**4. Discussion**

56 Pharmacology

Fig. 11. Photomicrographs showing c-Fos-positive neurons in the dorsal horn of L5 2 h after hindpaw injection of capsaicin alone (A), combined with CPCCOEt (B), with MCCG (C),

Table 1. Mean value of c-Fos-positive neurons in the dorsal horn of L5 2 h after s.c. injection of Glu receptors agonists and antagonists. The value in each group was represented mean ± S.E.M. obtained from at least 10 animals, and the difference of the means was analyzed with the Student's t-test. \* Significant difference at P< 0.05 between vehicle and capsaicin, or glutamate-treated group. # Significant difference at P< 0.05 between capsaicin and capsaicin+MK801, or capsaicin+CNQX, or capsaicin+CPCCOEt-treated group

with MSOP (D). A, B, C and D: ipisilateral side. Solid line indicates 100 μm .

We confirmed a large release of Glu immediately after the introduction of the catheter, followed by a rapid decrease, like in our previous study (Yonehara et al., 1987; Yonehara et al., 1992; Yonehara et al., 1995). Insertion of the polyethylene tube into the s.c. space of the rat instep did not evoke any inflammatory responses such as extravasation (Yonehara et al., 1995). All these data suggest that the basal levels of Glu in the s.c. perfusate were caused by neither acute noxious stimulation nor inflammation.

Topical application of capsaicin cream to the instep evoked a marked increase in Glu level in the s.c. perfusate, similar to the results in our previous study (Jin et al., 2006). In addition, electrical stimulation of the sciatic nerve or noxious heat stimulation (50C) also caused an increase of Glu level in the s.c. space, and this capsaicin-evoked Glu release was significantly decreased by daily high-dose pretreatment with capsaicin for three consecutive days (Jin et al., 2006).

The TRPV1 is located in a neurochemically heterogeneous population of small diameter primary afferent neurons (Tominaga et al., 1998). Furthermore, repeated exposure to highdose capsaicin selectively produces a prolonged influx of cations leading to desensitization of small-diameter sensory neurons to subsequent noxious stimulation (Yonehara et al., 1987; Lynn, 1990; Zhou et al., 1998; Caterina and Julius, 2001), while myelinated Afibers are insensitive to capsaicin (Jancso et al., 1977; Nagy et al., 1983; Michael and Priestly, 1999). These findings and the present results suggest that the activation of capsaicin-sensitive afferent fibers by capsaicin causes release of Glu from the peripheral endings via activation of peripheral TRPV1, particularly from those of small-diameter fibers possibly through a mechanism such as the axon-reflex pathway, or autocrine and/or paracrine. It is reasonable to speculate that axon-reflex mechanism is involved in capsaicin-induced Glu release observed in Figs. 1 and 2, as only nociceptive afferent fibers have the axon-reflex mechanism which is localized on superficial tissues exposed to noxious influences (Celander and Folkow, 1953).

Amount of capsaicin-induced Glu release was remarkably decreased by concomitant administration of ionotropic Glu receptors antagonists; MK801 and NBQX, and mGluR I antagonist; CPCCOEt in the hindpaw, but not by administration of group II and III mGluR antagonist; MCCG and MSOP. These results suggest that peripheral ionotropic Glu receptors and group I mGluR appear to play a role in mediating capsaicin-evoked increases in Glu release. The Glu release through the activation of TRPV1 could then further activate ionotoropic Glu receptors and group I mGluR on the same neuronal terminal or adjacent neighboring peripheral terminals. In this connection, there were evidences supporting the co-localization of peripheral NMDA and TRPV1 receptors on the same primary afferent terminal (Lam et al., 2003; Lam et al., 2004).

Activation of peripheral Glu receptors could lead to enhance the Glu release in the peripheral tissues and might alter TRPV1 receptor responsiveness to reinforce nociceptive responses. As it is necessary to investigate the interaction between TRPV1 and glutamate receptors by using specific receptor antagonists of TRPV1 in detail, the mechanism to account for the antagonism of peripheral Glu receptors contributes to inhibit capsaicininduced Glu release remains unanswered. However, it may be possible that glutamate receptors play a pivotal role for the activation of TRPV1 in the peripheral terminals. This

Interactions Between Glutamate Receptors and TRPV1 Involved in

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Cairns, B.E., Sessle, B.J., and Hu, J.W., 1998. Evidence that excitatory amino acid receptors

Carlton, S.M., Hargett, G.L., and Coggeshall, R.E., 1995. Localization and activation of

