Adrian Sculptoreanu

Abnormal excitability in capsaicin-responsive DRG neurons from cats with feline interstitial cystitis.

Interstitial cystitis (IC) is a painful disorder which affects urinary bladder function in cats and humans. We used patch clamp techniques to measure firing properties and K+ currents of dorsal root ganglion (DRG) neurons (L4-S3) from normal cats and cats with feline interstitial cystitis (FIC) to examine the possibility that the properties of primary afferent neurons are changed in cats with FIC. We found that capsaicin (CAPS)-responsive neurons from FIC cats were increased in size, had increased firing in response to depolarizing current pulses and expressed more rapidly inactivating K+ currents. CAPS-sensitive neurons from FIC cats were 28% larger than those from normal cats but were otherwise similar with respect to membrane potential and action potential (AP) threshold. CAPS-responsive neurons from normal cats fired 1.5 APs in response to a 600 ms depolarizing current pulse, 60-200 pA in intensity. The number of APs was increased 4.5 fold in FIC neurons. Neurons from FIC cats also exhibited after hyperpolarization potentials which were on the average 2x slower than those in normal cat neurons. In addition, there was a lack of K+ currents in the critical voltage range of action potential generation (between -50 to -30 mV). These changes were not detected in CAPS-unresponsive neurons from normal and FIC cats. Our data suggest that FIC afferent neurons exhibit abnormal firing which may be due to changes in the behavior of K+ currents and show that these changes are restricted to a subpopulation of CAPS-responsive neurons.

Protein kinase C contributes to abnormal capsaicin responses in DRG neurons from cats with feline interstitial cystitis

Interstitial cystitis (IC) is a painful disorder which affects urinary bladder function in cats and humans. We have used patch clamp techniques to examine the possibility that the properties of primary afferent neurons are changed in feline interstitial cystitis (FIC). We measured transient receptor potential vanilloid receptor 1 (TRPV1) responses to capsaicin (CAPS) in dorsal root ganglion (DRG) neurons (L4-S3) from normal cats and cats with FIC. We show that FIC neurons are increased in size and exhibit CAPS responses which are increased in amplitude and desensitize slowly. CAPS responses desensitized seven times slower in FIC neurons. Phorbol 12,13-dibutyrate (PDBu), an activator of PKC, slowed the desensitization of CAPS responses in normal cat bladder and non-bladder neurons, but had no effect in FIC neurons. Bisindolylmaleimide, an inhibitor of PKC, reversed the PDBu effects in normal cat neurons and normalized the desensitization of CAPS responses in FIC neurons. Our data suggest that FIC afferent neurons exhibit abnormal CAPS responses. The latter may be due to enhanced endogenous activities of PKC.

Neurokinins enhance excitability in capsaicin-responsive DRG neurons

Neurokinins released by capsaicin-responsive (C-R) dorsal root ganglia neurons (DRG) may control firing in these neurons by an autofeedback mechanism. Here we used patch clamp techniques to examine the effects of neurokinins on firing properties of dissociated DRG neurons of male rats. In C-R neurons that generated only a few action potentials (APs, termed phasic) in response to long depolarizing current pulses (600 ms), substance P (SP, 0.5 microM) lowered the AP threshold by 11.0+/-0.3 mV and increased firing from 1.1+/-0.7 APs to 5.2+/-0.6 APs. In C-R tonic neurons that fire multiple APs, SP elicited smaller changes in AP threshold (6.0+/-0.1 mV reduction) and the number of APs (11+/-1 vs. 9+/-1 in control). The effects of SP were similar to the effect of heteropodatoxin II (0.05 microM) or low concentrations of 4-aminopyridine (50 microM) that block A-type K(+) currents. A selective NK(2) agonist, [betaAla(8)]-neurokinin A (4-10) (0.5 microM), mimicked the effects of SP. The effects of SP in C-R phasic neurons were fully reversed by an NK(2) receptor antagonist (MEN10376, 0.5 microM) but only partially by a protein kinase C (PKC) inhibitor (bisindolylmaleimide, 0.5 microM). An NK(3)-selective agonist ([MePhe(7)]-neurokinin B, 0.5 microM), an NK(1)-selective agonist ([Sar(9), Met(11)]-substance P, 0.5 microM) or activation of PKC with phorbol 12,13-dibutyrate (0.5 microM) did not change firing. Our data suggest that the excitability of C-R phasic afferent neurons is increased by activation of NK(2) receptors and intracellular signaling mediated only in part by PKC.

