Gábor Petho

Investigation of the role of TRPV1 receptors in acute and chronic nociceptive processes using gene-deficient mice.

&NA; Capsaicin‐sensitive, TRPV1 (transient receptor potential vanilloid 1) receptor‐expressing primary sensory neurons exert local and systemic efferent effects besides the classical afferent function. The TRPV1 receptor is considered a molecular integrator of various physico‐chemical noxious stimuli. In the present study its role was analysed in acute nociceptive tests and chronic neuropathy models by comparison of wild‐type (WT) and TRPV1 knockout (KO) mice. The formalin‐induced acute nocifensive behaviour, carrageenan‐evoked inflammatory mechanical hyperalgesia and partial sciatic nerve lesion‐induced neuropathic mechanical hyperalgesia were not different in WT and KO animals. Acute nocifensive behaviour after intraplantar injection of phorbol 12‐myristate 13‐acetate, an activator of protein kinase C (PKC), was absent in TRPV1 KO animals showing that PKC activation elicits nociception exclusively through TRPV1 receptor sensitization/activation. Thermal hyperalgesia (drop of noxious heat threshold) and mechanical hyperalgesia induced by a mild heat injury (51 °C, 15 s) was smaller in KO mice suggesting a pronociceptive role for TRPV1 receptor in burn injury. Chronic mechanical hyperalgesia evoked by streptozotocin‐induced diabetic and cisplatin‐evoked toxic polyneuropathy occurred earlier and were greater in the TRPV1 KO group. In both polyneuropathy models, at time points when maximal difference in mechanical hyperalgesia between the two groups was measured, plasma somatostatin concentrations determined by radioimmunoassay significantly increased in WT but not in TRPV1 KO mice. It is concluded that sensitization/activation of the TRPV1 receptor plays a pronociceptive role in certain models of acute tissue injury but under chronic polyneuropathic conditions it can initiate antinociceptive counter‐regulatory mechanisms possibly mediated by somatostatin released from sensory neurons.

Nociceptor excitation by thermal sensitization--a hypothesis.

Publisher Summary This chapter discusses the nociceptor excitation by thermal sensitization. Sensitivity to noxious heat of the skin and oral cavity appears to be a useful protective mechanism. However, many deep tissues including skeletal muscle, dura, testis, and colon are also reported to be innervated by polymodal, mechano-heat-sensitive primary afferents. Discovery of heat-evoked ionic currents and of the heat-activated capsaicin receptor (VRl) and its homologue (VRL-1) in sensory neurons of the rat dorsal root ganglion (DRG) has shed important new light on the transduction mechanisms by which heat, inflammatory mediators, and chemical irritants may excite nociceptors and contribute to pain. There is growing evidence that several inflammatory mediators including BK (possibly PGE2 and histamine) and low pH as well as the sensitizing model agent capsaicin act on nociceptors, at least partly, by lowering the threshold of their heat transduction mechanisms so profoundly that body or lower tissue temperatures become a driving force of excitation and pain. This unifying theory is attractive for pharmaceutical research and development because diverse and multiple nociceptive mechanisms are converging onto one novel target, heat-activated ion channels, which, however, await final molecular identification.

Relative roles of protein kinase A and protein kinase C in modulation of transient receptor potential vanilloid type 1 receptor responsiveness in rat sensory neurons in vitro and peripheral nociceptors in vivo.

The function of the transient receptor potential vanilloid type 1 capsaicin receptor is subject to modulation by phosphorylation catalyzed by various enzymes including protein kinase C and cAMP-dependent protein kinase. The aim of this study was to compare the significance of the basal and stimulated activity of protein kinase C and cAMP-dependent protein kinase in transient receptor potential vanilloid type 1 receptor responsiveness in the rat in vitro by measurement of the intracellular calcium concentration in cultured trigeminal ganglion neurons and in vivo by determination of the behavioral noxious heat threshold. KT5720, a selective inhibitor of cAMP-dependent protein kinase, reduced the calcium transients induced by capsaicin or the other, much more potent transient receptor potential vanilloid type 1 receptor agonist resiniferatoxin in trigeminal sensory neurons and diminished the drop of the noxious heat threshold (heat allodynia) evoked by intraplantar resiniferatoxin injection. Chelerythrine chloride, a selective inhibitor of protein kinase C, failed to alter either of these responses, although it inhibited the effect of phorbol 12-myristate 13-acetate in the in vitro assay. Staurosporine, a rather nonselective protein kinase inhibitor, failed to reduce the capsaicin- and resiniferatoxin-induced calcium transients but inhibited the resiniferatoxin-evoked heat allodynia. Dibutyryl-cAMP and phorbol 12-myristate 13-acetate, activator(s) of cAMP-dependent protein kinase and protein kinase C, respectively, enhanced the effect of capsaicin in the calcium uptake assay while forskolin, an activator of adenylyl cyclase, augmented that of resiniferatoxin in the heat allodynia model. None of the protein kinase inhibitors or activators altered the calcium transients evoked by high potassium, a nonspecific depolarizing stimulus. It is concluded that basal activity of cAMP-dependent protein kinase, unlike protein kinase C, is involved in the maintenance of transient receptor potential vanilloid type 1 receptor function in somata of trigeminal sensory neurons but stimulation of either cAMP-dependent protein kinase or protein kinase C above the resting level can lead to an enhanced transient receptor potential vanilloid type 1 receptor responsiveness. Similar mechanisms are likely to operate in vivo in peripheral terminals of nociceptive dorsal root ganglion neurons.

