Effect Of Increasing Dosages Of Epinephrine On Bufo Marinus Heart Rate



      Epinephrine, also known as Adrenaline is a type of catecholamine hormone and neurotransmitter. Production of epinephrine stems from chromaffin cells synthesised from the amino acid tyrosine (Boron, Boulpaep 2010). Secreted exclusively by the adrenal medulla, epinephrine is released from chromaffin granules in response to stress stimuli of the sympathetic nervous system (Campbell, Reece and Meyers 2009). Such stimuli include hypoglycaemia and flight or fight. The release of epinephrine is regulated through nerve signals transported via involuntary neurons (Mefford, I.N, 1987). In response to stress stimuli, nerve signals from the hypothalamus travel via the splanchnic nerve. Synaptic terminals of preganglionic fibres release the neurotransmitter acetylcholine (ACh) acting on nicotinic acetylcholine receptors (nAChR) of chromaffin cells. Depolarization of nAChR causes an influx of calcium via voltage gated calcium channels. Chromaffin cells now in postganglionic form release epinephrine into the bloodstream functioning as simple neurohormones (Boron, Boulpaep 2010). The release of epinephrine has numerous roles such as increased rate of glycogen breakdown, mobilization of glycogen stores, stimulation of fatty acids from fat cells and several endocrine functions (Mefford, I.N, 1987).Epinephrine, also known as Adrenaline is a type of catecholamine hormone and neurotransmitter.

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      Another feature of secreting epinephrine is the potent ability to stimulate the cardiovascular system. Increased heart rate and cardiac output are two of the main effects from epinephrine. Epinephrine hormones which have blinded to ß1 (beta) adrenergic receptors increase heart rate by two main mechanisms (Nelson, Cox 2009). Epinephrine increases If voltage dependent currents (VDC) that increase steepness of depolarization of the pacemaker activity. Increased steepness of depolarization results in quicker time frame for the heart to reach threshold. A blinded Ic (VDC ) voltage dependent current in the sinoatrial node and atrioventricular node steepens depolarization which again allow the heart to reach threshold quicker (Dorland.2009). The aim of the study was to investigate such molecular mechanisms of epinephrine increasing the heart rate of a B. marinus.

Increasing amounts of epinephrine cause contraction strength to increase (positive inotropic effect) in ventricle and atrial muscle. In conjunction with voltage dependent currents, increasing levels of epinephrine causes four main changes to contraction strength. Increased amounts of Epinephrine binded to ICa (VDC) causes calcium release from the sarcoplasmic reticulum (SR) (Boron, Boulpaep 2010). The binded epinephrine then causes greater sensitivity of the sarcoplasmic calcium release to cytoplasmic calcium. Epinephrine also further enhances calcium pumping into the cytoplasm through stimulation of Sacro/endoplasmic Reticulum calcium ATPase (SERCA) (Endoh 1995). The combination of factors results in more calcium available for troponin C, a binding site on myosin (thick) filaments, resulting in greater contraction of the heart, hence greater heart rate (Boron, Boulpaep 2010).


      B. Marinus heart rate will increase relative to the dosage of Epinephrine (adrenalin) administered via Pasteur pipette.



      A B.marinus (aquatic cane toad) was obtained. Tissue and sternum were cut to expose cavity. The bent pin that was attached to the cotton string was tightened. Pericardium was lifted and cut from heart. Frog ringer solution was constantly applied to the toad's heart during the experiment. Three ECG wires were then attached to the frog. Powerlab was then started. A brief time period of 15 seconds baseline was recorded which served as the controlled variable. Epinephrine was then administered though a Pasteur pipette. A 90 second interval was given after each drop(s) of epinephrine. This was done to act as a control between each drop and allow epinephrine to affect heart rate. A 15 second interval was directly measured after the first interval for heart rate ECG. The heart was then undisturbed for 75 seconds. The process was repeated for 2, 3, 5, 10, 15, and 25 drops of epinephrine.

Data Analysis:

      The data was collected in a tabulated form. It was then converted into beats per minute. Data was then inserted into the Prism program. The data was analysed with a grouped bar graph from which a one-way ANOVA test was used to determine the P-value.