Carlton, S.M., Zhou, S., and Coggeshall, R.E., 1998. Evidence for the interaction of glutamate

Carlton, S.M., 2001. Peripheral excitatory amino acids. Current Opinion in Pharmacology, 1,

Carlton, S.M., and Hargett, G.L., 2007. Colocalization of metabotropic glutamate receptors in

Caterina, M.J., Schumacher, M.A., Tominaga, M., Rosen, T.A., Levine, J.D., and Julius, D.,

Caterina, M.J., and Julius, D., 2001. The vanilloid receptor: a molecular gateway to the pain

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Davidson, E.M., Coggeshall, R.E., and Carlton, S.M., 1997. Peripheral NMDA and non-

Davidson, E.M., and Carlton, S.M., 1998. Intraplantar injection of dextrorphan, ketamine or memantine attenuates formalin-induced behaviors. Brain Res. 785, 136-142. Du, J., Zhou, S., Coggeshall, R.E. and Carlton, S.M., 2003. *N*-methyl-d-aspartate-induced

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within the temporomandibular joint region are involved in the reflex activation of

glutamate receptors in unmyelinated axons of rat glabrous skin. Neurosci. Lett. 197,

idea is supported by the results that the intraplantar injection of ionotoropic Glu receptors and group I mGluR agonists evoked dose-dependent thermal hyperalgesia. Moreover, it is very interesting to note that injection of Glu receptors antagonists alone did not produce any changes on withdrawal latency, and intraplantar co-injection of ionotropic Glu receptors and group I mGluR antagonists with capsaicin not only antagonized capsaicin-induced hyperalgesia, but also resulted in remarkable longer withdrawal latency to heat irradiation.

Concerning the mechanism that ionotoropic Glu receptors and mGluR antagonists produced remarkable analgesic action in the presence of capsaicin, there is evidence that capsaicin injected into the rat temporomandibular joint evoked a dose-dependent increase in jaw muscle electromyographic activity. This capsaicin-evoked increase in electromyographic activity was attenuated by ipsilateral injection of NMDA receptor antagonists into the temporomandibular joint (Lam et al. 2005). This finding and our present results indicate that the activation of peripheral Glu receptors, especially ionotropic Glu receptors and group I mGluR could be indispensable in the mechanisms whereby capsaicin evokes nociceptive responses.

The ionotropic, and metabotropic subunits of Glu receptors are present in DRG cell bodies and on unmyelinated fibers in the glabrous skin of the mammalian foot (Carlton et al., 1995; Bhave et al., 2001; Carlton et al., 2001; Sato et al., 1993; Carlton et al., 2007). It is well established that the excitatory amino acids in the peripheral endings of small-diameter afferent fibers contribute to development and/or maintenance of pain in humans (Nordlind et al., 1993; Warncke et al., 1997) and in laboratory animals (Davidson et al., 1997; Cairns et al., 1998; Davidson et al., 1998). For example, peripherally applied NMDA and non-NMDA receptor antagonists attenuate or block nociceptive behaviors in several animal models of inflammation (Jackson et al., 1995; Lawand et al., 1997; Carlton et al., 1998).

In the present study, we examined the c-Fos expression in spinal cord dorsal horn following injection of drugs associated with glutamate receptors with/without capsaicin into the hindpaw. c-Fos is rapidly and transiently induced in cells of the spinal dorsal horn after noxious stimulation (Hunt et al., 1987, Strassman and Vos, 1993, Takemura et al., 2000), c-Fos has been widely used as a marker for analyzing nociceptive processing.

Our present data support the view that Glu receptors, in particular, ionotropic Glu receptors and group I mGluR existing in peripheral ending of capsaicin-sensitive afferent fibers play an important role on development and/or maintenance of pain following excitation of TRPV1. In addition, the formulation of the peripheral ionotoropic Glu receptors and group I mGluR antagonists that do not cross the blood brain barrier may be of potential benefit by reducing peripheral nociceptive excitability, and therefore they could provide a new therapeutic target to pain control in the periphery.