Bradykinin-induced nociceptor sensitization to heat is mediated by cyclooxygenase products in isolated rat skin

Bradykinin can excite C‐polymodal nociceptors and sensitize them to heat and it can also enhance prostaglandin synthesis, but it is unclear whether these effects are causally related. The role of cyclooxygenase products was investigated using two enantiomers of the cyclooxygenase inhibitor flurbiprofen of which S(+)‐ is more potent than R(–)‐flurbiprofen. Single‐unit activity was recorded from mechano‐heat‐sensitive, polymodal C‐fibers in the isolated rat skin‐saphenous nerve preparation. Bradykinin pretreatment (10 µm, 5 min) induced a 219 ± 26% increase in the number of spikes evoked by noxious heat stimulation and a drop in the heat threshold by 5.2 ± 0.6 °C in a fully reproducible manner. S(+)‐flurbiprofen (1 µm) abolished the bradykinin‐induced heat sensitization but did not alter the unconditioned heat response itself. Under R(–)‐flurbiprofen (1 µm) bradykinin still induced a significant heat sensitization which was reduced by 33 ± 21% (P = 0.11) of its previous extent; this effect may be due to the limited purity of the enantiomer preparation or to a cyclooxygenase‐independent action of flurbiprofen. The heat sensitization suppressed by S(+)‐flurbiprofen could be significantly restored (to 43 ± 12%) by addition of PGE2 plus PGI2 (10 µm both) to bradykinin. Neither S(+)‐ nor R(–)‐flurbiprofen had an influence on the magnitude of the excitatory effect of bradykinin. It is concluded that (i) cyclooxygenase products are the main mediators of nociceptor sensitization to heat following bradykinin treatment in the isolated rat skin; (ii) PGE2/I2 are essential but perhaps not the only relevant cyclooxygenase products involved and (iii) neither S(+)‐ nor R(–)‐flurbiprofen inhibit the unconditioned noxious heat response and the excitatory bradykinin response of the polymodal C‐nociceptors.

Effects of TRPV1 receptor antagonists on stimulated iCGRP release from isolated skin of rats and TRPV1 mutant mice

&NA; Capsaicin antagonists including ruthenium red, capsazepine and iodo‐resiniferatoxin (I‐RTX) have recently been shown to inhibit the activation by noxious heat of the capsaicin receptor (TRPV1) expressed in non‐neuronal host cells, and natively, in cultured dorsal root ganglion cells. Noxious heat has been shown to release immunoreactive calcitonin gene‐related peptide (iCGRP) from the isolated rat skin. In this model, ruthenium red, I‐RTX as well as capsazepine 10 &mgr;M caused no alteration in iCGRP release at 32 °C by themselves whereas capsazepine 100 &mgr;M doubled it reversibly. In wild‐type mice 100 &mgr;M capsazepine also stimulated iCGRP release while it was without effect in TRPV1 knockout littermates. In the rat skin, both ruthenium red and capsazepine (10/100 &mgr;M) reduced and abolished, respectively, capsaicin‐induced iCGRP release while I‐RTX (1/10 &mgr;M) was ineffective. Only ruthenium red 100 &mgr;M showed an unspecific effect inhibiting iCGRP release induced by KCl. Ruthenium red and capsazepine (10/100 &mgr;M) caused no significant alteration of iCGRP release induced by heat stimulation at 47 °C. Employing 45 °C stimulation intensity, capsazepine and I‐RTX (in the higher concentrations) showed a significant facilitatory effect on the heat response suggesting a partial agonistic action of the compounds. It is concluded that noxious heat‐induced iCGRP release in the isolated rat skin occurs through a mechanism that is not inhibited by TRPV1 antagonism reflecting a different pharmacological profile of noxious heat transduction in terminals of sensory neurons compared to that in cultured cell bodies and TRPV1‐transfected host cells.

