2000] Planta 210: 336-342). To understand whether this herbivore-induced signal cross-talk occurs at the level of transcript accumulation, we cloned the putrescine methyltransferase genes (NaPMT1 and NaPMT2) of N. attenuata, which are thought to represent the rate limiting step in nicotine biosynthesis, and measured transcript accumulations by northern analysis after various jasmonate, 1-MCP, ethephon, and herbivory treatments. Transcripts of both root putrescine N-methyltransferase (PMT) genes and nicotine accumulation increased dramatically within 10 h of shoot MeJA treatment and immediately after root treatments. Root ethephon treatments suppressed this response, which could be reversed by 1-MCP pretreatment. Moreover, 1-MCP pretreatment dramatically amplified the transcript accumulation resulting from both wounding and M. sexta herbivory. We conclude that attack from this nicotine-tolerant specialist insect causes N. attenuata to produce ethylene, which directly suppresses the nitrogen-intensive biosynthesis of nicotine.

Herbivore-induced ethylene burst reduces fitness costs of jasmonate- and oral secretion-induced defenses in Nicotiana attenuata

Specialist herbivores are known to alter their host's wound-induced responses but the beneficiaries of these alterations are unknown. Nicotiana attenuata plants release a burst of ethylene specifically in response to feeding by Manduca sexta larvae, which is known to suppress wound- and methyl jasmonate (MeJA)-inducible nicotine accumulation. The ethylene burst may be a mechanism by which M. sexta larvae feed "stealthily" on their host plants or, alternatively, it may allow the plant to optimize its defense response against this specialist herbivore by reducing costs of induction. We examined the impact of the ethylene burst on defense-related fitness costs that are readily observed when plants are treated with MeJA and grown in competition with untreated plants. We elicited nicotine induction (with MeJA), the ethylene burst (with the ethylene releasing compound, ethephon) and inhibited the plant's ability to perceive ethylene (with applications of an antagonist of ethylene receptors, 1-methylcyclopropene, 1-MCP). By simultaneously applying MeJA and ethephon we mimicked the plant's hormonal response to larval attack. We hypothesized that if the ethylene burst benefited the plant, the fitness costs of MeJA induction should be reduced by ethephon and restored if the plants were additionally treated with 1-MCP. In a second experiment, we applied larval oral secretion (OS) to elicit endogenous hormone production and predicted that the 1-MCP treatment should reduce the fitness of OS-treated plants. Our measures of plant fitness, namely the rate of stalk elongation and lifetime capsule production, supported these predictions. We conclude that the ethylene burst elicited by this specialist herbivore can reduce MeJA-induced fitness costs and increase the competitive strength of OS-treated plants. Suppressed nicotine production is likely to contribute to, but is not sufficient to explain, the observed fitness outcomes. The intensity of intra-specific competition and herbivore attack will likely determine the adaptive value of the M. sexta-elicited ethylene response.

Allocation of nitrogen to an inducible defense and seed production in Nicotiana attenuata.

