A Comparison of Nicotinic Receptor Stimulation and Heart Rate Responses by Lobeline and Nicotine in...

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Assessing Crayfish Heart Rate Responses After Nicotinic Receptor Stimulation by Nicotine and Lobeline Lukas Isenhart, Brittany Files, Cody Costley, Niariah Fields Birmingham-Southern College Department of Biology 12 May, 2016

Transcript of A Comparison of Nicotinic Receptor Stimulation and Heart Rate Responses by Lobeline and Nicotine in...

Page 1: A Comparison of Nicotinic Receptor Stimulation and Heart Rate Responses by Lobeline and Nicotine in Crayfish

Assessing Crayfish Heart Rate Responses After Nicotinic Receptor Stimulation by Nicotine and Lobeline

Lukas Isenhart, Brittany Files, Cody Costley, Niariah Fields Birmingham-Southern College

Department of Biology

12 May, 2016

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Abstract

Crayfish were administered 0.05mL dosages of 3μM nicotine [(S)-3-[1-Methylpyrrolidin-2-yl]pyridine] and lobeline [2-((2R,6S)-6-((S)-2-Hydroxy-2-phenylethyl)-1-

methylpiperidin-2-yl)-1-phenylethanone] to determine effects on heart rate as a method for comparing cardiac stress. This would suggest lobeline’s possible use as a nicotine replacement therapy (NLT) in humans. Data proved insignificant for experimental groups

of three crayfish per drug. Further study is needed to determine the viability of lobeline as an effective nicotinic-cholinergic competitive agonist for nicotine in nicotinic receptors,

which would prove beneficial to cardiovascular medicine and its treatment of nicotine and other drug addictions.

1 Introduction

The nonscientific community often directly associates nicotine with cancer and

other negative physiological effects because it is the main psychoactive and addictive

ingredient in cigar and cigarette smoke, hookah and e-cigarette vapor, snuff, and other

various tobacco products. As a stimulant, high doses of nicotine can prove lethal, but the

nicotine concentration necessary to reach this isn’t possible with commonly available

tobacco products like cigarettes or snuff (Brandon, et al. 2015). Use of nicotine during

pregnancy is associated with a fetus’s future respiratory dysfunction, obesity,

hypertension, and neurobehavioral defects as well as increased spontaneous abortions,

premature delivery rates, and decreased final trimester birth weights (Schraufnagel, et

al.). Nicotine has demonstrated an in-vitro association to cancer, but nicotine’s

carcinogenic effects have yet to be demonstrated in-vivo, suggesting that common

nicotine ingestion methods have little effect on future cancer development (Hass and

Kuebler 1997). Nicotine use in pharmacological treatments for developed adults is

relatively risk-free to overall health: it can be said that the additional chemical

compounds found in nicotine products – such as carbon monoxide and the resulting gases

of combusted pesticides, herbicides, and fungicides, among others – pose a higher risk to

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users. However, nicotine poses a threat to long-term cardiovascular health in adults by

increasing heart rate, myocardial contractility, and blood pressure, which inadvertently

lead to increased likelihood of heart attacks, angina, and strokes (Haass and Kuebler

1997).

Nicotine is an alkaloid commonly found in cultivated Nicotiana tabacum, which,

when ingested, leads to reward-motivated addictive behavior as a result of dopamine

release inflection and increased concentrations of extracellular dopamine near brain

reward systems (Goutier, et al. 2016). Nicotine interacts with cholinergic receptors,

including multiple nicotinic acetylcholine receptors that are found throughout the human

body, but their interaction within the brain and central nervous system suggests nicotine’s

importance as an agonist. Due to nicotine’s agonist characteristics on nicotinic receptors,

it is possible to employ chemicals of similar structure for use as competitive nicotine

blockers. This led to research into chemicals like lobeline, which has a high binding

affinity to the nicotinic cholinergic receptors and was considered useful for treating

nicotine addiction (Stead and Hughes 2012).

