Sensory neuron-specific actions of capsaicin: mechanisms and applications

TiPS

Stuart E&win and Jh-tos SzolcsAnyi

Capsaicin, the pungent ingredient in peppers of the Cilpsicrfnr family, has been used widely as a tool in studies on primary afferent sensory neurons. One well-known property of capsaicin is to excite some, but not all, sensory neurons and so elicit the sensation of ‘burning’ pain’. A major use of capsaicin has been to damage selectively this subpopulation of sensory neurons and to deplete candidate neurotransmitter substances like substance P and other sensory neuropepddes’. Despite this use of capsaicin as a tool, surprisingly little was known until recently about its mechanisms of action. In this review, we focus on two main areas: firstly some recent studies that illuminate the ways in which capsaicin can act on sensory neurons; and secondly the physiological effects and potential therapeutic applications of capsaidn-like compounds.

nociceptive neurons with conduction velocities in the ranges of both C- and A&fibres. These single fibres are responsive to noxious heat, noxious mechanical and noxious chemical stimuli. Warm receptors are also activated by capsaicin, but other C-fibres (high- and low-threshold mechanoreceptors, cold receptors), other A&fibres and A@fibres are unaffected. Some visceral nerves (interoceptors) are capsaicin scnsitive, but once again the effect is cell selective and restricted to slowly conducting afferents. Cap-

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saicin sensitivity appears to be a stable phenotypic property of adult mammalian neurons that IS retained even when the cells are maintained in cell culture for many weeks’. The close correlation between capsaicin sensitivity and polymodal nociceptnrs found in adult animals does not extend to newborn rats and mice3. In devefopiyg animals, cap?aicin ser;si+’.r.i:~ LS shared by other C-fib:e afferents. Nc;ertheless capsaicin sensitivity is restricted to afferent neurons and there is no suggestion that other peripheral neurons are sensitive at any stage of development’.“.5-?. All mammalian species examined either behaviourally or electrophysiologically have proved to be sensitive to capsaicin. However, this is not a general property of vertebrates as the agent is almost completely ineffective on sensory neurons from birds (pigeons and chick)3*H*9. Mechanisms of action The mechanisms that underlie the unique sensory neuronspecific actions of capsaicin have been revealed by a combination of electrophysiological and ion-flux studies. 0 Agonism. Recent studies on acutely isolated and cultured sensory neurons have provided a de-

depolarize to -) ~~fes~old.* Pi%%.h c-

DIRECT STIMULATION OF TRANSMITTER RELEASE

Recordings made from identified single nerve fibres have demonstrated that capsaicin acts with great cellular specificity”. In the skin of aduit rats, cats and rabbits, capsaicin excites polymodal

BLOCK LYSIS

Fig.

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Scheme of action

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capsaicin.

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iIF TRANSMITTER RELEASE

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tailed insight into the ionic basis of the action of capsaicin on visceral and somatic sensory neurons. Voltage-clamp experiments on nodose and dorsal root gang!ion neurons demonstrated that capsaicin evokes an inward, depolarizing current in some but not ail neurons’“~“. This inward current is associated with a conductance increase, which suggests that ion channels are opened by capsaicin (see below). The channel is relatively nonselective for cations, but anions such as Cl- do not contribute directly to the cell depolarization’,“. Both monovalent and divalent cations carry the current, with a permeability sequence Ca2+ > Mg2+ > K+ > Na+. In hysiological conditions both CaY+ and Na+ ions will flow into the cell and KC ions will flow out (Fig. 1). The net effect will be a current that depolarizes the cell towards -0 mV. The influx of Ca2+ has been followed in radiotracer flux experiments on cultured rat dorsal root ganglion neurons9. Capsaicin stimulates a large uptake of 45Ca2+, which accumulates within the neurons. It seems likely that mitochondria take up most of the Ca2+, since inhibitors of mitochondria! respiration, such as cyanide and dinitrophenol, can inhibit the accumulation. This interpretation is supported by electron ,microscopical studies which have shown swollen mitochondria and mitochondrial Ca2’ deposits in capsaicin-treated nerves and sensory ganglia 10*‘2 . The efflux of some other radioactive ions (22Na+, [ 14C]guanidinium and 86Rbf) is also evoked by capsaicin’. All these flux experiments show that capsaicin acts with an ECso of lOO300 nM, which agrees well with estimates from electrophysiological studies”*“. Single channel currents evoked by capsaicin have recently been recorded in isolated membrane patches from capsaicin-sensitive dorsal root ganglion neuronsI (Fig. 2~). These channels display the cation permeability expected from the recordings made in whole cells. The single channel conductance is about 100 pS at +60 mV and 20-30 pS at -60 mV even with identical NaCl solutions bathing both surfaces of the membrane. The available evidence shows that capsaicin acts directly to open

