Evidence for the existence of substance P autoreceptor in the membrane of rat dorsal root ganglion neurons

Substance P, a putative peptide neurotransmitter contained in primary sensory neurons, is suggested to play a major role in nociceptive transmission. In the present study, the existence of substance P autoreceptor in dorsal root ganglion neurons was identified with a method we developed recently and substance P-activated inward current in the dorsal root ganglion neurons and its ionic mechanism were also explored preliminarily. The majority of the cells examined (68/76, 89.5%) were sensitive to external application of substance P (0.01-10 microM) with a concentration-dependent inward current. This current was found to result from the opening of nonselective ion channel, preferring the Na+ channel. The substance P-activated current can be suppressed by Cd2+ (0.05 microM), which suggested Ca2+ may also be involved. Soon after the neurons had been identified to be endowed with substance P receptor with whole-cell patch-clamp technique, 17 cells were chosen for immunocytochemical staining to detect substance P-immunoreactivity. Seven neurons which were classified into small and intermediate size were found to reveal substance P-immunoreactivity. Using this method we have identified the existence of substance P autoreceptor in rat DRG neurons.     

Tachykinin receptors in the small intestine of the cane toad (Bufo marinus): a radioligand binding and functional study

In this study, we have used radioligand binding and functional techniques to investigate tachykinin receptors in the small intestine of the cane toad Bufo marinus. The radioligand [125I]Bolton-Hunter [Sar9,Met(O2)11]substance P (selective at mammalian NK-1 receptors) showed no specific binding. Specific binding of [125I]Bolton-Hunter substance P ([125I]BHSP) was saturable, of high affinity (Kd 0.3 nM) and was inhibited by SP (IC50 0.64 nM) > ranakinin approximately neurokinin A (NKA) > or = SP(5-11) > or = neuropeptide gamma > or = scyliorhinin II > scyliorhinin I > or = [Sar9]-SP > or = neurokinin B approximately physalaemin approximately carassin > SP(7-11) approximately eledoisin > or = SP(4-11) approximately SP(6-11). Binding was also inhibited by Gpp[NH]p > or = GTPgammaS > App[NH]p, indicating a G-protein coupled receptor. The order of potency of tachykinins and analogues in contracting the isolated lower small intestine was carassin (EC50 1.4 nM) > eledoisin approximately SP > or = physalaemin > or = ranakinin > SP(6-11) > scyliorhinin II > or = neuropeptide gamma > neurokinin B approximately NKA approximately scyliorhinin I > or = SP(4-11) > or = SP(5-11) > [Sar9]SP > SP(7-11). In both studies, the selective mammalian NK-1, NK-2 and NK-3 receptor agonists [Sar9,Met(O2)11]SP, [Lys5,Me-Leu9,Nle10]NKA(4-10) and senktide were weak or ineffective. There was a strong positive correlation between the pD2 and pIC50 values for mammalian tachykinins and analogues (r = 0.907), but not for the non-mammalian tachykinins, which were all full agonists but variable binding competitors. [Sar9,Met(O2)11]-SP(pD2 5.7) was approximately 25-fold less potent as an agonist than [Sar9]SP, which was itself 25-fold weaker than SP. Responses to SP were significantly reduced (n = 8, P<0.001) by the antagonist [D-Arg1,D-Trp7,9,Leu11]-SP (spantide; 1 microM). Highly selective NK-1 receptor antagonists including CP 99994 and GR 82334 (both 1 microM) were ineffective in both functional and binding studies. Tetrodotoxin (1 microM) did not inhibit contractile responses to SP, NKA and senktide. In summary, this study has shown the presence of one or more tachykinin receptor in the toad intestine. The binding site recognised by [125I]BHSP prefers SP and ranakinin. This toad "NK-1-like receptor" differs from the mammalian NK-1 receptor in having a low affinity for all mammalian NK-1 selective ligands, including antagonists. For some non-mammalian peptides, their high potency as contractile agonists relative to their poor binding affinity suggests the existence of other tachykinin receptors in the toad small intestine.     