Resource-based tradeoffs in the allocation of a limiting resource are commonly invoked to explain negative correlations between growth and defense in plants, but critical examinations of these tradeoffs are lacking. To rigorously quantify tradeoffs in a common currency, we grew Nicotiana attenuata plants in individual hydroponic chambers, induced nicotine production by treating roots with methyl jasmonate (MJ) and standardized leaf puncturing, and used 15N to determine whether nitrogen-based tradeoffs among nicotine production, growth, and seed production could be detected. Plants were treated with a range of MJ quantities (5, 45 or 250 μg plant-1) to effect a physiologically realistic range of changes in endogenous jasmonic acid levels and increases in nicotine production and accumulation; MJ treatments were applied to the roots to target JA-induced nicotine production, since nicotine biosynthesis is restricted to the roots. Leaf puncturing and 5 μg MJ treatments increased de novo nicotine synthesis and whole-plant (WP) nicotine pools by 93 and 66%, while 250 μg MJ treatments increased these values 3.1 and 2.5-fold. At these high rates of nicotine production, plants incorporated 5.7% of current nitrogen uptake and 6.0% of their WP nitrogen pools into nicotine. The 15N-labeled nicotine pools were stable or increased for the duration of vegetative growth, indicating that the N-nicotine was not metabolized and re-used for growth. Plants with elevated nicotine production grew more slowly and the differences in plant biomass gain between MJ-treated plants and controls were linearly related to the differences in nicotine accumulation. Despite the reductions in rosette-stage growth associated with nicotine production, estimates of lifetime fitness (cumulative lifetime seed production, mass/seed, seed viability) were not affected by any treatment. Only two treatments (leaf puncturing and 250 μg MJ) increased the allocations of 15N acquired at the time of induction to seed production. On average, plants used only 14.9% of their WP nitrogen pool for seed production, indicating that either the nitrogen requirements for seed production or the reproductive effort of these hydroponically-grown plants are low. To determine if seed production is strongly influenced by the amount of vegetative biomass attained before reproduction, the experiment was repeated with plants that had 44% of their leaf area (or 29% of their WP biomass) removed before MJ treatments with a removal technique that minimized the nicotine response. MJ treatments of these plants dramatically increased nicotine production and accumulation, but these plants also suffered no measurable fitness consequences from either the leaf removal or MJ treatments. We conclude that when N. attenuata plants are grown in these individual hydroponic chambers, their allocation to reproduction is sufficiently buffered to obscure the large increases in nitrogen allocations to an inducible defense. To determine whether soil-grown plants are similarly buffered, we grew two genotypes of plants in the high-nutrient soil from a 1-year-old burn in a piñyon-juniper forest (the plants' natural habitat) and in low-nutrient soil from an adjacent unburned area, and induced nicotine production in half of the plants with a 500 μg root MJ treatment. Plants grown in burned soils had an estimated lifetime fitness that was on average 2.8-fold greater than that of plants grown in unburned soils. MJ treatment reduced fitness estimates by 43% and 71% in the burned and unburned soils, respectively. We conclude that while hydroponic culture allows one to rigorously quantitate nitrogen allocation to growth, reproduction and defense, the allocation patterns of plants grown in hydroponic culture differ from those of plants grown in soil. Under hydroponic conditions, plants have low reproductive allocations and reproductive-defense tradeoffs are not detected. Reproductive-defense tradeoffs are readily discernible in soil-grown plants, but under these growing conditions, the nitrogen-basis for the tradeoff is difficult to quantify.

Fire, nitrogen, and defensive plasticity in Nicotiana attenuata.

Nicotiana attenuata is a post-fire annual that utilizes jasmonate-inducible nicotine production as an inducible chemical defense which, in turn, can utilize 6% of a plant' s nitrogen budget and be costly to seed production. We characterize the nitrogen pools of burned soils in the plant' s native environment (piñyon-juniper woodlands) and examine how variation in nitrogen source and supply rate influence the patterns of allocation to growth and inducible and constitutive nicotine production. Available soil nitrogen increases dramatically (40-fold) immediately after a fire and consists principally of ammonia which is subsequently oxidized to nitrate during post-fire succession. We simulate these changes in nitrogen availability in hydroponic culture and use allometric techniques to characterize changes in allocation. In two experiments, we alter (1) nitrate supply rates 8-fold and (2) the ratio of ammonia:nitrate under consistent nitrogen supply rates. In both experiments, we increase the allocation to nicotine by treating roots with methyl jasmonate (MJ), the methyl ester of the plant' s internal wound signal, jasmonic acid, which increases nicotine production in the roots after shoot herbivory. MJ treatments decrease whole plant (WP) growth, increase root:shoot ratio, and increase WP nicotine pools in all nitrogen environments. Overall, source and supply rate of nitrogen have no effect on either the constitutive or induced allometric relationships of nicotine accumulation and growth. This remarkable homeostasis in allocation patterns contradicts a key prediction of carbon nutrient (C/N) theory. With 15N-pulse-chase techniques, we demonstrate that plants preferentially utilize ammonia for nicotine production over nitrate when both nitrogen sources are available. This preferential use of ammonia may allow N. attenuata to reduce the biochemical costs of producing nicotine in the post-fire environment.