331

a resiniferatoxin

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capsaicin -..-closed

.....open

(1 )

SPA ‘-_

50ms

resiniferatoxin ....-closed .....open (1 )

Fig. 2. a: Structures of resiniferatoxin and capsaicin. b: %a’ ’ uptake into neonatal rat cultured dorsal root aanalion neurons. 0. resiniferatoxm: q caosaicin. c: Sinole-channel currents evoked by-resniferatoxin and capsaicin in an outside-out parch 6om a neonatal rat dorsal root ganglion neuron. Holding polential-80 mV. This patch contains several (> 3) channels; the current levels for a// channels closed and one channel open are indicated. Occasional openings of a second channel can be seen.

ion channels. No second messenger systems are necessary for capsaicin to act, although the influx of Ca’+ can stimulate Ca’+sensitive enzymes and so induce a secondary rise in the levels of intracellular mediators such as cGMP (Ref. 14). l EIfects on voltage-activated currents. High (0.2-300 PHI) concentrations of capsaicin can inhibit the activity of voltagegated Na+ and K+ channels in a range of including neurons guinea-pig and chick dorsal root ganglion neurons”“5. Also, capsaicin has been reported to increase reversibly the rate of inactivation of voltage-gated Ca2+ channels in guinea-pig dorsal root ganglion neurons’“. These effects of capsaicin appear to be unrelated to its cell-specific agonist

actions and the underlying mechanisms are unknown. Recent experiments on rat dorsal root ganglion neurons have revealed a dramatic cell-specific effect on voltage-gated Ca?+ currents. Ca’+ currents were greatly reduced in amplitude or abolished by a relatively brief (30 s) challenge with The inhibition apcapsaicin’7. peared to be long lasting (> 30 min) and occurred only in neurons that showed an agonist response to capsaicin. Furthermore it appeared to be dependent on the agonist-induced entry of Ca”, since no such effect was noted The when Ba’+ replaced Ca”. most plausible interpretation of this result is that, by arlowing Ca’+ to enter the cell, cap >aicin causes an increase in intracellular Cal+ concentration, which triggers a long-term inactivation of voltage-

3.2

sible for the intcr,lction

C-qstk-i~r 11561iterrrotoxin .A frequent u5e of capsaicm in neurob~ological in~es~~~~tions is to damage the sensitive sensory neurons. This neurotoxic action usually requires high concentrations of capsaicin and the effectivencs?; of the treatment depends on the age of the animals. Sensory neuronti from newborn animals (rats and mice) appear Co be more sensitive than those from adults and the axons also appear to be more susceptible to damage than the cell soma’. After neonatal treatment secondary changes like reorganization in the somatosensorv afferent pathway mav occur’. The initial mechanisms fbr neurotoxicity are essentially the same as those for agonism (see Fig. 1). Caps&in and related compounds such as resiniferatoxin {see below) damage neurons by two basic mechanisms (Ref. 19 and P. Hogan, unpublished). The first is related to an influx of Ca’+ into the cell that is not dependent on the presence of any other external ion. The way in which CaZ+ damages the cell is unclear, but probably involves the activation of Ca” -dependent enzymes and a long-lasting impairment of mitochondria. The second mechanism involves the influx of Na’ through the capsaicin-activated channels and also requires the passive movement of Cl- ions into the cell through other ionic pathways, The net effect is the uptake of NaCl; this is folJowed by an influx of water and so the cells swell and are damaged by osmotic lysis. Cells in culture can be killed by either of the mechanisms alone but the damage is more rapid when both operate as is the case in physiological solutions. These mechanisms are similar to those that are responsible for glutamateinduced neurotoxicity via NMDA receptors in CNS neurons”‘. A membrane receptor for capsaicin Several fines of evidence suggest that capsaicin acts on a specific membrane component: The cellular specificity and marked species differences in action imply that some unique cellular component(s) are responl