Substance P-induced inward current in identified auditory efferent neurons in rat brain stem slices

The effects of substance P (SP) on whole cell currents were studied in neurons of the medial olivocochlear efferent system (MOCS) in the ventral nucleus of the trapezoid body (VNTB) of brain stem slices from neonatal rats. Each neuron was identified by retrograde labeling with Fast Blue injected into the cochlea. Bath application of SP (0.1-10 microM) reversibly induced an apparent inward current in 49 of 63 labeled neurons when voltage clamped at near resting voltages. This apparent inward current was consistent with the SP-induced membrane depolarization observed in current-clamp mode. The SP-induced change in current was dose dependent with a half-maximal response dose of 200 nM. It was mimicked by [Cys3,6, Tyr8, Pro9]-SP, a neurokinin (NK1) receptor selective agonist, whereas [Succinyl-Asp6, MePhe8]-SP 6-11 (Senktide), a NK3 receptor agonist, had no detectable effect. The SP effect was not blocked by 10(-6) M tetrodotoxin (TTX) and persisted when the perfusate contained 30 mM tetraethylammonium (TEA) or 100 microM Cd2+ or was in a 0-Ca solution. In a TTX-containing solution, SP caused a voltage-dependent decrease of membrane conductance, and the SP-evoked current reversed at a potential at around -105 mV. The predicted K+ equilibrium potential was -93.8 mV under the experimental conditions. The SP-induced inward current was attenuated by 66% when the perfusate contained 3 mM Cs+. We conclude that the apparent inward current is partly caused by SP decreasing an outward current normally maintained by the inward rectifier K+ channels in these cells. In the presence of Cs solution in the recording pipette and with a perfusate containing 3 mM Cs+, 0.1 mM Cd2+ and 10(-6) M TTX, a residual SP-induced inward current was observed at test voltages ranging from -120 to 40 mV. This subcomponent reversed its polarity at approximately 20 mV. This inward current was reduced substantially (but not abolished) when all NaCl in the external solution was replaced by TEA-Cl. The results indicate that SP also opens an unknown cation channel, which the available data suggests may be relatively nonselective. The results suggest that MOCS neurons are subject to modulation by SP, which depolarizes the cell membrane by decreasing the activity of inward rectifier K+ channels as well as concurrently activating a separate cation conductance. It also was found that in MOCS neurons responsive to both SP and norepinephrine, the norepinephrine effect was abolished by TTX, suggesting that an interneuronal population excited by norepinephrine converges selectively onto SP-sensitive MOCS neurons in the VNTB.     

Lack of induction of substance P gene expression by hypoxia and absence of neurokinin 1-receptor mRNAs in the rat carotid body

Peripheral chemoreceptors are commonly thought to respond to hypoxia by releasing neurotransmitters from the type 1 cells of the carotid body; these molecules then bind to post-synaptic receptors on the carotid sinus nerve. The tachykinin substance P (SP) may act as an important neurotransmitter/neuromodulator in hypoxic chemotransmission in peripheral arterial chemoreceptors. In order to elucidate the role of SP in modulating hypoxic chemotransmission, we have used quantitative in situ hybridization histochemistry, to determine the effect of hypoxia on SP gene induction, and the localization of neurokinin 1 (NK-1) receptor mRNA in the carotid body and petrosal ganglia complex in rats at 21 days post-natal age. For comparison, we also determined: (1) the effect of hypoxia on tyrosine hydroxylase (TH) gene induction and (2) the localization of the mRNA encoding the D2-dopamine receptor. SP mRNA was not detected in the rat carotid body during normoxia and its expression was not induced after a 1 h of exposure to hypoxia (10% O2/90% N2), a stimulus that was sufficient to cause a significant increase (P < 0.01) in TH mRNA levels in the carotid body. Both SP and TH mRNAs were abundantly expressed in multiple cells in the petrosal and the jugular ganglia. However, these mRNAs were not co-localized and SP and TH mRNA levels were not affected by hypoxia in these ganglia. Although D2-dopamine receptor mRNA was abundantly expressed in the rat carotid body, we found no evidence of NK-1 receptor mRNA in the carotid body. In contrast, both NK-1 receptor mRNA and D2-dopamine receptor mRNA were present in petrosal ganglion cells. In the rat, SP does not appear to modulate hypoxic chemotransmission by being made in and released from type 1 cells in the carotid body, and neither does SP modulate the activity of type 1 cells by binding to NK-1 receptors on these cells.     