Effect of Regenerated Roots and Shoots on Nicotine Production in Tobacco Tissue Culture

The accumulation of nicotine in regenerated shoot and root tissues of Nicotiana tabacum L. cv MD 872 grown from callus culture has been examined by high pressure liquid chromatography (HPLC) procedures. The presence of roots on callus tissue dramatically increased the accumulation of nicotine in both callus without shoots and in callus which had regenerated shoots. When shoots were regenerated, the concentration of nicotine that accumulated in callus decreased whether roots were present or not. When varying concentrations of the nicotine precursors ornithine and putrescine were fed to callus derived tissue with and without regenerated roots, the rooted tissue was able to take up the precursors and accumulate nicotine in the shoots in amounts greater than controls. The half-life turnover rate for ’H-nicotine absorbed by the roots and transported into shoots was approximately 48 hours. These experiments indicate that although the root is the major site of synthesis of nicotine in tobacco, the accumulation of nicotine in various parts of the plant is regulated by the interaction of both root and shoot tissue.

Down-Regulation of the Plasmalemma H+ Pump Activity by Nicotine-Induced Intracellular Alkalinization: a Balance between Base Accumulation, Biochemical pH-Stat Response and Intracellular pH Increase

H+ efflux was also evident in the presence of K + in the external medium, whereas no nicotine effect on net H + efflux was detected in the absence of K + (in spite of the larger accumulation of nicotine in the tissue) in agreement with a down-regulation of the activity of the K + -dependent plasmalemma H + -ATPase by alkaline intracellular pH values. The increase in vacuolar pH resulting from nicotine accumulation was small compared to the base load calculated from the vacuolar buffer capacity and the intracellular dissociation of nicotine. Conversely, the nicotine-induced increase in cytoplasmic pH was considerably larger than expected on the basis of the cytoplasmic buffer capacity and of the theoretical accumulation of nicotine in the experimental conditions adopted. A balance sheet between nicotine accumulation, intracellular alkalinization and malate system response was drawn up, and the seeming discrepancies observed were discussed.

Within-plant relationships among wounding, jasmonic acid, and nicotine implications for defence in Nicotiana sylvestris.

In Nicotiana sylvestris Spegazzini and Comes (Solanaceae), we examined the relationships among wounding, endogenous leaf jasmonic acid (JA) pools, and whole-plain (WP) nicotine accumulation over a range of wounding intensities and spatial distributions, in order to explore optimal defence (OD) theory predictions. We quantitatively wounded one or four leaves and then quantified: (1) JA in damaged and undamaged leaves 90 min after wounding; (2) WP nicotine concentration after 5 d (the times when JA and nicotine attain the largest wound-induced concentrations). We find: (1) statistically significant, positive relationships on a leaf-by-leaf basis among the number of leaf punctures, endogenous leaf JA, and WP nicotine accumulation; (2) that young, undamaged leaves have a higher concentration of JA than do older, undamaged leaves, and produce a greater amount of JA per puncture than older leaves, but that all leaves have the same JA content (ng JA per leaf); and (3) that a damaged leaf produces less JA when other leaves in the canopy are wounded than when it is the onh wounded leaf in the canopy, but that when it is the only wounded leaf, the phylotactically adjacent, undamaged leaves do not increase their JA concentrations. The observation that younger leaves produce more JA per puncture than do older leaves is consistent with OD theory predictions. The observation that a small amount of damage localized to a single leaf is as effective as a larger amount of damage dispersed across the canopy in increasing leaf JA and WP nicotine accumulation shows the plant's ability to differentiate between dispersed and localized damage. Because the quantity of JA in a wounded leaf 90 min after wounding is a reliable indicator of the WP nicotine response to wounding, this trait provides insight into how plants integrate information about environmental insults and tailor their defence responses.

Accumulation of nicotine in human hair during long-term controlled exposure to a low concentration of nicotine vapour.