Lobeline is an alkaloid found in Lobelia inflata, and, like nicotine, modulates

dopamine release and increases extracellular dopamine concentrations in reward centers

of the brain via its agonistic relationship with nicotinic receptors (Buchhalter, et al.

2008). Because of this, lobeline was used as a smoking cessation aid and nicotine

replacement therapy (NRT) until 1993, when the FDA found that lobeline lacked

sufficient efficacy in antismoking treatments (Stead and Hughes 2012). This is largely

due to improper use of controls in multiple studies, but also because the effective dose of

lobeline is nearly toxic to humans. Despite this, lobeline still has applications in

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respiratory health and there has been a revival of studies that delve into its use as an

addiction treatment for nicotine, amphetamines, alchohol, and cocaine because of its

effects on dopamine reuptake.

Because lobeline has potential to be used as an NRT, it is important to analyze its

efficacy in model organisms to suggest a reason for its further application to human

physiology. In rat models, lobeline has a significant impact on quelling

methamphetamine- induced behavior by limiting dopamine reuptake and by increasing

dopamine release from presynaptic storage vesicles (Dwoskin, Linda, and Crooks 2002).

Also in rats, lobeline displays similar cognitive effects to nicotine in terms of memory

retention (Decker, Majchrzak, and Arnerić 1993). Nicotine performs similarly to lobeline,

particularly in crayfish where nicotine significantly increased chemosensitivity (He,

Tucket, and English 1999).

Little is known about nicotine and lobeline’s effect on the heart rates of crayfish,

and therefore this study is important to the scientific community and possibly to the

cardiovascular realm of human medicine in providing a more effective method of

pharmacologic treatment for nicotine addiction. This study treated crayfish with lobeline

and nicotine solutions and used initial and resulting heart rate in beats per minute (bpm)

as a method for comparing the vascular health of the crayfish. Crayfish were used for

their simplistic heart anatomy as well was for ease of electrode implantation to measure

changes in heart rate. Because cardiovascular stress can lead to multiple long-term

conditions in smokers, it is essential to use model organisms like crayfish to compare the

cardiac stresses associated with nicotine or lobeline use. It was hypothesized that nicotine

and lobeline would have equivalent effects on heart rate due to the similarity of their

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nicotinic-antagonist properties (H0: bpmNicotine = bpmLobeline), but statistically significant

variation (Ha: bpmNicotine =/= bpmLobeline) could suggest that one drug is better suited to

addiction therapies by minimizing cardiac stress.

2 Materials and Methods

2.1 Initial Crayfish Preparation

Seven crayfish were removed from tanks of similar ion concentrations before being

wiped dry. The crayfish were then inverted and sexed by identifying longer swimmerettes

that extended upward above the back legs. Males possessed these swimmerrettes while

females did not. After sexing, the claws were glued for safety, covered with pieces of

aquarium tubing, and labeled. Crayfish were housed

in large, oxygenated beakers then covered to prevent

their escape and to minimize aggression and stress.

2.2 Preparation of Lobeline and Nicotine Doses

Using Ringer’s solution, 3 μM solutions of both

lobeline and nicotine were created and refrigerated

during storage. The solutions were gently swirled

before use.

2.3 Preparation of Impedance Converter Electrodes

The following methods follow Andrew T. Gannon’s procedure for electrode implantation

(Gannon, 2005). Two insulated copper wires, each 30cm long, were stripped at their ends

to reveal 1cm of exposed copper. A thicker gauge wire was then used as a guide to pull

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the electrode wiring through an aquarium tube so that both ends extend 6cm from the

tubing. These wires were then taped to the tubing to prevent dislocation. Two dental dam

squares, each 1cm x 1cm, were then

pierced with a syringe needle and

threaded onto each wire at one end by

inserting the wire into the needle point.

One end of each wire was then attached

to the impedance converter, which was

set to an AC Short time balance,

calibrated at 0.5Ω, and balanced in preparation for electrode implantation. Repeat for

every crayfish.