Should unipolar leads be implanted in the atrium? A Holter electrocardiographic comparison of threshold adapted unipolar and high sensitive bipolar sensing

1. The effects of nifedipine on both levcromakalim-induced membrane currents and unitary currents in pig proximal urethra were investigated by use of patch-clamp techniques (conventional whole-cell configuration and cell-attached patches). 2. Nifedipine had a voltage-dependent inhibitory effect on voltage-dependent Ba2+ currents at - 50 mV (Ki=30.6 nM). 3. In current-clamp mode, subsequent application of higher concentrations of nifedipine (> or =30 microM) caused a significant depolarization even after the membrane potential had been hyperpolarized to approximately -82 mV by application of 100 microM levcromakalim. 4. The 100 microM levcromakalim-induced inward current (symmetrical 140 mM K+ conditions, -50 mV) was inhibited by additional application of three different types of Ca antagonists (nifedipine, verapamil and diltiazem, all at 100 microM). In contrast, Bay K 8644 (1 microM) possessed no activating effect on the amplitude of this glibenclamide-sensitive current. 5. When 100 microM nifedipine was included in the pipette solution during conventional whole-cell recording at -50 mV, application of levcromakalim (100 microM) caused a significant inward membrane current which was suppressed by 5 microM glibenclamide. On the other hand, inclusion of 5 microM glibenclamide in the pipette solution prevented levcromakalim from inducing an inward membrane current. 6. The levcromakalim-induced K+ channel openings in cell-attached configuration were suppressed by subsequent application of 5 microM glibenclamide but not of 100 microM nifedipine. 7. These results suggest that in pig proximal urethra, nifedipine inhibits the glibenclamide-sensitive 43 pS K+ channel activity mainly through extracellular blocking actions on the K+ channel itself.     

Accessory cell requirements for T lymphocyte activation and interferon-gamma production in peripheral lymph nodes

Studies were performed on the central antidiuretic actions via the tachykinin NK-3 receptor in the rat hypothalamic paraventricular nucleus (PVN). Microinjections of the selective tachykinin NK-3 receptor agonist senktide (2-200 pmol) into the PVN resulted in prolonged inhibition of urine output in water-loaded rats, its effect being dose-dependent. The antidiuretic action of senktide was blocked by pretreatment with the vasopressin V2 receptor antagonist OPC-31260 (1 mg/kg, i.v.), but not by microinjection of the angiotensin II AT-1 receptor antagonist losartan (1 nmol) into the PVN. NK-3 receptor mRNA was strongly detected in the magnocellular part of the PVN and the supraoptic nucleus (SON) of the hypothalamus as detected by in situ hybridization histochemistry. Moreover, [3H]senktide binding sites were also detected in the PVN and the SON by receptor autoradiography. These findings suggest that NK-3 receptors in the PVN may be involved in water regulation by stimulation of vasopressin secretion from the posterior pituitary gland, and that vasopressin caused water reabsorbtion via the kidney V2 receptor.     

Inflammatory mediators at acidic pH activate capsaicin receptors in cultured sensory neurons from newborn rats

Whole cell membrane currents induced by the inflammatory mediators, bradykinin, 5-hydroxytryptamine (5-HT) and prostaglandin E2, were investigated in capsaicin-sensitive dorsal root ganglion (DRG) neurons from newborn rats grown on a monolayer of hippocampal glia without nerve growth factor (NGF). When firmly attached to an underlying cell, the neurons survived >14 days without growing extensive processes. A majority of the small diameter neurons ( approximately 80%) exhibited sensitivity to capsaicin (3-6 muM) and this was enhanced in solution of low pH. In acidic extracellular solution (pH 6.1), the combination of bradykinin (10 microM), 5-HT (10 microM) and prostaglandin E2 (1 microM) induced an inward membrane current in all capsaicin-sensitive DRG neurons (n = 43). The current exceeded the sustained, low pH-induced membrane current by 205 +/- 53 (SE) pA. The combination of acidic inflammatory mediators was ineffective in cells that were insensitive to capsaicin. In capsaicin-sensitive neurons, the inflammatory mediators when applied singly or in any combination of two, induced no membrane currents or small current at pH 7.3 and 6.1. Capsazepine (10 microM), the capsaicin antagonist, completely inhibited the facilitatory action of inflammatory mediator combination but not the sustained inward current induced by acidic extracellular solution (pH 6.1 or 5.5). It is suggested that the inflammatory mediators, bradykinin,5-HT, and prostaglandin E2 together act as endogenous mediators at capsaicin receptors to generate an inward current when the ion channel is protonized.     

