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=The Effects of Capsaicin on Mammalian Tissues=

Abstract
Capsaicin, the chief ingredient in chili peppers as well as other spicy foods, produces a “burning sensation” after coming into contact with mammalian tissues. This “burning sensation”, results from the binding of capsaicin to a receptor known as the vanilloid receptor subtype 1 (VR1) and subsequent stimulation of that sensory neuron. The existence of the sensory receptor specific to capsaicin is established via expression cloning. A mechanism for capsaicin-evoked responses as well as how it elicits a sensation of pain upon sensory neurons is proposed. Capsaicin-induced responses were found to go hand in hand with the activity accompanying a vanilloid receptor following stimulation by a fast increase in temperature (Nagy 1999). This helps to explain why ingestion of the molecule from red hot chili peppers, elicits the burning sensation of pain. The addition of hydrogen ions upon the extracellular medium of the cell was found to enhance capsaicin’s effect on the sensory neuron. Overexposure to capsaicin could result in inactivation and desensitization of the sensory neuron and in some cases, death of the cell (Wood 1988). The desensitization of the neuron that is caused by capsaicin has led to the molecule being involved in frequent studies involving the mechanisms of pain sensation and perception (Caterina 1997; Marsh 2003).

Background
That “burning sensation” that’s usually experienced after ingesting some red hot chili peppers or other spicy foods is actually not the result of the spicy food causing a burn or physical damage to the skin or tissue. The sensation of pain experienced is actually caused when capsaicin, the central component of many spicy foods, interacts with a specific group of sensory neurons known as nociceptors (Caterina 1997; Heyman 2003). These sensory neurons are very small in diameter and are known to be very sensitive to external stimuli such as heat and physical tears within tissues. They are also known to be very sensitive to a group of compounds that capsaicin belongs to, known as vanilloids. Upon binding with the sensory receptor on the nociceptor, capsaicin activates the sensory neuron and causes it to send signals to the brain. These signals are then interpreted by the brain as pain, and as a result, inflammation by the body at the site of capsaicin treatment usually follows (Caterina 1997). For awhile, the actual existence of a receptor specific to the capsaicin molecule was debatable due to the fact that there had been no viable evidence of its isolation from the sensory neuron (Peterson 2003b). Not too long, between the years of 1992-1997, the existence of the capsaicin receptor was successfully verified thanks to expression cloning of the sensory receptor through the use of microscopic fluorescent calcium imaging (Peterson 2003b; Caterina 1997). The strategy implemented for the expression cloning of the receptor was based off of capsaicin’s ability to cause an influx of calcium ions into the sensory neuron upon stimulation of the nociceptor. Fluorescent calcium-sensitive dye Fura-2 was used to microscopically mark the sites that exhibited capsaicin-induced changes in calcium content on the intracellular plasma membrane as shown in Figure 1 (Caterina 1997). Due to the site of capsaicin action being commonly referred to as the vanilloid receptor, the newly identified capsaicin receptor was given the name, vanilloid receptor subtype 1 aka VR1. The vanilloid receptor was experimentally determined to contain binding sites for capsaicin on both sides of the plasma membrane due to the fact that the capsaicin-induced responses from an isolated patch of a sensory neuron were virtually the same on both the extracellular and intracellular sides of the sensory neuron. VR1 is also fundamentally comparable to a group of receptors that belong to the TRP family of ion channels due to the receptor sharing many similarities with them (Caterina 1997).