Direct evidence for activation and desensitization of the capsaicin receptor by N-oleoyldopamine on TRPV1-transfected cell, line in gene deleted mice and in the rat

Effects of the endogenous lipid N-oleoyldopamine (OLDA) were analyzed on the rTRPV1-expressing HT1080 human fibrosarcoma cell line (HT5-1), on cultured rat trigeminal neurons, on the noxious heat threshold of rats and on nocifensive behavior of TRPV1 knockout mice. The EC(50) of capsaicin and OLDA on (45)Ca accumulation of rTRPV1-expressing HT5-1 cells was 36 nM and 1.8 microM, respectively. The efficacy of OLDA was 60% as compared to the maximum response of capsaicin. OLDA (330 nM to 3.3 microM) caused a transient increase in fluorescence of fura-2 loaded cultured small trigeminal neurons of the rat and rTRPV1-transfected HT5-1 cells measured with a ratiometric technique. Repeated application of OLDA and capsaicin caused similar desensitization in the Ca(2+) transients both in cultured neurons and rTRPV1-transfected HT5-1 cells. In the rat intraplantar injection of OLDA (5 nmol) decreased the noxious heat threshold by 6-9 degrees C and this response was strongly inhibited by the TRPV1 antagonist iodoresiniferatoxin (0.05 nmol intraplantarly (i.pl.)). In wild-type mice OLDA (50 nmol i.pl.) evoked paw lifting/licking which was significantly less sustained in TRPV1 knockout mice. It is concluded that on TRPV1 capsaicin receptors OLDA is 50 times less potent than capsaicin and it might serve as an endogenous ligand for TRPV1 in the rat, but more likely in humans.

Noradrenergic and peptidergic sympathetic regulation of cutaneous microcirculation in the rat

Cutaneous microcirculatory changes were measured by laser-Doppler flowmetry in response to electrical stimulation of sympathetic efferent fibres of the rats saphenous nerve. After perineural capsaicin (2%) pretreatment, electrical stimulation of the peripheral stump of the cut saphenous nerve evoked a reduction in blood flow (vasoconstriction) followed by a minimal enhancement. This late vasodilatation was further reduced by resiniferatoxin (1 microg/kg i.v.), and vasoconstriction was abolished by guanethidine (8 mg/kg i.v.), indicating the involvement of sensory and sympathetic fibres in the respective responses. The vasoconstrictor response was analysed after blockade of antidromic vasodilatation by combined capsaicin-resiniferatoxin pretreatment. alpha-Adrenoceptor antagonists (1 mg/kg phentolamine, 0.5 mg/kg prazosin and 1 mg/kg GYKI-12743 (RS-2-(3)N-(2-benzo;1,4i-dioxanyl)-methylamino(propyl)-3(2H) -piridazinone hydrochloride) inhibited, but did not eliminate the blood flow reduction evoked by 3 Hz stimulation. At 10 Hz stimulation significant inhibition was obtained only with GYKI-12743. No inhibition was observed with propranolol (10 microg/kg) on any occasion. A functional neuropeptide Y antagonist, alpha-trinositol (D-myo-inositol-1,2,6-trisphosphate, PP56; 50 mg/kg i.v.), markedly diminished the vasocontrictor response remaining after treatments with the alpha-adrenoceptor blocking agents. Inhibition was more pronounced at 10 Hz. Since 3 Hz corresponds to an average, and 10 Hz approaches the maximal firing rate of the sympathetic efferents, these results emphasise the significant role of neuropeptide Y in regulation of the cutaneous microcirculation by sympathetic fibres under physiological circumstances, particularly during high activity.