OFQ reverses the kappa-opioid receptor-mediated depression of calcium current in rat dorsal root ganglion neurons

Since the characterization of orphanin FQ (OFQ), the endogenous ligand of ORL1 receptor, much work has focused on its physiological functions. OFQ was reported to antagonize the effect of opioid-induced antinociception, although its mechanism remains obscure. In the present study, whole-cell patch clamp recording technique was used to observe if OFQ can reverse the inhibition of calcium current produced by the kappa-opioid agonist U50,488H (U50) in acutely dissociated rat DRG neurons. The concentrations of OFQ and U50 were 50 nM and 10 microM, respectively. Among 49 cells recorded, the calcium channel currents of 37 (75.5%) cells were inhibited by U50, of which 30 (81.1%) cells could be reversed by OFQ. It was interesting to note the similarity between OFQ and the well characterized anti-opioid peptide CCK-8 in that it reversed kappa-opioid receptor agonist induced suppression on calcium channel current, while by itself showed a calcium channel suppressive effect. Thus OFQ may be regarded as another anti-opioid peptide.     

Profiles of the fatty acids in the plasma membrane of human brain tumors

The ionic channels and signal transduction pathways underlying the 5-hydroxytryptamine (5-HT)-induced hyperpolarization in neurons of the rat dorsolateral septal nucleus (DLSN) were examined by using intracellular and voltage-clamp recording techniques. Application of 5-HT (1-50 microM) caused a hyperpolarizing response associated with a decreased membrane resistance in DLSN neurons. The hyperpolarization induced by 5-HT was blocked by Ba2+ (1 mM) but not by tetraethylammonium (TEA, 3 mM), glibenclamide (100 microM) and extracellular Cs+ (2 mM). 8-Hydroxy-di-n-propylamino tetralin (8-OH-DPAT; 3 microM), a selective agonist for the 5-HT1A receptor, mimicked 5-HT in producing the hyperpolarization. The 5-HT hyperpolarization was blocked by NAN-190 (5 microM), a 5-HT1A receptor antagonist. CP93129 (100 microM), a 5-HT1B receptor agonist, and L-694-247 (100 microM), a 5-HT1B/1D receptor agonist, also produced hyperpolarizing responses. The order of agonist potency was 8-OH-DPAT > CP93129 > or = L-694-247. (+/-)-2,5-Dimethoxy-4-iodoamphetamine hydrochloride (DOI, 100 microM), a 5-HT2 receptor agonist, and RS67333 (100 microM), a 5-HT4 receptor agonist, caused no hyperpolarizing response. The voltage-clamp study showed that 5-HT caused an outward current (I5-HT) in a concentration-dependent manner. I5-HT was associated with an increased membrane conductance. I5-HT reversed the polarity at the equilibrium potential for K+ calculated by the Nernst equation. I5-HT showed inward rectification at membrane potentials more negative than-70 mV. Ba2+ (100 microM) blocked the inward rectifier K+ current induced by 5-HT. I5-HT was irreversibly depressed by intracellular application of guanosine 5'-O-(3-thiotriphosphate)(GTP-gamma S) but not by guanosine 5'-O-(2-thiodiphosphate) (GDP beta S). These results suggest that in rat DLSN neurons activation of 5-HT1A receptors causes a hyperpolarizing response by activating mainly the inward rectifier K+ channels through a GTP-binding protein.     