Mechanism of Action
The molecular target of capsaicin induced action was identified as a non-specific ion channel thanks to the techniques applied from expression cloning of the cell (Peterson 2003a). This ion channel is mainly permeable to calcium ions and is usually triggered by stimulation of toxic heat along with capsaicin. Exposure of capsaicin either on the skin or the mucous membrane causes violent irritation in humans (Fitzgerald 2003; Savidge 2001). Once the capsaicin molecule binds with the receptor on the sensory neuron, it initiates an increase in the conductance of sodium and calcium ions as well as causing the neuron to bring forth a flow of inward current and experience depolarization (Maggi 2003; Marsh 2003). The depolarization of the neuron triggers the activation of the ion channel and increases the membrane’s permeability to calcium cations (Bleakman 1990; Marsh 2003). It has also been proposed that capsaicin may increase the excitability of a sensory neuron by inhibiting the permeability of the plasma membrane to potassium ions (Dray 2002b). Nonetheless, the activation of the ion channel causes an influx of calcium ions into the plasma membrane. This influx of calcium ions then stimulates the sensory neuron into sending sensory signals to the brain and spinal cord. The brain then translates and interprets this sensation as pain. The influx of calcium ions into the plasma membrane can also lead to a state known as secondary hyperpolarization resulting from a secondary increase in the conductance of potassium ions. Another side effect that usually results from the increase in the amount of intracellular calcium ions is the impairment of intracellular organelles which sets the stage for capsaicin’s subsequent harmful and toxic effects (Wood 1988). One distinguishable characteristic of a neuron that has been poisoned from capsaicin’s toxic effects is that of swollen mitochondria. The deviations in calcium ion levels may also result in obstruction of axoplasmic transport by capsaicin molecules (Marsh 2003). There has also been evidence of capsaicin being capable of activating at least two different types of inward current which differ kinetically and contain different reversal potentials. Activation of either of the inward currents was determined to be dependent upon the concentration of the capsaicin, the cell that capsaicin came into contact with and the rate and length of time that the capsaicin was administered into the neuron. Most of the cells that capsaicin came into contact with exhibited only a one of these currents (Liu 1999). The events that occur following activation of the capsaicin receptor are similar to the events that occur following activation of TRP ion channels (Caterina 1997). Activation of sensory neurons by capsaicin can also initiate the release of neuropeptides from the cell. The release in neuropeptides is most likely a result of the influx in calcium ions from the stimulated ion channel however this occurrence may also be tied in with voltage triggered calcium channels (Bleakman 1990; Dray 2002a). One other important point to note is that, just like other vanilloid receptors, complete activation of the capsaicin receptor is usually dependent upon the binding of more than just one agonist molecule. Also, the excitation of the neuron by capsaicin molecules can be blocked by competitive and non-competitive vanilloid receptor antagonists such as capsazepine and ruthenium red, respectively (Bleakman 1990; Peterson 2003b). The inflammation of the cell that occurs, following capsaicin activation sometimes alters the sensitivity of the neuron (Nicholas 1999). The inflammation causes an upsurge in the sensitivity of some neurons causing them to become hypersensitive to external stimuli, whereas other sensory neurons, that are usually either inactive or unresponsive, are now altered into functioning as nociceptors and becoming receptive to external stimuli (Dray 2002b). The long term effects of capsaicin treatment upon the sensory neuron include weakening of the neuron’s sensitivity to further doses of capsaicin and as well as other external stimuli. The neuron’s ability to transmit chemosensitive and thermal pain also experiences a decline. Signs of ultra-structural damage become present upon the sensory neuron and lasts for many weeks after exposure to capsaicin molecules. The small B-type dorsal root ganglion neurons within the neonate start to deteriorate and their ability to transmit pain experiences permanent damaged after prolonged exposure from capsaicin molecules (Marsh 2003).