Role of bradykinin in the hyperaemia following acid challenge of the rat gastric mucosa

1 This study examined whether the hyperaemia following acid challenge of the rat gastric mucosa involves bradykinin, a peptide formed in response to tissue injury. 2 Gastric mucosal blood flow in urethane‐anaesthetized rats was assessed by the hydrogen gas clearance method. Infusion of a bradykinin solution (10 μm) into the gastric wall augmented gastric mucosal blood flow by a factor of 2.3, an effect that was prevented by the bradykinin B2 antagonist Hoe‐140 (icatibant; 100 μmol kg−1, i.v.). 3 I.v. injection of bradykinin (20–60 nmol kg−1) caused a 2.3‐3.5 fold increase in blood flow through the left gastric artery as measured by the ultrasonic transit time shift technique. The hyperaemic effect of bradykinin in this gastric artery was also prevented by Hoe‐140 (100 μ mol kg−1, i.v.). 4 Gastric acid backdiffusion was evoked by perfusing the stomach with 15% ethanol, to break the gastric mucosal barrier, in the presence of luminal acid. Depending on the concentration of acid (0.05 and 0.15 M HC1), this procedure increased gastric mucosal blood flow by a factor of 1.6‐2.8 and caused formation of gross damage in 1.5‐3% of the glandular mucosa. Hoe‐140 (100 μmol kg−1, i.v.) failed to alter the moderate vasodilatation seen in the presence of 0.05 M HC1 but significantly (P < 0.05) attenuated the marked hyperaemia and enhanced the gross mucosal damage observed in the presence of 0.15 M HC1. 5 These data show that bradykinin is able to enhance gastric mucosal blood flow via activation of B2 receptors. It appears as if this kinin is formed during severe acid challenge of the rat gastric mucosa and participates in the hyperaemic reaction to gastric acid backdiffusion.

Effects of analgesics on the plantar incision-induced drop of the noxious heat threshold measured with an increasing-temperature water bath in the rat.

The behavioural noxious heat threshold i.e. the lowest temperature evoking nocifensive behaviour was previously shown to decrease in short-lasting, but not in sustained, inflammatory thermal hyperalgesias. The aim of this study was to examine whether the surgical incision-induced lasting heat hyperalgesia involves a drop of the heat threshold and to assess the effects of conventional opioid and non-opioid analgesics in this model. One of the hind paws of rats was immersed into a water bath whose temperature was near-linearly increased from 30 degrees C until the animal withdrew its paw from the water. The corresponding bath temperature was considered as the behavioural noxious heat threshold. Hyperalgesia to heat was induced by a standardized plantar surgical incision performed under pentobarbital anaesthesia which led to a 5-7 degrees C decrease of the noxious heat threshold for seven days. Morphine, diclofenac, and paracetamol administered intraperitoneally 18 h after incision dose-dependently inhibited the drop of heat threshold with minimum effective doses of 0.3, 1, and 100 mg/kg, respectively, as assessed 20, 30 and 40 min after treatment. Thermal hyperalgesia was also decreased by intraplantar treatment with morphine (10 microg) or diclofenac (100 microg). In conclusion, the incision-induced sustained thermal hyperalgesia in rats involves a drop of the heat threshold suggesting that mechanisms of postsurgical pain are distinct from those of pure inflammatory pain. The thermal antihyperalgesic actions of systemically and/or locally applied morphine, diclofenac and paracetamol could be detected with high temporal resolution and sensitivity in this model.

Mediation by CCKB receptors of the CCK‐evoked hyperaemia in rat gastric mucosa

1 Cholecystokinin octapeptide (CCK‐8) and gastrin‐17 augment gastric mucosal blood flow in the rat. The present study examined whether the gastric vasodilator effect of these peptides is mediated by CCKA or CCKB receptors. 2 Intravenous injection of CAM‐1481 (1 mg kg−1), a dipeptoid antagonist of CCKA receptors, or CAM‐1028, a dipeptoid CCKB receptor antagonist (1 mg kg−1), had no effect on basal gastric mucosal blood flow as determined by the clearance of hydrogen in urethane‐anaesthetized rats. 3 Intravenous infusion of CCK‐8 or gastrin‐17 (8–200 pmol min−1) increased gastric mucosal blood flow in a dose‐dependent fashion. The CCKB receptor antagonist, CAM‐1028, significantly attenuated the hyperaemic response to CCK‐8 and gastrin‐17 whereas the CCKA receptor antagonist, CAM‐1481, did not antagonize CCK‐8 but caused a slight attenuation of the vasodilator response to gastrin‐17. 4 The selectivity of the two antagonists was proved by the findings that CAM‐1028, but not CAM‐1481, inhibited gastric acid secretion evoked by CCK‐8 or gastrin‐17 (CCKB receptor assay) while CAM‐1481, but not CAM‐1028, inhibited the CCK‐8‐induced contraction of guinea‐pig isolated gall bladder strips (CCKA receptor assay). 5 These data show that the actions of CCK‐8 and gastrin‐17 to increase mucosal blood flow in the rat stomach are primarily mediated by CCKB receptors.