Tachykinin peptides affect differently the second messenger pathways after binding to CHO-expressed human NK-1 receptors

The human NK-1 receptor transfected in Chinese hamster ovary (CHO) cells was studied with use of different tachykinin analogs: Substance P, [Pro9]SP, [Sar9, Met(O2)11]SP, [Gly9 psi (CH2CH2) Leu10]SP, Ac-Arg-septide, septide, [Gly9 psi (CH2CH2) Gly10]SP, NKA, [pGlu6]SP(6-11) and [Lys5]NKA(4-10). Binding experiments with [3H][Pro9]SP discriminated two classes of peptides with either high affinity (K iota in the nanomolar range) for the human NK-1 receptor or with low affinity (K iota in the micromolar range); this second group of peptides included NKA and [pGlu6]SP(6-11). In spite of these differences, both peptide families evoked potent stimulation of phosphatidylinositol hydrolysis (EC50 in the nanomolar range). In contrast, only NK-1 agonists, with high affinity, stimulated with great potency cyclic AMP formation (EC50 from 8 to 50 nM), whereas the second family of peptides were only weak agonists (EC50 in the micromolar range). RP 67580, CP 96345 and GR 94800, a NK-2 antagonist, were either competitive or uncompetitive inhibitors of inositol phosphates or cyclic AMP formations induced by [Pro9]SP, septide or NKA, independently of the agonist or the response studied. Thus, NKA, the presumed NK-2 endogenous peptide that may be co-released with SP, and the enzymatically produced C-terminal fragment of SP, [pGlu6]SP(6-11), may trigger specific pharmacological responses via the NK-1 receptor, at nanomolar concentrations, and thus regulate the action of SP at the NK-1 receptor.     

Slow closed-state inactivation: a novel mechanism underlying ramp currents in cells expressing the hNE/PN1 sodium channel

To better understand why sensory neurons express voltage-gated Na+ channel isoforms that are different from those expressed in other types of excitable cells, we compared the properties of the hNE sodium channel [a human homolog of PN1, which is selectively expressed in dorsal root ganglion (DRG) neurons] with that of the skeletal muscle Na+ channel (hSkM1) [both expressed in human embryonic kidney (HEK293) cells]. Although the voltage dependence of activation was similar, the inactivation properties were different. The V1/2 for steady-state inactivation was slightly more negative, and the rate of open-state inactivation was approximately 50% slower for hNE. However, the greatest difference was that closed-state inactivation and recovery from inactivation were up to fivefold slower for hNE than for hSkM1 channels. TTX-sensitive (TTX-S) currents in small DRG neurons also have slow closed-state inactivation, suggesting that hNE/PN1 contributes to this TTX-S current. Slow ramp depolarizations (0.25 mV/msec) elicited TTX-S persistent currents in cells expressing hNE channels, and in DRG neurons, but not in cells expressing hSkM1 channels. We propose that slow closed-state inactivation underlies these ramp currents. This conclusion is supported by data showing that divalent cations such as Cd2+ and Zn2+ (50-200 microM) slowed closed-state inactivation and also dramatically increased the ramp currents for DRG TTX-S currents and hNE channels but not for hSkM1 channels. The hNE and DRG TTX-S ramp currents activated near -65 mV and therefore could play an important role in boosting stimulus depolarizations in sensory neurons. These results suggest that differences in the kinetics of closed-state inactivation may confer distinct integrative properties on different Na+ channel isoforms.     

Time-dependent outward currents through the inward rectifier potassium channel IRK1. The role of weak blocking molecules