Nitro-Oleic Acid Inhibits Firing and Activates TRPV1- and TRPA1-Mediated Inward Currents in Dorsal Root Ganglion Neurons from Adult Male Rats

Nitro-oleic acid (OA-NO2), an electrophilic fatty acid by-product of nitric oxide and nitrite reactions, is present in normal and inflamed mammalian tissues at up to micromolar concentrations and exhibits anti-inflammatory signaling actions. The effects of OA-NO2 on cultured dorsal root ganglion (DRG) neurons were examined using fura-2 Ca2+ imaging and patch clamping. OA-NO2 (3.5–35 μM) elicited Ca2+ transients in 20 to 40% of DRG neurons, the majority (60–80%) of which also responded to allyl isothiocyanate (AITC; 1–50 μM), a TRPA1 agonist, and to capsaicin (CAPS; 0.5 μM), a TRPV1 agonist. The OA-NO2-evoked Ca2+ transients were reduced by the TRPA1 antagonist 2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N-(4-isopropylphenyl) acetamide (HC-030031; 5–50 μM) and the TRPV1 antagonist capsazepine (10 μM). Patch-clamp recording revealed that OA-NO2 depolarized and induced inward currents in 62% of neurons. The effects of OA-NO2 were elicited by concentrations ≥5 nM and were blocked by 10 mM dithiothreitol. Concentrations of OA-NO2 ≥5 nM reduced action potential (AP) overshoot, increased AP duration, inhibited firing induced by depolarizing current pulses, and inhibited Na+ currents. The effects of OA-NO2 were not prevented or reversed by the NO-scavenger carboxy-2-phenyl-4,4,5,5-tetramethylimidazolineoxyl-1-oxyl-3-oxide. A large percentage (46–57%) of OA-NO2-responsive neurons also responded to CAPS (0.5 μM) or AITC (0.5 μM). OA-NO2 currents were reduced by TRPV1 (diarylpiperazine; 5 μM) or TRPA1 (HC-030031; 5 μM) antagonists. These data reveal that endogenous OA-NO2 generated at sites of inflammation may initially activate transient receptor potential channels on nociceptive afferent nerves, contributing to the initiation of afferent nerve activity, and later suppresses afferent firing.

Neurokinin 2 receptor-mediated activation of protein kinase C modulates capsaicin responses in DRG neurons from adult rats.

Patch‐clamp techniques and Ca2+ imaging were used to examine the interaction between neurokinins (NK) and the capsaicin (CAPS)‐evoked transient receptor potential vanilloid receptor 1 (TRPV1) responses in rat dorsal root ganglia neurons. Substance P (SP; 0.2–0.5 μm) prevented the reduction of Ca2+ transients (tachyphylaxis) evoked by repeated brief applications of CAPS (0.5 μm). Currents elicited by CAPS were increased in amplitude and desensitized more slowly after administration of SP or a selective NK2 agonist, [Ala8]‐neurokinin A (4–10) (NKA). Neither an NK1‐selective agonist, [Sar9, Met11]‐SP, nor an NK3‐selective agonist, [MePhe7]‐NKB, altered the CAPS currents. The effects of SP on CAPS currents were inhibited by a selective NK2 antagonist, MEN 10,376, but were unaffected by the NK3 antagonist, SB 235,375. Phorbol 12,13‐dibutyrate (PDBu), an activator of protein kinase C (PKC), also increased the amplitude and slowed the desensitization of CAPS responses. Phosphatase inhibitors, decamethrin and α‐naphthyl acid phosphate (NAcPh), also enhanced the currents and slowed desensitization of CAPS currents. Facilitatory effects of SP, NKA and PDBu were reversed by bisindolylmaleimide, a PKC inhibitor, and gradually decreased in magnitude when the agents were administered at increasing intervals after CAPS application. The decrease was partially prevented by prior application of NAcPh. These data suggest that activation of NK2 receptors in afferent neurons leads to PKC‐induced phosphorylation of TRPV1, resulting in sensitization of CAPS‐evoked currents and slower desensitization. Thus, activation of NK2 autoreceptors by NKs released from the peripheral afferent terminals or by mast cells during inflammatory responses may be a mechanism that sensitizes TRPV1 channels and enhances afferent excitability.