#### **5. References**

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idea is supported by the results that the intraplantar injection of ionotoropic Glu receptors and group I mGluR agonists evoked dose-dependent thermal hyperalgesia. Moreover, it is very interesting to note that injection of Glu receptors antagonists alone did not produce any changes on withdrawal latency, and intraplantar co-injection of ionotropic Glu receptors and group I mGluR antagonists with capsaicin not only antagonized capsaicin-induced hyperalgesia, but also resulted in remarkable longer withdrawal latency to heat irradiation. Concerning the mechanism that ionotoropic Glu receptors and mGluR antagonists produced remarkable analgesic action in the presence of capsaicin, there is evidence that capsaicin injected into the rat temporomandibular joint evoked a dose-dependent increase in jaw muscle electromyographic activity. This capsaicin-evoked increase in electromyographic activity was attenuated by ipsilateral injection of NMDA receptor antagonists into the temporomandibular joint (Lam et al. 2005). This finding and our present results indicate that the activation of peripheral Glu receptors, especially ionotropic Glu receptors and group I mGluR could be indispensable in the mechanisms whereby capsaicin evokes nociceptive

The ionotropic, and metabotropic subunits of Glu receptors are present in DRG cell bodies and on unmyelinated fibers in the glabrous skin of the mammalian foot (Carlton et al., 1995; Bhave et al., 2001; Carlton et al., 2001; Sato et al., 1993; Carlton et al., 2007). It is well established that the excitatory amino acids in the peripheral endings of small-diameter afferent fibers contribute to development and/or maintenance of pain in humans (Nordlind et al., 1993; Warncke et al., 1997) and in laboratory animals (Davidson et al., 1997; Cairns et al., 1998; Davidson et al., 1998). For example, peripherally applied NMDA and non-NMDA receptor antagonists attenuate or block nociceptive behaviors in several animal models of

In the present study, we examined the c-Fos expression in spinal cord dorsal horn following injection of drugs associated with glutamate receptors with/without capsaicin into the hindpaw. c-Fos is rapidly and transiently induced in cells of the spinal dorsal horn after noxious stimulation (Hunt et al., 1987, Strassman and Vos, 1993, Takemura et al., 2000), c-

Our present data support the view that Glu receptors, in particular, ionotropic Glu receptors and group I mGluR existing in peripheral ending of capsaicin-sensitive afferent fibers play an important role on development and/or maintenance of pain following excitation of TRPV1. In addition, the formulation of the peripheral ionotoropic Glu receptors and group I mGluR antagonists that do not cross the blood brain barrier may be of potential benefit by reducing peripheral nociceptive excitability, and therefore they could provide a new

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**1. Introduction** 

molecular pharmacology of these compounds.

(+)RNA/(-)DNA duplex (Ghosh et al., 1997).

**2. Mechanism of action** 

**3** 

*USA* 

**Molecular Pharmacology of** 

Brian D. Herman and Nicolas Sluis-Cremer *University of Pittsburgh, Department of Medicine,* 

*Division of Infectious Diseases, Pittsburgh,* 

**Nucleoside and Nucleotide HIV-1 Reverse Transcriptase Inhibitors** 

In 1985, 3'-azido-thymidine (AZT, zidovudine) was identified as the first nucleoside analog with activity against human immunodeficiency virus type 1 (HIV-1) (Mitsuya et al., 1985, 1987; Mitsuya & Broder, 1986), the etiologic agent of acquired immunodeficiency syndrome (Barre-Sinoussi et al., 1983; Gallo et al., 1984). This seminal discovery showed that HIV-1 replication could be suppressed by small molecule chemotherapeutic agents, and provided the basis for the field of antiviral drug discovery. Zidovudine was approved by the United States of America Food and Drug Administration for the treatment of HIV-1 infection in 1987. In the 26 years since, an additional seven nucleoside or nucleotide analogs have been approved, while several others are in clinical development. This chapter will provide a summary of the

Retroviruses such as HIV-1 carry their genomic information in the form of (+)strand RNA, but are distinguished from other RNA viruses by the fact that they replicate through a double-stranded DNA that is integrated into the host cell's genomic DNA (Temin & Mizutani, 1970; Baltimore, 1970; DeStefano et al., 1993). While the conversion of viral RNA into double-stranded DNA intermediate is a complex process, all chemical steps are catalyzed by the multi-functional viral enzyme reverse transcriptase (RT). HIV-1 RT exhibits two types of DNA polymerase activity, an RNA-dependent DNA polymerase activity that synthesizes a (-)strand DNA copy of the viral RNA, and a DNA-dependent DNA polymerase activity that generates the (+)strand DNA (Peliska & Benkovic, 1992; Cirno et al., 1995). RT also has ribonuclease H activity that degrades the RNA in the intermediate

Once metabolized by host cell enzymes to their triphosphate forms (described in more detail below), nucleoside analogs inhibit HIV-1 reverse transcription. As such, they are typically referred to as nucleoside RT inhibitors (NRTI). NRTI-triphosphates (NRTI-TP) inhibit RTcatalyzed proviral DNA synthesis by two mechanisms (Goody et al., 1991). First, they are