Hair from 5 subjects were exposed in dynamic exposure chambers to air nicotine vapour for 72 hr or 12 months at concentrations of 200 or 5 micrograms/m3, respectively. Nicotine in the chamber air and human hair was determined by GC/ MS. A linear accumulation of nicotine in hair was found with time for all hairs during the long-term, low concentration exposure, with individual hair nicotine uptake rate constants ranging from 0.70 to 3.75 x 10(-3) m3/g x hr. The corresponding hair nicotine uptake rate constants during short-term, high concentration exposure, were significantly higher, ranging from 1.35 to 15.11 x 10(-3) m3/g x hr, showing, however, a highly significant linear correlation with the individual long-term exposure rate constants, r2 = 0.9961. It is indicated that long-term hair nicotine uptake rate constants calculated from controlled exposure experiments with pure nicotine vapour are adequate for estimation of individual long-term hair accumulation of nicotine from environmental tobacco smoke even at variable and intermittent exposure. Although higher than the long-term uptake rate constants, the short-term uptake rate constants seem well fitted for a differentiation between different types of hair in their ability to adsorb nicotine also during long-term exposures. The short-term uptake rate constants might also be useful parameters for establishing a reliable cut-off limit in the hair concentration of nicotine between smokers and non-smokers which otherwise seems to be overlapping.

Quantification, correlations and manipulations of wound-induced changes in jasmonic acid and nicotine in Nicotiana sylvestris

Jasmonic acid (JA) is thought to be part of a signal-transduction pathway which dramatically increases de-novo nicotine synthesis in the roots and increases whole-plant (WP) nicotine pools in response to the wounding of the leaves in Nicotiana sylvestrisSpegazzini and Comes (Solanaceae). We report the synthesis of a doubly labeled JA ([1, 2-13C]JA) and use it as an internal standard to quantify by gas chromatography-mass spectrometry the changes in root and shoot JA pools in plants subjected to differing amounts of standardized leaf wounding. Wounding increased JA pools 10-fold locally in damaged leaves within 90 min and systemically in the roots (3.5-fold) 180 min after wounding. If JA functions as an intermediary between stimulus and response, quantitative relationships among the stimulus, JA, and the response should exist. To examine these relationships, we varied the number of punctures in four leaves and quantified both the resulting JA in damaged leaves after 90 min and the resulting WP nicotine concentration after 5 d. We found statistically significant, positive relationships among number of leaf punctures, endogenous JA, and WP nicotine accumulation. We used two inhibitors of wound-induced nicotine production, methyl salicylate and indole-3-acetic acid, to manipulate the relationships between wound-induced changes in JA and WP nicotine accumulation. Since wounding and the response to wounding occur in widely separated tissues, we applied inhibitors to different plant parts to examine their effects on the local and systemic components of this response. In all experiments, inhibition of the wound-induced increase in leaf JA 90 min after wounding was associated with the inhibition of the nicotine response 5 d after wounding. We conclude that wound-induced increases in leaf JA are an important component of this long-distance signal-transduction pathway.

Transdermally administered nicotine accumulates in gastric juice.

Transdermal nicotine patches (Nicorette 15 mg.16 h-1) were administered to 7 healthy volunteers. Nicotine concentrations in gastric juice were monitored for 8 h via a naso-gastric tube and so was nicotine in saliva and plasma. Nicotine accumulated in gastric juice, the average concentration being 60.6-times higher than in plasma. In saliva, too, the concentration was higher than in plasma, the average ratio being 10.5. These results strongly suggested ion-trapping of nicotine base in the acidic gastric juice and possibly also in the acinar cells, followed by active secretion. It is hypothesised that accumulation in saliva occurs via a similar mechanism. Pretreatment with omeprazole did not increase the pH to a sufficiently high degree to test the hypothesis that the accumulation of nicotine in gastric juice was pH dependent. Transdermal administration of nicotine produced a high intragastric concentration. The clinical consequence of this effect of long-term nicotine replacement therapy during smoking cessation is unclear.

