Transient Receptor Potential Channels and Local Anesthetics

16 May

Transient receptor potential (TRP) channels are widely distributed  in mam- malian tissue. The TRP family includes at least 20 related  cation channels that  are responsive  to a range of chemical and  physical stimuli  (Clapham et al. 2001). The functions  of TRP channels in sensory neurons  include re- sponding to painfully hot temperatures as well as to moderate warming and cooling and, perhaps, to noxious cold. Selectively expressed in many primary nociceptors,  TRPV1 mediates the response  to painful heat, to extracellular acidosis and to capsaicin, the pungent  component  in hot peppers (Caterina et al. 2000). TRPV1 is probably a tetramer  that  forms a nonspecific cation

channel, depolarizing the cell by allowing influx of Na+ and Ca2+ ions when it opens. The depolarization further activates other voltage-dependent channels,

leading to an increase in excitability (lowering of threshold)  for intermedi- ate steady depolarizations  or to a membrane  that  is strongly refractory  to stimulation  (i.e., a “depolarization  block”) for larger depolarizations.  Entry of Ca2+  through  the open  TRPV1 channel  can stimulate  further  increases in intracellular  Ca2+, e.g., via Ca2+-induced  Ca2+  release from intracellular stores. In addition,  intracellular  Ca2+  may be released in response  to cap- saicin that acts directly on membranes  of the endoplasmic reticulum, an or- ganelle that  stores calcium and may contain  TRPV1 channels  (Karai et al.

2004). In distal terminals  of skin, muscle, and joints and in central termi- nals in the dorsal horn of the spinal cord, activation of TRPV1 elevates in- tracellular Ca2+, resulting in the release of glutamate and substance P from the primary nociceptor  terminals  and the subsequent  activation of multiple

ionotropic  and  metabotropic  receptors  on postsynaptic  membranes.  Some of these activations may have synergistic interactions,  e.g., interactions  be- tween NK-1 receptors  for substance  P and N -methyl-d-aspartate (NMDA)- type glutamate  receptors.  These and other  interactions  may induce activa- tion of signal transduction pathways in spinal neurons that have far-reaching acute and chronic actions (Ji and Strichartz 2004), likely important for cen- tral sensitization that contributes  strongly to enduring pain from injury and inflammation.

Different local anesthetics appear to have different effects on TRPV1. For ex- ample, lidocaine and prilocaine suppress the capsaicin-induced increase in in-

tracellular Ca2+ mediated by recombinant TRPV1 receptors (Hirota et al. 2003), and bupivacaine  inhibits  the capsaicin-induced  current  in isolated sensory neurons, although tetracaine actually enhances this current (Komai and Mc- Dowell 2005). The reasons underlying this difference are not known, and the sensitivity of other TRP channels to local anesthetics have not been investi-

gated. Intracellular Ca2+ is elevated by many local anesthetics in several types of cells (Gold et al. 1998b; Johnson et al. 2002), very possibly through  their

ability to uncouple mitochondria and thereby release this organelle’s stored Ca2+ into the cytoplasm (Chance et al. 1968, 1969). Consequently, a number of Ca2+-dependent  processes are stimulated  by local anesthetics,  including calmodulin-dependent reactions  that  turn  on  kinases  and  inactivate  Ca2+ channels. Thus it is possible that part of the inhibition  by local anesthetics of both TRPV1 receptors and other ion channels is due not to a direct action at those sites but through the recruitment  of inhibitory pathways activated by such elevated Ca2+.

Local anesthetics appear to have a potentiating  effect on the long-lasting, impulse-inhibiting  actions of vanilloids. Both in isolated nerves (Shin et al.

1994) and  in rat  sciatic nerve  block in vivo (Kohane  et al. 1999), partial reduction  of action potentials  by local anesthetics is furthered  by the addi- tion of capsaicin, possibly through  the LA-induced elevation of intracellu- lar Ca2+, which is known to mediate the desensitization  of TRPV1 receptors

in sensory neurons  (Cholewinski et al. 1993). Capsaicin is currently  being used therapeutically for the relief of certain neuropathic  pains (Rowbotham et al. 1995, 1996), and  a related,  high-potency  vanilloid, resiniferatoxin,  is being developed for analgesia via selective blockade of TRPV1-containing pri- mary nociceptors  (Kissin et al. 2002, 2005a, b). Both of these applications involve the coadministration of local anesthetics  to block the generation  of impulses during initial application. Interactions between local anesthetics and the vanilloid receptor are thereby clinically important as well as scientifically interesting.