Outward currents through the inward rectifier K+ channel contribute to repolarization of the cardiac action potential. The properties of the IRK1 channel expressed in murine fibroblast (L) cells closely resemble those of the native cardiac inward rectifier. In this study, we added Mg2+ (0.44-1.1 mM) or putrescine (approximately 0.4 mM) to the intracellular milieu where endogenous polyamines remained, and then examined outward IRK1 currents using the whole-cell patch-clamp method at 5.4 mM external K+. Without internal Mg2+, small outward currents flowed only at potentials between -80 (the reversal potential) and approximately -40 mV during voltage steps applied from -110 mV. The strong inward rectification was mainly caused by the closed state of the activation gating, which was recently reinterpreted as the endogenous-spermine blocked state. With internal Mg2+, small outward currents flowed over a wider range of potentials during the voltage steps. The outward currents at potentials between -40 and 0 mV were concurrent with the contribution of Mg2+ to blocking channels at these potentials, judging from instantaneous inward currents in the following hyperpolarization. Furthermore, when the membrane was repolarized to -50 mV after short depolarizing steps (> 0 mV), a transient increase appeared in outward currents at -50 mV. Since the peak amplitude depended on the fraction of Mg(2+)-blocked channels in the preceding depolarization, the transient increase was attributed to the relief of Mg2+ block, followed by a re-block of channels by spermine. Shift in the holding potential (-110 to -80 mV), or prolongation of depolarization, increased the number of spermine-blocked channels and decreased that of Mg(2+)-blocked channels in depolarization, which in turn decreased outward currents in the subsequent repolarization. Putrescine caused the same effects as Mg2+. When both spermine (1 microM, an estimated free spermine level during whole-cell recordings) and putrescine (300 microM) were applied to the inside-out patch membrane, the findings in whole-cell IRK1 were reproduced. Our study indicates that blockage of IRK1 by molecules with distinct affinities, spermine and Mg2+ (putrescine), elicits a transient increase in the outward IRK1, which may contribute to repolarization of the cardiac action potential.     

Interactions between excitatory amino acids and tachykinins in the rat spinal dorsal horn

Whole-cell patch-clamp technique of freshly isolated rat spinal dorsal horn (DH) neurons, intracellular recording from DH neurons in a slice preparation, and high performance liquid chromatography with fluorimetric detection of release of endogenous glutamate and aspartate from spinal cord slice following activation of primary afferent fibers were employed to investigate interactions between excitatory amino acids (EAA) and tachykinins [substance P (SP) and neurokinin A (NKA)]. Potentiation of N-methyl-D-aspartate (NMDA)-, quisqualate (QA)- and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-, but not kainate-induced currents by SP and NKA was found. Spantide II, a claimed novel nonselective tachykinin antagonist, effectively blocked the SP (2 nM)-induced potentiation of the responses of DH neurons to NMDA. In the presence of glycine (0.1 microM), the SP-evoked increase of the NMDA-induced current was prevented. However, 7-chlorokynurenic acid (2 microM), a competitive antagonist at the glycine allosteric site of the NMDA receptor, led to the reestablishment of the SP effect. Brief high frequency electrical stimulation of primary afferent fibers produced a long-lasting potentiation of presumed monosynaptic and polysynaptic excitatory postsynaptic potentials and sustained enhanced release of endogenous glutamate (218.3 +/- 66.1%) and aspartate (286.3 +/- 58.0%). Possible functional implications of the observed phenomena are discussed in relation to transmission and integration of sensory information, including pain.     

The presence of Human Papillomavirus (HPV) in microinvasive in situ spinocellular carcinoma of the oral cavity. Preliminary report

To determine whether functional Ca2+ channels are present in vestibular dark cells, changes in intracellular Ca2+ concentration ([Ca2+]i) due to K+ applications were measured using the Ca(2+)-sensitive dye (fura-2) and patchclamp whole-cell recordings were made in dark cells isolated from the ampullae of the semicircular canal of the guinea pig. Exchange of the external solution with a buffer medium containing a high K+ concentration (80 mM K+ or 150 mM K+) caused a concentration-dependent increase in [Ca2+]i in vestibular dark cells. Application of 1 microM nifedipine as a Ca2+ channel antagonist completely blocked the increase in [Ca2+]i. Further treatment with 10 microM BAY K 8644 as a Ca2+ channel agonist caused an increase in [Ca2+]i. In the patch-clamp whole-cell recordings a 1-s depolarizing pulse given into the dark cell in the presence of a high barium concentration (50 mM Ba2+) induced an inward current. In determining the current-voltage relationship, a current was detected at a potential that depolarized at-50 mV and was maximal at +10 mV. This inward current was completely blocked by 1 mM La3+ as a Ca2+ channel antagonist. These findings suggest the presence of voltage-dependent Ca2+ channels in dark cells, which have a presumed function in the regulation of [Ca2+]i in the vestibular endolymph.     