with capsaicin. of capsaicino The presence activated single channels in isolated patches of neuronal membrane suggests that capsaicin interacts directly with a membrane protein. o The responsiveness to capsaicin is under cellular control and is regulated by nerve growth factor (NG#, a protein that is known to influence the expression of certain neuronal characteristics. This regulation has been demonstrated in cultured dorsal root ganglion neurons from adult rats which, unlike neurons from newborn animals, do not need NGF for survival. Capsaicin sensitivity is retained for weeks in culture if NGF is included in the medium. If, however, NGF is removed the response to capsaicin is lost over the next three to four days. This loss is reversible and capsaicin sensitivity can be regained if NGF is added back to the culture medium. l Studies with cagsaicin analogues have demonstrated that small changes in structure can have profound effects on the agonist activity of the compounds’. 0 Photoaffinity probes based on capsaicin evoke long-lasting agonism on rat dorsal root ganglion neuronP. While these properties point to the existence of a specific capsaicin receptor whose expression is subject to cellular regulation, its physiological function and molecular identity are unclear. A ~ezu probe fur the capsaicin receptor The IIkelihood of characte~zing the receptor biochemically has improved greatly in the last two years with the identifizatien of resiniferatoxin’” ts a potent capsaicin-like agent’“*?“,‘“. Resiniferatoxin is a natural product found in some species of plants of the genus E~cpfzorbin;its structure shows some similarity to phorbol esters as wr I! as to capsaicin (Fig. 2a). Resiniferatoxin has been known for scme time to be a potent in~amnlato~ agent but the basis for thi: activity remained uncertain. It i. now clear that the toxin acts 11*.e capsaicin and, unlike the Fherbol esters, does not operate by the activation of protein kinase C. Capsaicin and

resiniferatoxin induce identical behavioural changes, such as hypothermia, when administered ia vim, and cross-desensitization between the hypothermic effects of the two agents has been demonstratedZ5. Furthermore, studies on single cells have shown that resiniferatoxin evokes the same changes in membrane permeability as capsaicin’” by opening the same ionic channels (Fig. 2~). The important difference is that resiniferatoxin is active at ~0~10000 times lower concentrations than capsaicin both irk u~oo’~,” and irr vitro. For example, ion-flux experiments on cultured dorsal root ganglion neuronsfV have shown that resiniferatoxin acts with an ECso of 1-2 nM (see Fig. 2b). The availability of a high affinity ligand like resiniferatoxin provides a valuable probe to begin to identify the capsaicin receptor by both protein isolation and molecular cloning techniques. Already the tritiated toxin has been used to demonstrate the presence of specific, saturable binding sites in dorsal root ganglion membranes but not in membranes from other brain areas”. Functions of capsaicin-sensitive neurons A major function of the capsaicin-sensitive sensory neurons is, of course, to respond to noxious stimuli. The release of peptides, such as substance I’, from the peripheral terminals of the same axons is also responsible for the neurogenic component of inflammation3s7. This suggests a plausible model for events at the peripheral nerve terminals. Noxious stimuli associated with tissue damage excite the nerves which in turn release mediators that contribute to the local inflammatory response. In addition to these two related functions, evidence is emerging for an efferent function for caps&in-sensiiive neurons on peripheral target tissues”7. For example, dilation of blood vessels in skin can be evoked by antidromic stimulation of afferent nerves at !ow frequencies (0.025-0.2 Hz) and this property is lost if animals are pretreated with capsaicin. The frequency of stimulation necessary to produce this effect is below that necessary to elicit the sensation of pain. The physiological