      A significant increase in heart rate during epinephrine treatments one and two compared with baseline conditions. (P<0.05, See Figure1)


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The overall findings from both trial one and two show substantial increased in heart rate over base line. The results also seem to indicate increasing epinephrine dosages correlates with increasing heart rate levels. The data also shows a flat line period for dosages 10, 15 and 25 drops from trial two. As epinephrine is administered, heart rate seems to remain constant (still significantly above baseline) in the presence of increasing epinephrine. Trial one also seems to have a similar flat line pattern between 5 drops to 25 drops. The only slight increase was for the15 drop epinephrine treatment with a slight increase in heart rate.

The underlying molecular mechanism behind epinephrine increasing heart rate is through mimicking positive intropic effects. One such positive intropic effect is the opening of calcium channels. Epinephrine acting as an adrenergic agonist acts on ß1 adrenoceptors which in turn activates as subunit of GS heterotrimeric G proteins. The activation of as heterotrimeric proteins increases heart rate through two pathways. as opens firstly raises intracellular levels of cyclic adenosine mono-phosphate (CAMP) and stimulation of protein kinase A. Raised level of cAMP also directly leads to the opening of numerous L type calcium channels in the plasma membrane. This in turn allows calcium into the myocardial cell. The influx of calcium during action potentials allows for stronger and shorter contractions thus increased heart rate (Boron, Boulpaep 2010). The opening of calcium channels through cAMP enhancement however is not totally understood. It has been speculated that phosphorylation of the calcium protein channel results in influx of calcium into the myocardial cell. Influx of calcium influences cAMP which triggers the release stored calcium ions in the SR. This is then speculated to increase action potentials (Endoh 1995). The findings from this study also seem to further support this theory of increasing epinephrine triggering greater cAMP activity and calcium release.

The epinephrine caused action potentials now produce a chronotropic effect within the sinoatrial node. ß1 and L type channels adrenoceptors stimulate If (VDC), diastolic sodium currents and ICa (VDC) via T type calcium channels. The net effect of stimulation is an increase rate of diastolic depolarization and a negative shift in threshold (Endoh 1995). The combination of both results in shortening of the diastole and heart rate increase.

The findings from both trials also seem to indicate flat line phenomena where increasing epinephrine levels of higher dosages fail to increase heart rate. Nacimiento (1963) found the average resting heart rate for B.marinus was 40b/min at 25 degrees, similar to the findings from this study. Wahlqvist and Campbell (1987) studied autonomic influences on B.marinus heart rate and suggested maximum heart rate was in the region of 50-60 b/min.

The aim of the study was to investigate such molecular mechanisms of epinephrine increasing the heart rate of a B. marinus. The study showed a significant increase in heart rate after being administered with increasing amounts of epinephrine. The study was also the first to show the heart rate of a B.marinus reaching maximum heart rate induced through epinephrine. The study also seemed to support pervious findings in regards to resting and maximum B.marinus heart rate and evidence for epinephrine role in phosphorylation of the calcium protein channels. Future studies could focus on how epinephrine is directly involved in the phosphorylation of the calcium protein channels or perhaps change focus and study the possibility of epinephrine binding to other adrenoceptors such as a (alpha) family and possible benefits for society.


  1. Dorland's illustrated medical dictionary.2009, “Epinephrine”, 31ST edition, pp641.
  2. Campbell, Reece and Meyers.2009, “Adrenal hormones: Response to stress.” Biology Australian edition, 8th edition, pp 1006 -1008.
  3. Boron, Boulpaep.2010, “Adrenergic receptors”, pp 563, “Positive intropic agents increase myocardial contractility”, pp 552, Medical Physiology, 2nd edition.
  4. Nelson, Cox.2009, “G protein coupled receptors and second messengers.” Lehninger: Principles of biochemistry, 5th edition, pp 423-425
  5. Nacimiento, A.C, 1963, “On circulation and hypothermia in the toad”, Archives Italiennes de Biologie, vol 101: pp 296-305.
  6. Wahlqvist, I and Campbell, G, 1988, “Autonomic influences on heart rate and blood pressure in the toad, bufo marinus at rest and during exercise”, Journal of experimental biology, vol 134, pp377-396.
  7. Karch, S.B, 1988,”Coronary artery spasm induced by intravenous epinephrine overdose”, Department of emergency medicine, university medical centre, Las Vegas.
  8. Mefford, I.N, 1987, “ Epinephrine in mammalian brain”, Progress in Neuropsychopharmacology and Biological Psychiatry, vol 12, pp 365-388.
  9. Endoh, M, 1995, “The effects of various drugs on the Myocardial Inotropic response”, General Pharmacology, vol 26, pp 1-31.
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