2.4 Surgical Insertion of Electrodes

Using a wooden block with four attached

metal rings, the crayfish were restrained

using rubber bands while paying

attention to not harm the eyestalk,

antennae, antennules, and rostrum. For

best restraint tightly cover the cranial

cephalothorax, chelipeds, and telson. A

dremel tool with an abrading stone

attachment was then used to pierce the

cephalothorax of the crayfish without

piercing the epidermis. While drilling, the exposed tissue will change from white to light

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red when the epidermis is reached. Holes were drilled in the left and right carapace,

laterally adjacent to the heart. A third hole was then similarly drilled caudal to the heart at

the carapace nearest the abdomen. Taking great care both to avoid harming the heart and

prevent the loss of hemolymph by piercing the pericardium or gonads, the wires were

inserted lateral to the heart. A steady reading for heart rate by the impedance converter

suggests that this is done correctly. Rebalance the impedance converter as needed. Using

minimal superglue, the wires were glued in place and covered with dental dam squares,

adding another drop of superglue to the wire above the dental dam for added security.

The caudal opening was closed by gluing the circumference of the hole and by applying a

square of dental dam.

2.5 Dosage Data Collection

Given the stressful nature of the implantation, an aggressive crayfish heart rate was

minimized by allowing the crayfish three hours of dark, covered rest before beginning the

doses. The crayfish were restrained as previously mentioned and given an additional five

minutes of covered rest to reach a submissive resting heart rate. Due to similar

favorability for nicotinic active sites, nicotine and lobeline experienced similar dosages,

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each requiring 3μM solutions and 0.05mL doses per three-minute period. These doses

were applied using a needle syringe inserted through the dental dam of the caudal

carapace. The doses accumulated over a 15-minute period to reach a total dosage of

0.25mL. This dose schedule applied for six dosing crayfish (N1, N2, N3, L1, L2, L3),

while a seventh crayfish (Test) was initially used for a dose-intensity analysis of nicotine

on heart rate, which was compared to outside data to determine the lethality of this assay.

Heart rate was analyzed using Loggerpro software and an impedance converter.

Rebalance the impedance converter as needed. The seven crayfish were subsequently

euthanized by applying a 0.3mL dosage of water saturated with potassium chloride (KCl)

through the caudal carapace to cause cardiac arrest. Using Microsoft Excel software,

lobeline and nicotine analyses were graphed along a polynomial trend line and a two-

tailed heteroscedastic test was performed, using a P-value of 0.05 to differentiate between

accepting the null or alternate hypotheses.

3 Data

Table 1. Individual and average weight of nicotine, lobeline, test, and total experimental

groups.

Identification Sex Weight (g)

Test M 33.0

N1 M 30.8

N2 F 39.3

N3 M 39.2

Nic. Average 36.4

L1 M 33.6

L2 M 41.3

L3 M 40.8

Lob. Average 38.6

Total Average 36.9

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Lobeline and nicotine doses did not account for the weights of individual crayfish,

instead relying on equivalent dosages across test subjects (Table 1.). Most crayfish

weighed over 33.0g, suggesting developmental maturity (Table 1.). There is no

significant correlation between crayfish sizes and their sex, as only one female crayfish

was tested (Table 1.).

Table 2. Total applied dosages for nicotine and lobeline experimental groups.

Time

(m)

Nicotine Dosage

(3 μM)

Lobeline Dosage

(3 μM)

0 0.00 mL 0.00 mL

3 0.05 mL 0.05 mL

6 0.10 mL 0.10 mL

9 0.15 mL 0.15 mL

12 0.20 mL 0.20 mL

15 0.25 mL 0.25 mL

Table 3. Individual heart rate responses by dosage for nicotine and lobeline crayfish groups. (N=7).