Protein kinase C epsilon contributes to basal and sensitizing responses of TRPV1 to capsaicin in rat dorsal root ganglion neurons

Phosphorylation of the vanilloid receptor (TRPV1) by protein kinase C epsilon (PKCɛ) plays an important role in the development of chronic pain. Here, we employ a highly defective herpes simplex virus vector (vHDNP) that expresses dominant negative PKCɛ (DNPKCɛ) as a strategy to demonstrate that PKCɛ is essential for: (i) maintenance of basal phosphorylation and normal TRPV1 responses to capsaicin (CAPS), a TRPV1 agonist and (ii) enhancement of TRPV1 responses by phorbol esters. Phorbol esters induced translocation of endogenous PKCɛ to the plasma membrane and thereby enhanced CAPS currents. These results were extended to an in‐vivo pain model in which vHDNP delivery to dorsal root ganglion neurons caused analgesia in CAPS‐treated, acutely inflamed rat hind paws. These findings support the conclusion that in addition to receptor sensitization, PKCɛ is essential for normal TRPV1 responses in vitro and in vivo.

Nitro-oleic acid targets transient receptor potential (TRP) channels in capsaicin sensitive afferent nerves of rat urinary bladder.

Nitro-oleic acid (9- and 10-nitro-octadeca-9-enoic acid, OA-NO(2)) is an electrophilic fatty acid nitroalkene derivative that modulates gene transcription and protein function via post-translational protein modification. Nitro-fatty acids are generated from unsaturated fatty acids by oxidative inflammatory reactions and acidic conditions in the presence of nitric oxide or nitrite. Nitroalkenes react with nucleophiles such as cysteine and histidine in a variety of susceptible proteins including transient receptor potential (TRP) channels in sensory neurons of the dorsal root and nodose ganglia. The present study revealed that OA-NO(2) activates TRP channels on afferent nerve terminals in the urinary bladder and thereby increases bladder activity. The TRPV1 agonist capsaicin (CAPS, 1 μM) and the TRPA1 agonist allyl isothiocyanate (AITC, 30 μM), elicited excitatory effects in bladder strips, increasing basal tone and amplitude of phasic bladder contractions (PBC). OA-NO(2) mimicked these effects in a concentration-dependent manner (1 μM-33 μM). The TRPA1 antagonist HC3-030031 (HC3, 30 μM) and the TRPV1 antagonist diaryl piperazine analog (DPA, 1 μM), reduced the effect of OA-NO(2) on phasic contraction amplitude and baseline tone. However, the non-selective TRP channel blocker, ruthenium red (30 μM) was a more effective inhibitor, reducing the effects of OA-NO(2) on basal tone by 75% and the effects on phasic amplitude by 85%. In bladder strips from CAPS-treated rats, the effect of OA-NO(2) on phasic contraction amplitude was reduced by 65% and the effect on basal tone was reduced by 60%. Pretreatment of bladder strips with a combination of neurokinin receptor antagonists (NK1 selective antagonist, CP 96345; NK2 selective antagonist, MEN 10,376; NK3 selective antagonist, SB 234,375, 1 μM each) reduced the effect of OA-NO(2) on basal tone, but not phasic contraction amplitude. These results indicate that nitroalkene fatty acid derivatives can activate TRP channels on CAPS-sensitive afferent nerve terminals, leading to increased bladder contractile activity. Nitrated fatty acids produced endogenously by the combination of fatty acids and oxides of nitrogen released from the urothelium and/or afferent nerves may play a role in modulating bladder activity.