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relevance of this activity is suggested by the finding that skin blood flow is reduced after capsaicin pretreatment of nerves. Other suggested efferent roles for capsaicin-sensitive neurons are in the motor control of viscera, such as bronchoconstriction and increased mucociliary movement in airways, increased peristalsis of the gastrointestinal tract, constriction of the iris and contraction of the urinary bladder’. The likely basis for all these effector roles of afferent neurons is the action of peptides released from the peripheral afferent terminals’. Physiological effects and therapeutic potential Capsaicin has two, apparently contradictory effects. The first is stimulation. related to nerve Application of capsaicin can (1) evoke the sensation of pain and cause hyperalgesia. (2) activate autonomic reflexes, e.g. changes in blood pressure, and (3) release peptides and other putative transmitters from nerve terminals and so induce events such as bronchoconstriction and inflammation. The second, subsequent effect is that capsaicin can act as an antinociceptive and anti-inflammatory agent*‘. The duration of this important effect can range from hours to weeks and probably represents different levels of capsaicin action. Some long-lasting effects are undoubtedly associated with axonal degeneration and are usually seen when high concentrations of capsaicin are used. Antinociceptive and anti-inflammatory effects lasting only a few hours, however, show characteristics of functional changes without degeneration. For example, after i.a. injection, polymodal nociceptors can become unresponsive to one kind of stimulation (e.g. heat) but still respond to another (e.g. mechanical)‘s. Furthermore, after topical or syste:nic treatment the first nociceptive response to a noxious chemical challenge is unchanged but there is rapid fatigue to repeated cha11enges3,29. Such findings rule out a simple depolarization block of action potentials as an explanation for all the antinociceptive events. The central and peripheral terminals of afferent neurons are also important sites of action for capsaicin-induced antinociception””

333 and anti-inflammatory effects. The long-lasting inhibition of voltagegated Ca’+ channels” could reduce the amount of transmitter released by subsequent nerve activity even though the levels of putative transmitters are not depleted. An alteration of mitochondrial function induced by the accumulated Ca*+ might also contribute to the impaired function of capsaicinsensitive neurons. The sequence of events induced by capsaicin may simply reflect different degrees of agonism. In such a model, relatively weak stimulation would lead to a specific inhibition of neuronal function, while a high level of stimulation would cause cell damage. It may be possible to develop partial agonists that retain the beneficial analgesic effects but show little or no painful excitatory actions. The properties of known capsaicin analogues”,“’ suggest that this goal may be achievable. The potential therapeutic uses of capsaicin-like compounds will be as diverse as the functions of sensory neurons. Analgesia is one obvious possibility and preliminary studies have shown that topical application of capsaicin can have a beneficial effect in the treatment of post-herpetic neuralgia3’. The anti-inflammatory actions of capsaicin suggest another related area of use. Capsaicin analogues may also be effective in treating disorders that involve increased or inappropriate release of peptides from peripheral sensory terminals (e.g. asthma, psoriasis3*). Finally, compounds may be used to pctentiate some efferent functions of sensory neurons by stimulating peptide release. Such an action of capsaicin is probably responsible for its beneficial effects in the protection of gastric mucosa against both acid-induced”” and ethanol-induced lesions”’ and in the acceleration of e ithelial g growth in wounded skin3.. The excitatory and neurotoxic effects of capsaicin and resiniferatoxin are likely to preclude their genera1 use, but analogues with reduced excitatory activity may well have important thcrapcstic roles in a variety of clinical conditions. References

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