Pharmacokinetics of a 16-Hour Transdermal Nicotine Patch

A nicotine transdermal delivery system (NTDS) designed to deliver 15 mg/16 hours (Nicotrol®) was applied to 40 healthy smoking volunteers in 3 separate studies: A single administration to different body sites, a multiple-dose study with 16-hour repeated application for 6 days, and a single-dose study with a 16-hour versus a 24-hour application period. The resulting plasma nicotine concentration profiles were similar after application to the arm or the hip with maximum nicotine concentrations of approximately 13 µg/L attained after 8 to 10 hours. The nicotine half-life after removal of the patch was approximately 4 hours. When applying the NTDS daily for a 16-hour period, nicotine stored in a shallow skin compartment was emptied during the nonapplication night-time period, and the repeat-dose study over 6 days showed no accumulation of plasma nicotine. Compared with an average nicotine intake of 15 mg/16 hours when applying NTDS according to recommendation, wearing it for 24 hours resulted in an additional nicotine release of only 2 to 3mg. This profile of nicotine release lends support to the idea of Nicotrol® being a daytime (16-hour) delivery system. Pharmacokinetic simulation indicated that the transdermal system itself determines the flux so an increase in cutaneous blood flow would have no major influence on the nicotine plasma concentration-time profile.

Allometric Limits to the Induced Accumulation of Nicotine in Native Tobacco

Abstract The induced defenses of plants are, by definition, examples of adaptive phenotypic plasticity; however, they have rarely been considered as such due largely to the difficulty of defining the character states of these dynamic chemical responses. Nicotine functions as an inducible defense in Nicotiana sylvestris and is one of the few induced defenses which is understood on a whole‐plant basis. Nicotine biosynthesis is restricted to the roots and increases after wounding or herbivore attack to the shoot. Newly synthesized nicotine is transported throughout the plant; induced plants are better protected from subsequent herbivore attack in laboratory trials. Root nicotine production is activated by an endogenously‐produced signal, jasmonic acid (JA), which is synthesized in response to leaf damage and either directly through transport or indirectly via another, as yet unknown signal, increases JA pools and nicotine biosynthesis in the roots. The addition of JA or its methyl ester (MJ) to plants stimulates nicotine production in the same way that leaf damage does. In this study I use MJ to elicit the induced defense without wounding and describe a new character state for this induced defense: the allometrically determined setpoint. Plants were induced with 45 μg of MJ, once or in different combinations of 2 or 3 times during a 21‐day period of vegetative growth, allowed 7 days between elicitations for the response to relax, and harvested during one of 4 harvests in order to quantify biomass and whole‐plant nicotine pools. For plants induced only once, the largest induced change in whole‐plant nicotine pools occurred during the second induction period, when elicitation increased nicotine pools by 5.99 mg, approximately 87% more than either the first or third induction periods. However, plants induced once had the same whole‐plant nicotine pools at the end of the experiment regardless of whether they were induced during the highly responsive second period or during the less responsive first and second induction periods. Sequential elicitations consistently increased nicotine pools, but after 3 inductions, the nicotine pools were not significantly higher than those in plants which had been induced twice; plants can only increase their nicotine pools in response to 2 inductions when grown under these conditions. These results suggest that this plant has allometrically‐determined setpoints for this induced defense which are determined by the number of times a plant's wound‐signal cascade is stimulated, rather than by the quantity of nicotine synthesized during an induction episode.

Effects of octadecanoid metabolites and inhibitors on induced nicotine accumulation inNicotiana sylvestris