Association with Heat
Capsaicin’s association with a “burning sensation” is consistent with the fact that the interactions of capsaicin with the vanilloid receptors are essentially identical to the activity brought up by heat-mediated responses. This was proved when stimulation of VR1 expressing cells, to a fast rise in temperature created a large inward current that was similar in magnitude to the inward current produced capsaicin came into contact with these cells. As expected, exposure of a sensory neuron from both capsaicin and heat result in the activation of ion channels (Caterina 1997; Ringkamp 2001). Signs of external modifications were present from both capsaicin-induced and heat-mediated responses. Coupled with the fact that VR1 expressing neurons tend to be activated when temperatures are raised to a level that is capable of producing pain, the activity that followed from both heat and capsaicin-induced responses hinted at the likelihood of both being controlled by a single factor (Caterina 1997). Figure 2 displays some of the similarities between the characteristics of heat and capsaicin-evoked responses (Kirchstein 1997). Further experiments have shown that the heat-sensitive capsaicin receptors are stimulated very quickly by heat that is especially very harmful and toxic (Kirschstein 1997; Ringkamp 2001). One other important thing to note is that sensitivity to both heat and capsaicin-induced responses was initially thought to be present only in unmyelinated nociceptors. Evidence for the presence of capsaicin-sensitive sensory receptors within myelinated nociceptors was later revealed from the results of research on the sensory neurons within primates. Most of these capsaicin-sensitive receptors were also sensitive to stimulation from fast increases in temperature (Kirshstein 1997; Nagy 1999). Interestingly, there are many sensory receptors that exhibit sensitivity to capsaicin and heat, yet remain unresponsive to stimulation from mechanical stimuli (Ringkamp 2001). In some cases, certain groups of sensory neurons have been reported to be sensitive to stimulation from capsaicin and yet unresponsive to stimulation from heat (Kirshstein 1997). Examples of neurons that exhibit these characteristics include some A-fiber nociceptors and dorsal root ganglion cells that are insensitive to heat stimulation. However, the occurrence for heat-insensitive sensory neurons to become responsive to stimulation from capsaicin molecules does not happen very often. Most of the time, sensory neurons that were unresponsive to stimulation from heat were also insensitive to capsaicin stimulation. This was especially the case for large diameter neurons (Heyman 2003; Kirshstein 1997). On the other hand, there have also been cases of some sensory neurons being sensitive to stimulation from heat and yet unresponsive to stimulation from capsaicin. An example of such would be the VRL1 receptor (Ringkamp 2001). It is likely that the transduction proteins that are receptive to certain external stimuli are randomly spread throughout the peripheral terminals of the axon. This is consistent with the claim that capsaicin induced action seems to work specifically on the central and peripheral terminals of sensory fibers without affecting the rest of the neuron (Fitzgerald 2003; Heyman 2003). A neuron’s sensitivity to a particular stimulus may also depend on the density of the operating channels within the membrane (Ringkamp 2001).

Proton Dependency
Both protons and capsaicin are known to elicit pain in humans after coming into contact with human tissues (Baumann 1999). Although the addition of protons to the receptor was not capable of singlehandedly stimulating the capsaicin receptor, it was still found capable of influencing the effect of capsaicin-evoked responses from the sensory neuron (Caterina 1997). To illustrate this point the effect of protons on capsaicin-induced responses on an isolated group of dorsal root ganglion neurons (DRG) is experimentally observed. First of all, capsaicin is capable of stimulating DRG sensory neurons by enticing an inward current. The magnitude of the current induced by capsaicin increased significantly with increasing amounts of proton content in the extracellular medium. For example, lowering the pH of the external medium of a DRG cell from a pH of 7.3 to 6.3 boosted the capsaicin-evoked current considerably as shown in Figure 3 (Peterson 2003b). One important thing to note is that absolutely no inward current was produced when the cell was placed within an acidic solution alone without any excitation from capsaicin. When the application of capsaicin and elevated acidity was tested sequentially, with the acidic solution coming first, followed by capsaicin at a biological pH of 7.3, the result produced no change on the inward current evoked by capsaicin in an acidic solution. There was also no change in this inward current when the order of the acidic solution and capsaicin at a pH of 7.3 was added in vice-versa. The magnitude of the capsaicin-induced currents began to gradually decrease when the neuron was administered with capsaicin over and over again at a biological pH as well as an acidic pH level. This, in effect, showed a sign of capsaicin’s desensitizing effect. Cells that were insensitive to stimulation from capsaicin at a biological pH remained insensitive to stimulation from capsaicin even when in the presence of an acidic solution. These findings help in proving that the elevation of the capsaicin-induced current is not a direct result of a leak current brought about by protons (Peterson 2003b). Ultimately, capsaicin was found to have a more significant effect on the stimulation of vanilloid receptors if the proton concentration in the external medium was increased during treatment of the molecule with a sensory neuron (Caterina 1997). Two different types of capsaicin-induced currents were thought to be activated by the addition of protons in DRG neurons. One of them is activated by small elevations in the amount of proton content in the external medium. This current was found to rapidly become activated and inactivated. The mechanism for this current involves an increase in the permeability, of voltage-gated calcium channels to sodium ions, by protons. One other interesting characteristic about this current is that it can be found in both sensory and non-sensory neurons. In contrast to this first current, the other current can only be activated by protons that are high in concentration and is also only found within sensory neurons. Other contrasting qualities of this current include its activation and inactivation being much slower than the first. The second current persists for longer periods of time and is generated from the activation of a non-specific cation channel. The ion fluctuations brought about by this current seem very similar to the ion fluctuations brought about by capsaicin-induced currents. Both currents also seem to affect the same population of cells thus, indicating the possibility of both currents acting on the same ion channel (Peterson 2003b). In the end, protons do not necessarily interact in conjunction with capsaicin but rather help in facilitating the inward current induced by capsaicin and other algesic compounds in sensory neurons. Proton moderation of capsaicin-induced currents and/or the activation of proton-induced currents are also thought to contribute in the neuron’s sensation of pain (Peterson 2003b).