Distribution of the tetrodotoxin-resistant sodium channel PN3 in rat sensory neurons in normal and neuropathic conditions

The novel sodium channel PN3/alpha-SNS, which was cloned from a rat dorsal root ganglion (DRG) cDNA library, is expressed predominantly in small sensory neurons and may contribute to the tetrodotoxin-resistant (TTXR) sodium current that is believed to be associated with central sensitization in chronic neuropathic pain states. To assess further the role of PN3, we have used electrophysiological, in situ hybridization and immunohistochemical methods to monitor changes in TTXR sodium current and the distribution of PN3 in normal and peripheral nerve-injured rats. (1) Whole-cell patch-clamp recordings showed that there were no significant changes in the TTXR and TTX-sensitive sodium current densities of small DRG neurons after chronic constriction injury (CCI) of the sciatic nerve. (2) Additionally, in situ hybridization showed that there was no change in the expression of PN3 mRNA in the DRG up to 14 d after CCI. PN3 mRNA was not detected in sections of brain and spinal cord taken from either normal or nerve-injured rats. (3) In contrast, immunohistochemical studies showed that major changes in the subcellular distribution of PN3 protein were caused by either CCI or complete transection of the sciatic nerve. The intensity of PN3 immunolabeling decreased in small DRG neurons and increased in sciatic nerve axons at the site of injury. The alteration in immunolabeling was attributed to translocation of presynthesized, intracellularly located PN3 protein from neuronal somata to peripheral axons, with subsequent accumulation at the site of injury. The specific subcellular redistribution of PN3 after peripheral nerve injury may be an important factor in establishing peripheral nerve hyperexcitability and resultant neuropathic pain.     

Nociceptin/orphanin FQ-induced nociceptive responses through substance P release from peripheral nerve endings in mice

We have studied the in vivo signaling mechanisms involved in nociceptin/orphanin FQ (Noci)-induced pain responses by using a flexor-reflex paradigm. Noci was 10,000 times more potent than substance P (SP) in eliciting flexor responses after intraplantar injection into the hind limb of mice, but the action of Noci seems to be mediated by SP. Mice pretreated with an NK1 tachykinin receptor antagonist or capsaicin, or mice with a targeted disruption of the tachykinin 1 gene no longer respond to Noci. The action of Noci appears to be mediated by the Noci receptor, a pertussis toxin-sensitive G protein-coupled receptor that stimulates inositol trisphosphate receptor and Ca2+ influx. These findings suggest that Noci indirectly stimulates nerve endings of nociceptive primary afferent neurons through a local SP release.     

Activation of Ca2+-dependent currents in dorsal root ganglion neurons by metabotropic glutamate receptors and cyclic ADP-ribose precursors

Cultured dorsal root ganglion neurons were voltage clamped at -90 mV to study the effects of intracellular application of nicotinamide adenine dinucleotide (betaNAD+), intracellular flash photolysis of caged 3',5'-cyclic guanosine monophosphate (cGMP), and metabotropic glutamate receptor activation. The activation of metabotropic glutamate receptors evoked inward Ca2+-dependent currents in most cells. This was mimicked both by intracellular flash photolysis of the caged axial isomer of cGMP [P-1-(2-nitrophenyl)ethyl cGMP] and intracellular application of betaNAD+. Whole cell Ca2+-activated inward currents were used as a physiological index of raised intracellular Ca2+ levels. Extracellular application of 10 microM glutamate evoked the activation of Ca2+-dependent inward currents, thus reflecting a rise in intracellular Ca2+ levels. Similar inward currents were also activated after isolation of metabotropic glutamate receptor activation by application of 10 microM glutamate in the presence of 20 microM 6-cyano-7-nitroquinoxaline-2,3-dione and 20 microM dizocilpine maleate (MK 801), or by extracellular application of 10 microM trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid. Intracellular photorelease of cGMP, from its caged axial isomer, in the presence of betaNAD+ was also able to evoke similar Ca2+-dependent inward currents. Intracellular application of betaNAD+ alone produced a concentration-dependent effect on inward current activity. Responses to both metabotropic glutamate receptor activation and cGMP were suppressed by intracellular ryanodine, chelation of intracellular Ca2+ by bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid, and depletion of intracellular Ca2+ stores, but were insensitive to the removal of extracellular Ca2+. Therefore both cGMP, possibly via a mechanism that involves betaNAD+ and/or cyclic ADP-ribose, and glutamate can mobilize intracellular Ca2+ from ryanodine-sensitive stores in sensory neurons.     