We examined the effects of inhibitors of the octadecanoid pathway (n-propyl gallate, acetosalicylic acid, salicylhydroxamic acid, methyl salicylate, and antipyrine) on wound- and jasmonate-induced nicotine accumulation and compared the nicotine-inducing ability of exogeneous additions of linolenic acid (18:3) and its methyl ester, linoleic acid (18:2), abscisic acid, traumatic acid, and methyl dihydrojasmonate to the nicotine-inducing ability of exogenous additions of methyl jasmonate (MJ). The first four of these inhibitors significantly reduced wound-induced nicotine accumulation when applied in a lanolin paste to wounded tissues immediately after wounding at concentrations of 89-90µg/plant. When methyl salicylate and propyl gallate were mixed individually with MJ, neither inhibited MJ-induced nicotine synthesis, which suggests that the inhibitors block jasmonate synthesis or release from stored pools and not its effects. Linolenic acid or its methyl ester applied to undamaged plants or damaged plants (to either damaged or undamaged leaves) or to the roots of hydroponically growing plants did not induce nicotine accumulation or increase nicotine accumulation above levels found in damaged plants. Similarly, traumatic acid, linoleic acid, and abscisic acid did not induce nicotine accumulations. Methyl dihydrojasmonate, which is biosynthetically derived from linoleic acid, had 12-56% of the nicotine-inducing acitivity of MJ when added to the roots of hydroponically grown plants. The signal transduction pathway mediating wound-induced nicotine production therefore shares many features of the pathway eliciting wound-induced proteinase inhibitor production but differs in not being regulated at the lipase step in jasmonic acid production and not being responsive to abscisic acid.

Immunological "Memory" in the Induced Accumulation of Nicotine in Wild Tobacco

Nicotine, an inducible defense in Nicotiana sylvestris, is produced in the roots and transported throughout the plant after leaf damage. Root nicotine production is activated by an endogenously produced signal, jasmonic acid (JA), which is synthesized in response to leaf damage and either directly through transport or indirectly via another signal, increases JA pools in the roots. The addition of JA or its methyl ester (MJ) to the roots of hydroponically grown plants stimulates nicotine production in the same way that leaf damage does. In this paper we use MJ to stimulate the induced defense without damaging plants in order to examine the hypothesis that these plants have an immunological memory of prior inductions. Such a memory can be measured as a change in either the speed or the amount of induced nicotine production or accumulation that results from prior inductions. Memory is an important component of the induced defenses of animals and is predicted to evolve in systems where an initial attack is a reliable predictor of future attacks. We induced 200 plants 1, 2, or 3 times during an 18—d period of rosette growth, allowed 6 d between inductions for the relaxation of the response between inductions and quantified changes in biomass, whole—plant nicotine pools and de novo rates of nicotine production from 15N—labeled nitrate acquired at the time of induction. Induced changes in nicotine pools and the rates of nicotine production varied across the three induction periods: the highest rates and induced pools occurred during the second induction period, but the highest concentrations were found during the first induction period. The rates of de novo nicotine production from 15N—labeled nitrate were dramatically increased by MJ stimulations during all three induction periods. We found no evidence for alterations in the rates of nicotine production in plants not currently exposed to MJ; 6 d after an induction, plants had relaxed their rates of nicotine production to levels that were not significantly different from those found in plants without a prior induction history. Moreover, the maximum pools of 15N— labeled nicotine were equivalent among all induced plants regardless of prior induction history. In summary, one or two prior inductions did not markedly affect nicotine production from recently acquired nitrate as measured in either induced or uninduced plants. However, the ecologically more relevant measure of the speed of induction is the change in whole—plant nicotine pools, and plants with one and two prior inductions increased their whole—plant nicotine pool significantly faster during the third induction period than did plants not previously induced. Plants with two prior inductions attained significant increases in their nicotine pools 2 d earlier than did plants with one or no prior inductions. Moreover, the effect was additive: plants with two prior inductions were faster than plants with one. The effects of prior inductions on the speed of the induced response did not translate into increases in the magnitude of the response at the end of the 18—d experiment. The lack of change in the magnitude of the induced response may reflect the methods used in our experiment more than the plant's capabilities. The whole—plant induced response is limited by the amount of signal transported to the roots, and since we used the same amount of MJ in all stimulations, our experiment would not detect memory in the production of the signal in the damaged leaf or its transport to the roots. These results demonstrate that plants, like animals, alter their induced defense in response to their prior experiences.