Desensitization and Death of the Cell
Excessive administration of capsaicin can cause deterioration in the sensitivity of the sensory neuron (Priestley 2003). Prolonged exposure to it for a long period of time could even result in death and destruction of small sensory neurons such as DRG neurons and C fibers (Fitzgerald 2003). Examples of capsaicin-induced cell death are shown in Figure 4a and 4b (Caterina 1997). Figure 4a shows the percentage of dead cells that came about after coming into contact with capsaicin for 7 hours at a temperature of 37 degrees Celsius. Figure 4b shows an image of pcDNA3 and VR1 cells before (left) and after (right) being exposed to capsaicin at a concentration of 3M for 4 hours (Caterina 1997). As portrayed in Figure 4b, treatment of a sensory neuron with a sufficiently large concentration of capsaicin molecules could end up impairing the sensitivity of the neuron for a longer period of time and/or cause death of the cell (Caterina 1997; Cholewinski 2003). This is consistent with the fact that the addition of hydrogen ions significantly enhances the effect that capsaicin induces upon the cell. It is this characteristic of capsaicin that led to further study of the molecule in order to achieve a better understanding of how pain is perceived by the human body. Its ability to impair a neuron’s sensitivity to pain has also led to its use as a possible therapeutic tool involving pain relief (Peterson 2003b). It is also important to note that at low concentrations, capsaicin impairs only the neuron’s sensitivity to further exposure from other capsaicin molecules without degrading its sensitivity to other external stimuli (Dray 2002b; Maggi 2003). The desensitization of the sensory neuron was found to be caused by and completely dependent upon the presence of extracellular calcium ions (Cholewinski 2003). However, the neuron’s loss in sensitivity to capsaicin molecules following exposure to it at low concentrations occurred despite the lack of calcium ions present on the extracellular surface. A possible explanation to a nociceptor’s loss in sensitivity to capsaicin molecules alone may be highlighted by the events that occur after capsaicin comes into contact with one of the neuron’s peripheral nerves. Peripheral nerves coming into contact with capsaicin could result in a blockage of the conduction of the sensory fibers. The blocked conduction would thus cause the signal transduction at the nerve endings to weaken severely and cause a subsequent degradation in the neuron’s sensitivity to additional capsaicin molecules. The desensitization would be dependent on the concentration of the capsaicin molecule and on the time of the neuron’s exposure to it. Osmotic changes which come as a result of a buildup of intracellular ions are also thought to contribute to the desensitization of a neuron (Dray 2002a).

Conclusion
In Conclusion, capsaicin’s ability to evoke a “burning sensation” on human tissues was verified as being a result of its interactions with vanilloid receptors on sensory neurons. These interactions were revealed to be virtually identical to heat-evoked responses from sensory neurons and to also be influenced by the concentration of protons in the extracellular medium. Repeated administrations of capsaicin lead to the desensitization of the neuron to further treatment from capsaicin. Under different circumstances, prolonged exposure to capsaicin resulted in the neuron’s loss in sensitivity to other external stimuli as well as death of the cell for some neurons. There are still however, some aspects about the activity of capsaicin that remain unclear. Future study of the molecule could possibly lead to a better understanding of how pain is perceived as well as the development of new drugs designed to alleviate pain (Caterina 1997).