Heart Rate (bpm)

Time (m) Test N1 N2 N3 L1 L2 L3

0 73 61 67 72 52 63 56

3 204 87 79 84 114 90 82

6 x 96 96 81 120 96 85

9 x 96 99 95 114 95 106

12 x 99 92 94 102 101 99

15 x 109 95 99 108 103 101

Doses of 0.05mL of 3μM nicotine and lobeline solutions resulted from the analysis of a

test crayfish to determine the immediate effects of a 0.25mL dose of 3μM nicotine (Table

1., Table 2.). The test crayfish experienced a potentially harmful heightened heart rate

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response and abdomen hyperactivity, but heart rate normalized after 15 minutes (Table

3.).

Figure 1. Average heart rate responses at three-minute intervals due to 0.05 mL nicotine doses. Error bars denote standard deviation. (N=3).

The polynomial regression had a high coefficient of determination for nicotine (0.9593)

and displayed a relatively low peak for average heart rate at 101bpm + 7.2 (Fig. 1.)

y = -0.1798x2 + 4.7183x + 68.393R² = 0.9593

50

60

70

80

90

100

110

120

0 5 10 15 20

He

art

Ra

te (

bp

m)

Time (m)

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Figure 2. Average heart rate responses at three-minute intervals due to 0.05 mL lobeline doses. Error bars denote standard deviation. (N=3).

Compared to that of nicotine, the polynomial regression for lobeline had a lower

coefficient of determination (0.87813) and displayed a higher peak for average heart rate

at 104bpm + 3.6 (Fig. 1.) A T-test performed between the data sets resulted in a P-value

of 0.6108, supporting the null hypothesis.

4 Discussion

Given a P-value over 0.05, the null hypothesis was supported, suggesting that

lobeline-stimulated heart rate had no statistical significance in comparison to nicotine-

stimulated heart rate. Despite lobeline’s higher average heart rate peak at 104 bpm + 3.6,

lobeline did not have a greater effect on heart rate than nicotine (Fig. 2.). This suggests

that nicotine and lobeline can be used interchangeably in drug treatment therapies when

not considering other cardiological effects attributed to each drug. This said, crayfish

visibly experienced greater stress in lobeline trials, especially in regard to abdomen

shaking and quivering at higher dosages. In the test subject, where dosages were used to

determine lethality, larger upfront dosages appeared to have a much higher resulting heart

y = -0.421x2 + 8.7526x + 62.807R² = 0.8781

50

60

70

80

90

100

110

120

0 5 10 15 20

He

art

Ra

te (

bp

m)

Time (m)

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rate than in the 15-minute procedure. This study does not effectively demonstrate that one

drug is preferential for administration to crayfish and thus cannot suggest any new

association for nicotine and lobeline in terms of heart rate responses. If two identical

people were given equal doses of nicotine and lobeline they would be expected to have

the exact same responsive heart rate.

Obvious issues need to be fixed within this study, including a necessary increase

in sample size and a sample set of same-sex crayfish that are similar in size. More tests

are needed to verify these results; perhaps nicotine and lobeline have different cardiac

responses that weren’t observed within the confines of such a small data set. Additional

measures also need to be addressed as far as the handling and preparation of crayfish for

this procedure, as crayfish had a relatively high die-off rate as a result of the lateral

placement of the electrode wires, which may have pierced the pericardium in some

crayfish during implantation. These crayfish may have been too small to support the

procedure. A larger gauge electrode wire is also recommended, as the crayfish easily

pulled out their wires, leading to repeated procedures that caused unnecessary stress to

the crayfish.

5 Conclusion

When administered directly to the heart of crayfish, lobeline and nicotine did not have a

statistically significant variation in output heart rate at various dosages, suggesting no

new applicable knowledge to the realm of NRTs for people. Future trials are needed to

further support this claim.

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6 Acknowledgments

We would like to thank the Department of Biology at Birmingham-Southern College for

providing materials and laboratory space as well as Dr. Andrew T. Gannon for assistance

with his crayfish electrode implantation technique.

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