Effects of ralfinamide, a Na+ channel blocker, on firing properties of nociceptive dorsal root ganglion neurons of adult rats

Recent studies revealed that ralfinamide, a Na(+) channel blocker, suppressed tetrodotoxin-resistant Na(+) currents in dorsal root ganglion (DRG) neurons and reduced pain reactions in animal models of inflammatory and neuropathic pain. Here, we investigated the effects of ralfinamide on Na(+) currents; firing properties and action potential (AP) parameters in capsaicin-responsive and -unresponsive DRG neurons from adult rats in the presence of TTX (0.5 microM). Ralfinamide inhibited TTX-resistant Na(+) currents in a frequency- and voltage-dependent manner. Small to medium sized neurons exhibited different firing properties during prolonged depolarizing current pulses (600 ms). One group of neurons fired multiple spikes (tonic), while another group fired four or less APs (phasic). In capsaicin-responsive tonic firing neurons, ralfinamide (25 microM) reduced the number of APs from 10.6+/-1.8 to 2.6+/-0.7 APs/600 ms, whereas in capsaicin-unresponsive tonic neurons, the drug did not significantly change firing (8.4+/-0.9 in control to 6.6+/-2.0 APs/600 ms). In capsaicin-responsive phasic neurons, substance P and 4-aminopyridine induced multiple spikes, an effect that was reversed by ralfinamide (25 microM). In addition to effects on firing, ralfinamide increased the threshold, decreased the overshoot, and increased the rate of rise of the AP. To conclude, ralfinamide suppressed afferent hyperexcitability selectively in capsaicin-responsive, presumably nociceptive neurons, but had no measurable effects on firing in CAPS-unresponsive neurons. The action of ralfinamide to selectively inhibit tonic firing in nociceptive neurons very likely contributes to the effectiveness of the drug in reducing inflammatory and neuropathic pain as well as bladder overactivity.

Neurokinins inhibit low threshold inactivating K+ currents in capsaicin responsive DRG neurons

Neurokinins (NK) released from terminals of dorsal root ganglion (DRG) neurons may control firing of these neurons by an autofeedback mechanism. In this study we used patch clamp recording techniques to determine if NKs alter excitability of rat L4-S3 DRG neurons by modulating K(+) currents. In capsaicin (CAPS)-responsive phasic neurons substance P (SP) lowered action potential (AP) threshold and increased the number of APs elicited by depolarizing current pulses. SP and a selective NK(2) agonist, [betaAla(8)]-neurokinin A (4-10) also inhibited low threshold inactivating K(+) currents isolated by blocking non-inactivating currents with a combination of high TEA, (-) verapamil and nifedipine. Currents recorded under these conditions were heteropodatoxin-sensitive (Kv4 blocker) and alpha-dendrotoxin-insensitive (Kv1.1 and Kv1.2 blocker). SP and NKA elicited a >10 mV positive shift of the voltage dependence of activation of the low threshold currents. This effect was absent in CAPS-unresponsive neurons. The effect of SP or NKA on K(+) currents in CAPS-responsive phasic neurons was fully reversed by an NK(2) receptor antagonist (MEN10376) but only partially reversed by a PKC inhibitor (bisindolylmaleimide). An NK(1) selective agonist ([Sar(9), Met(11)]-substance P) or direct activation of PKC with phorbol 12,13-dibutyrate, did not change firing in CAPS-responsive neurons, but did inhibit various types of K(+) currents that activated over a wide range of voltages. These data suggest that the excitability of CAPS-responsive phasic afferent neurons is increased by activation of NK(2) receptors and that this is due in part to inhibition and a positive voltage shift in the activation of heteropodatoxin-sensitive Kv4 channels.

Protein kinase C is involved in M1-muscarinic receptor-mediated facilitation of L-type Ca2+ channels in neurons of the major pelvic ganglion of the adult male rat.

We used patch clamp recording techniques to determine if muscarinic signaling mechanisms are present in dissociated autonomic neurons obtained from the major pelvic ganglion, which provides the cholinergic innervation of the urinary bladder and other pelvic organs. The M1 specific agonist, McN-A-343 (2–30 μM) enhanced Ca2+ currents in approximately 37% of neurons (by 50–80%). This enhancement was reduced by atropine (5–10 μM) or a PKC inhibitor (bisindolylmaleimide, 50–200 nM). In responsive neurons Ca2+ currents were also enhanced by the phorbol ester, phorbol-12, 13-dibutyrate (50–300 nM) and the dihydropyridine agonist Bay K 8644 (5 μM) and had kinetics of activation and inactivation as expected for L-type Ca2+ channels. We conclude that in a subpopulation of MPG neurons, M1-mediated activation of PKC phosphorylates and enhances L-type Ca2+ channel activities. This muscarinic facilitatory mechanism in MPG neurons may be the same as the M1-mediated facilitation of transmitter release reported previously at the nerve terminals in the urinary bladder.