Involvement of both L- and N-type voltage-dependent Ca2+ channels in KCl- and veratridine-evoked transmitter release from non-adrenergic, non-cholinergic nerves in the rabbit iris sphincter muscle

To determine which types of voltage-dependent Ca2+ channels mediate tachykinin release in the isolated rabbit iris sphincter muscle, we examined the effects of several Ca2+ channel modulators on contractions induced by either an elevation of the extracellular KCl concentration or application of the Na+ channel activator veratridine. Contractions caused by either 45.9 mM KCl or veratridine (10 microM) were inhibited by spantide (10 microM), a tachykinin receptor antagonist, and capsaicin (10 microM), a tachykinin-depleting agent, but were not changed by atropine. Nicardipine, an L-type Ca2+ channel blocker, inhibited contractions induced by KCl and veratridine in a concentration-dependent manner. omega-Conotoxin GVIA (1 microM), an N-type Ca2+ channel blocker, inhibited only contractions induced by lower concentrations of KCl, both when applied alone and when combined with nicardipine. Bay K 8644, an L-type Ca2+ channel activator, caused a spantide- and nicardipine-sensitive contraction in muscles partially depolarized with 15.9 mM KCl, and enhanced contractions induced by 15.9 mM KCl and veratridine (2 microM). omega-Agatoxin IVA (0.3 microM), a P-type voltage-dependent Ca2+ channel blocker, did not affect contractions induced by either KCl or veratridine. Contractions induced by exogenous substance P were not modified by any of the Ca2+ channel blockers or by Bay K 8644. These results suggest that, in the rabbit iris sphincter muscle. L- and N-type voltage-dependent Ca2+ channels are involved in neurotransmitter release from tachykininergic nerves elicited by high KCl and by veratridine.     

Expression and regulation of the neuropeptide Y Y2 receptor in sensory and autonomic ganglia

The Y2 subtype of neuropeptide tyrosine (NPY) receptors (Y2R) and some neuropeptides have been studied with in situ hybridization in sensory and autonomic neurons of rat and monkey. Between 10% and 20% of the lumbar dorsal root ganglion (DRG) neuron profiles (NPs) contain Y2R mRNA in the rat and monkey. In rat DRGs Y2R mRNA is expressed in calcitonin gene-related peptide (CGRP)-positive, medium-sized, and large neurons, that is in a complementary fashion to the Y1R that is located in small CGRP neurons. In monkey DRGs Y2R mRNA is expressed mainly in small neurons. Peripheral axotomy up-regulates the Y2R in small and large DRG neurons in both species. Y2R and NPY mRNAs are colocalized in many large neurons in axotomized rat DRGs. Y2R mRNA is expressed in 50% of the NPs in the nodose ganglion with a modest increase after axotomy. Y2R mRNA is detected in a few NPs in normal rat superior cervical ganglia, with a marked increase after transection of the carotid nerves. No Y2R mRNA-positive, but many (approximately 30%) weakly Y1R mRNA-positive NPs were found in the sphenopalatine ganglion. Finally, Y2R mRNA levels increase in rat spinal motoneurons after axotomy. Thus, under normal circumstances NPY may act on Y1 and Y2Rs expressed, respectively, in small and large CGRP-positive DRG neurons in the rat. Y2R may be an important receptor in the viscero-sensory neurons. Y2Rs may be particularly important after axotomy serving as presynaptic and/or autoreceptors on rat DRG, superior cervical ganglion, and nodose ganglion neurons and as presynaptic receptors in monkey DRG neurons.