Carbachol and Cholecystokinin Enhance Accumulation of Nicotine in Rat Pancreatic Acinar Cells

Nicotine is a possible risk factor for chronic pancreatitis and pancreatic cancer. To study the loci where nicotine might exert its effect, we examined interactions between nicotine and rat pancreatic acini. When pancreatic acini were incubated with [3H]nicotine, [3H]nicotine levels in pancreatic acini were increased in time-and dose-dependent manners, and the t1/2 for dissociation of [3H]nicotine was 63.8 min. At 4 degrees C, the association of [3H]nicotine was 33% of the association at 37 degrees C. Unlabeled nicotine had no significant effect on the accumulation of [3H]nicotine. In addition, surface-bound [3H]nicotine was not detected when acini were washed in a low-pH solution or when they were trypsinized. These results suggest that the accumulation of nicotine may be a biological phenomenon and that [3H]nicotine does not bind to surface receptors of acinar cells, but accumulates intracellularly. The addition of verapamil (0.1 mM) or 12-O-tetradecanoylphorbol-13-acetate (1 microM) had no effect on [3H]nicotine association, while 4-bromo-A23187 (2 microM) or EGTA (10 mM) significantly increased the accumulation of [3H]nicotine. Carbachol and cholecystokinin significantly enhanced the accumulation of [3H]nicotine in a dose-dependent manner. Taken together, the increasing effects of carbachol and cholecystokinin on the accumulation of nicotine may explain, at least in part, the mechanisms involved in the multiplicative effects of the combination of two risk factors, smoking habit and high-fat or high-protein diets, on human pancreatic diseases.

Korrelation des Cotininspiegels in Fruchtwasser, Nabelarterienblut und mütterlichem Blut

The aim of the present study was to determine the correlation of cotinine levels in amniotic fluid and in fetal and corresponding maternal blood samples. Amniotic fluid samples (N = 130) were taken during second trimester amniocentesis, umbilical artery blood samples (N = 75) at birth, both together with corresponding maternal blood samples. Self-reported smokers showed maternal serum cotinine levels > 15 ng/ml in 93%, self-reported nonsmokers levels < 15 ng/ml in 89%. Correlation of corresponding values for cotinine was 0.81-0.92. Cotinine values were increased in fetal blood and amniotic fluid in comparison to maternal serum levels. Despite the fact that pathophysiology is not fully understood, an accumulation of nicotine and its metabolites both in the fetus and in the amniotic fluid appears to be evident.

Allocation of 15N from Nitrate to Nicotine: Production and Turnover of a Damage‐Induced Mobile Defense

Nicotine is thought to be an excellent example of a mobile" (sensu Coley et al. 1985) plant defense metabolite: it is synthesized in the roots, transported to leaves, and reported to be metabolically labile, with a half—life of &lt;24 h. In a companion paper (Ohnmeiss and Baldwin 1994), we demonstrated that nondestructive damage dramatically increased the whole—plant allometric accumulation of nicotine in Nicotiana sylvestris (Solanaceae) and that damaged plants accumulated a larger proportion of their total nitrogen in nicotine pools than did undamaged plants. These damage—induced accumulations could arise from either increased nicotine production and/or decreased turnover with unaltered production. Here we ask how much of the change in whole—plant nicotine pools that is induced by damage is a result of increased nicotine production, with nitrogen derived from 15NO3 that was acquired, reduced, and assimilated after damage. In three experiments, we examine the rates of induced and constitutive nicotine production over 2 d, over 8 d under two nitrate supply rates, and over the lifetime of the plant. By examining 15N—labeled nicotine pool sizes at different times after damage, we estimate the magnitudes of nicotine turnover and its importance in the induced changes in whole—plant nicotine pools. Two days after damage, damaged plants had accumulated 4.8 times the nicotine pool of undamaged plants, and 57% of the increase in the nicotine pool of damaged plants was synthesized with N from 15NO3 acquired after damage. Damage increased the rate of nicotine—N15 production from 2.0 mg/h in undamaged plants to 6.3 mg/h in damaged plants. No evidence for nicotine turnover was found in either damaged or undamaged plants. The 8—d experiment conducted under two different nitrate supply rates confirmed these results. Furthermore, the 8—d experiment documented that N from 15NO3 acquired after damage was allocated to nicotine production in constant proportions within damaged and undamaged plants, independently of nitrate supply rate; 3.0—3.1 and 5.7—6.1% of the total 15N pool in undamaged and damaged plants, respectively, was used for nicotine production. Therefore, leaf damage doubles the allocation of recently acquired nitrogen to nicotine production in plants that do not differ in nitrate uptake or growth. In an experiment lasting 41 d after damage, we found that the damage—induced increase in total nicotine pools quantified after 5 d remained unchanged over the lifetime of the plants and that in undamaged plants the nicotine—15N pool produced after 5 d was similarly unchanged at 41 d. In damaged plants the nicotine—15N pool produced after 5 d decreased by 33.3% after 41 d. This decrease in the nicotine—15N pools of damaged plants could be due to either metabolism or volatilization. We conclude that nicotine is not the metabolically labile secondary metabolite that it has been previously reported to be and that the changes in pool sizes induced by damage and nitrogen stress are principally due to changes in production and not turnover. Previous studies examined the metabolism of exogenously fed nicotine, rather than the metabolism of endogenously produced nicotine, and this difference in experimental protocol may account for the differences in results. These results demonstrate that the induced patterns of nicotine accumulation are not consistent with the predictions of the carbon/nutrient theory and are consistent with those of the optimal defense theory.