Mechanisms of Spinal and Epidural Anesthesia

The mechanism by which local anesthetics block impulses in peripheral nerves through inhibition of voltage-gated Na+ channel is well established. In contrast, the overall mechanism for spinal and epidural anesthesia is almost certainly more complex than simply the blockade of impulses in nerve roots, involving

pre- and postsynaptic receptors as well as intracellular pathways (Butterworth and Strichartz 1990). Both epidural and spinal (intrathecal)  delivery of local anesthetics results in their presence in the spinal cord, as well as in the spinal roots (Gokin and Strichartz 1999), allowing the possibility of altering both synaptic activity and impulse conduction and affecting the responses of cells

contained within the spinal cord, including interneurons, astrocytes and mi- croglia. At an even further  level of resolution,  local anesthetics can interact with membrane phospholipids and proteins and thereby affect various cellular activities. For example, local anesthetics can affect several subtypes of PKC (Mikawa et al. 1990; Nivarthi et al. 1996), PKA (Gordon et al. 1980), G pro- teins (Hagelüken et al. 1994), and the various receptors  that  activate them (Hitosugi et al. 1999). As noted above (Sect. 2.4 and 2.5), the effects of LAs on membrane-associated enzymes and second-messenger  systems operating in the cytoplasm are extensive. One means of assessing the actions of local anesthetics on integrated responses in spinal cord, such as might occur during a spinal or epidural blockade, is to measure their effects on intracellular signal transduction pathways that are activated by many different inputs and thus report the overall “activation status” of cells in the spinal cord. This approach is described in the following section.

MAP Kinases and Pain Processing in the Spinal Cord

The mitogen-activated protein kinase (MAPK) enzymes are a family of serine- threonine  protein kinases that are activated in response to many stimuli and play important roles in cellular signal transduction, both acutely by protein phosphorylation and chronically by affecting gene transcription. MAPKs were originally implicated in the regulation of mitosis, proliferation, differentiation, and survival of mammalian cells. ERKs 1 and 2 are the best-studied members of the MAPK family that transduce extracellular stimuli into intracellular re- sponses. ERK activation in spinal dorsal horn neurons  is driven by afferent activity of primary  nociceptors  (Ji et al. 1999). An increase in excitation in the superficial dorsal horn, caused by transmitters glutamate and substance P (Conn and Pin 1997), stimulates PKA and PKC, leading to the activation of ERK to pERK by its downstream  phosphorylation, and subsequently  to all

those processes that follow ERK signaling. Elevation of cytoplasmic Ca2+ also appears essential for noxious stimulation-induced ERK activation (Lever et al.

2003). All the above-listed factors have been shown to enhance nociceptive

processing in the spinal cord dorsal horn, one biochemical correlate of so- called “central sensitization” (Ji and Woolf 2001). Bupivacaine has been shown to suppress neuronal ERK activation in spinal cord slices by a variety of sub- stances, including those that act directly on dorsal horn neurons, giving insight into potential mechanisms for spinal anesthesia.

Presynaptic or Postsynaptic Sites for LA Inhibition

Synaptic transmission  in the spinal cord may be inhibited  directly by LAs through  the modification  of postsynaptic  receptors  as well as the blockade

of presynaptic  calcium channels that must function to stimulate the release of transmitters (see Sect. 2.3, above). Bupivacaine can inhibit  the elevation in dorsal horn  neurons  of pERK by excitatory ionotropic  receptor  agonists such  as capsaicin,  S-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate, and  NMDA, but  with no LA effects on ERK activa- tion by GPCRs such as bradykinin,  mGluR 1/5, and NK-1, by immunohis- tochemistry  using spinal slices as assayed (Yanagidate and Strichartz 2006). Receptors for capsaicin, acting on the ligand-gated vanilloid-sensitive tran- sient receptor potential channel, TRPV1, are exclusively located at presynap- tic terminals of nociceptive afferents. Thus, bupivacaine almost certainly has

presynaptic  actions, blocking Na+  channels, Ca2+  channels, and TRPV1 re- ceptor channels. On the other hand, AMPA and NMDA are classically located on postsynaptic  sites, but also can be found on presynaptic endings. Presy-

naptic  NMDA autoreceptors  appear  to facilitate the release of substance  P and glutamate in a positive-feedback network (Liu et al. 1997). In contrast, AMPA receptors  have an excitatory  postsynaptic  action,  but  also a strong

inhibitory  presynaptic  action  on  glutamate  release from  primary  afferent terminals  in the superficial dorsal horn (Lee et al. 2002). Therefore, the in- hibitory  effects of bupivacaine  on NMDA-induced pERK might be at both pre- and postsynaptic sites and AMPA-induced pERK must be occurring pre-

dominantly  through  activation  of postsynaptic  receptors.  Ionotropic  gluta- mate receptors are known targets of local anesthetics (Nishizawa et al. 2002; Sugimoto et al. 2003). Other studies also showed the “presynaptic”  actions of LAs to inhibit  Ca2+  channels  in dorsal root  ganglion neurons,  channels

that  are essential for the release of neurotransmitter during  depolarization (Rane et al. 1987). Therefore, LAs will act at many locations to alter func- tions during  spinal and epidural  anesthesia,  and any understanding of LA actions  in the spinal  cord  should  include  their  effects on a variety of ion channels,  membrane-related enzymes, and  intracellular  second  messenger pathways.

Local anesthetics at the neuraxis often provide analgesia synergistic with the actions of other drugs. Examples of the latter include Ca2+ channel blockers and opiates.

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