The Allometry of Nitrogen to Growth and an Inducible Defense under Nitrogen‐Limited Growth

As plants become nitrogen—limited in their growth, carbon/nutrient (C/N) theory predicts a decreasing allocation of nitrogen to nitrogen—intensive defense compounds, while optimal defense (OD) theory predicts an increasing allocation to defense; CN ratio of the plant does not change, while OD theory does. We compare C/N and OD theory predictions for the inducible nicotine production of a native tobacco, Nicotiana sylvestris. We define allocation as the slope of the ln—ln allometric relationship between whole—plant nicotine and nicotine—free biomass in undamaged and damaged plants, harvested every other day in eight consecutive harvests. Relative growth, nitrogen, and nitrogen in nicotine accumulation rates (RGRs, RNARs, and RNicARs) were calculated to estimate the instantaneous investment of nitrogen in nicotine and the growth of plant tissues. Nicotine accumulation continues during nitrogen stress, resulting in a 3.29—fold increase in the nicotine pools of undamaged plants, and is accelerated in response to leaf damage, resulting in 4.06 fold increase in the nicotine pools of damaged plants. Nitrogen pool size did not change after day 2, and both undamaged and damaged plants allocated an increasing proportion of their nitrogen pool to nicotine throughout the experiment. Damage increased the allocation of nitrogen to nicotine; the slope of the ln nicotine vs ln nicotine—free biomass regression for damaged plants was significantly greater (16.9%; P &lt; .041) than the slope for undamaged plants as measured during the period of damage—induced nicotine accumulation (days 2—10 damage). Allocation of nitrogen to nicotine accumulating is clearly not a passive function of the nitrogen in excess of growth requirements, and damage increases this allocation. Trade—offs between biomass and nicotine production over the course of the experiment between damaged and undamaged plants were not apparent. However, the short—term estimates indicate otherwise. On day 2, there were a significant decrease in RGR, with a correlated increase in RNicAR in response to damage, suggesting that there was a short—term trade—off between allocation to growth and nicotine accumulation or other induced processes. By the end of the experiment, each damaged plant had accumulated 0.595 mg N in nicotine above that accumulated by undamaged plants, and additional investment representing 1.94% of the total nitrogen pool. However 2 d after damage, damaged plants allocated 0.131 mg more N to nicotine than undamaged plants, representing a short—term investment of 17.7% of the nitrogen potentially made available by the decrease in RGR (.0740 mg). Plants invest substantial portions of their nitrogen in an induced defense, but our estimates do not include the nitrogen costs of production, storage, transport, detoxification, or the indirect cost of this induced defense.