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The transient receptor potential vanilloid type 1 (TRPV1) channel is a ligand-gated cation channel expressed by sensory nerves. P2Y receptors are G protein-coupled receptors that are also expressed by TRPVl-positive sensory neurons. Therefore, we studied interactions between P2Y receptors and TRPV1 function on kidney projecting sensory neurons. Application of Fast Blue (FB) to nerves surrounding the renal artery retrogradely labeled neurons in dorsal root ganglia of rats. Whole cell recording was performed on FB-labeled neurons maintained in primary culture. Capsaicin was used to activate TRPV1. Four types of kidney projecting neurons were identified based on capsaicin responses: 1) desensitizing (35%), 2) nondesensitizing (29%), 3) silent (3%), and 4) insensitive (30%). Silent neurons responded to capsaicin only after ATP (100 [micro]M) pretreatment. ATP reversed desensitization in desensitizing neurons. Insensitive neurons never responded to capsaicin. UTP, a P2Y purinoceptor 2 ([P2Y.sub.2])/[P2Y.sub.4] receptor agonist, reversed capsaicin-induced TRPV1 desensitization. 2-methyl-thio-ATP (2Me-S-ATP), a [P2Y.sub.1] receptor agonist, did not change desensitization. MRS 2179 and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), drugs that block [P2Y.sub.1] receptors, did not block ATP-induced resensitization of TRPV1. Suramin, a [P2Y.sub.2] receptor antagonist, blocked resensitization caused by UTP. Immunocytochemical studies showed that FB-labeled neurons coexpressed [P2Y.sub.2] receptors and TRPV1. We conclude that [P2Y.sub.2] receptor activation can maintain TRPV1 function perhaps during sustained episodes of activity of kidney projecting sensory neurons. dorsal root ganglia; capsaicin; desensitization; retrograde labeling doi: 10.1152/ajpregu.00235.2009.

The enteric nervous system (ENS) controls gut function. P2X receptors and nicotinic acetylcholine receptors (nAChRs) are ligand-gated cation channels that mediate fast synaptic excitation in the ENS. Close molecular coupling in enteric neuronal membranes contributes to a mutually inhibitory interaction between these receptors; this effect is called cross-inhibition. We studied the molecular mechanisms responsible for cross-inhibition. Whole cell patch-clamp techniques were used to measure P2X-and nAChR-mediated currents in cultured enteric neurons and HEK-293 cells. In cultured myenteric neurons, ACh (3 mM) and ATP (1 mM) coapplication evoked an inward current that was only 57 [+ or -] 6% (P < 0.05) of the predicted current that would have occurred if the two populations of channels were activated independently. In HEK-293 cells coexpressing [[alpha].sub.3][[beta].sub.4] nAChR/[P2X.sub.2] receptors, coapplication of ATP and ACh caused a current that was 58 [+ or -] 7% of the predicted current (P < 0.05). To test the importance of P2X subunit COOH-temainal tail length on cross-inhibition, [P2X.sub.3] and [P2X.sub.4] subunits, which have shorter COOH-terminal tails, were studied. Cross-inhibition with [[alpha].sub.3][[beta].sub.4] nAChRs and [P2X.sub.3] or [P2X.sub.4] subunits was similar to that occurring with [P2X.sub.2] subunits. P2X receptor or [[alpha].sub.3][[beta].sub.4] nAChR desensitization did not prevent receptor cross-inhibition. These data indicate that the [[alpha].sub.3][[beta].sub.4]-P2X receptor interaction is not restricted to [P2X.sub.2] subunits. In addition, active and desensitized conformations of the P2X receptor inhibit nAChR function. These molecular interactions may modulate the function of synapses that use ATP and ACh as fast synaptic transmitters in the ENS. enteric nervous system; synaptic transmission; ligand-gated ion channels

5-[HT.sub.4] receptor agonists facilitate synaptic transmission in the enteric nervous system, and these drugs are used to treat constipation. In the present study, we investigated the effects of the [5-HT.sub.4] receptor agonist, renzapride, on rundown and recovery of fast excitatory postsynaptic potentials (fEP-SPs) during and after trains of stimulation and on transmitter release from individual myenteric neuronal varicosities. Intracellular electrophysiological methods were used to record fEPSPs from neurons in longitudinal muscle myenteric plexus preparations of guinea pig ileum in vitro. During trains of supramaximal electrical stimulation (10 Hz, 2 s), fEPSP amplitude declined (time constant = 0.6 [+ or -] 0.1 s) from 17 [+ or -] 2 mV to 0.7 [+ or -] 0.3 mV. Renzapride (0.1 [micro]M) did not change the time constant for fEPSP rundown, but it decreased the time constant for recovery of fEPSP amplitude after the stimulus train from 7 [+ or -] 2 s to 1.6 [+ or -] 0.2 s (P < 0.05). 5-HT (0.1 [micro]M) also increased fEPSPs and facilitated recovery from rundown. The adenylate cyclase activator, forskolin (1 [micro]M), mimicked the actions of renzapride and 5-HT, whereas H-89, a protein kinase A (PKA) inhibitor, blocked the effects of renzapride. We used nicotinic acetylcholine receptor containing outside-out patches obtained from myenteric neurons maintained in primary culture to detect acetylcholine release from single varicosities. Renzapride (0.1 [micro]M) increased release probability two-fold. We conclude that [5-HT.sub.4] receptors activate the adenylyl cyclase-PKA pathway to increase acetylcholine release from single varicosities and to accelerate recovery from synaptic rundown. These responses may contribute to the prokinetic actions of [5-HT.sub.4] receptor agonists. fast synaptic transmission; enteric nervous system; electrophysiology; renzapride; single nicotinic acetylcholine receptor channels

Increased sympathetic nervous activity (SNA) elevates venomotor tone in deoxycorticosterone acetate (DOCA)-salt hypertension. We studied the mechanisms by which the SNA increases venomotor tone in DOCA-salt hypertension by making in situ intracellular recordings of venous smooth muscle cell (VSMC) membrane potential (Era) and measurement of outside diameter (OD) in mesenteric veins (MV) and mesenteric arteries (MA) of anesthetized rats. We also studied norepinephrine (NE)- and endothelin-1 (ET-1)-induced increases in MA or MV perfusion pressure (PP) in vitro. [E.sub.m] in DOCA-salt MV was depolarized compared with sham MV. Prazosin hyperpolarized VSMC [E.sub.m] in DOCA-salt but not in sham MV. NE concentration-response curves (CRCs) for OD decreases in MV from DOCA-salt rats were left-shifted with an increased maximum response ([E.sub.max] ) compared with sham MV. NE CRCs for OD decreases in MA were right-shifted with reduced [E.sub.max] in DOCA-salt compared with sham rats. ET-1 CRCs were similar in DOCA-salt and sham MV but were right-shifted with reduced [E.sub.max] in DOCA-salt MA. NE CRCs for MAPP increases were left-shifted without a change in [E.sub.max] in DOCA-salt rats. NE did not change MVPP. MAPP and MVPP for ET-1 CRCs were similar in sham and DOCA-salt rats, but [E.sub.max] for MAPP was reduced in DOCA-salt rats. Hematoxylin staining revealed hypertrophy in DOCA-salt MA but not in MV. We conclude that there is increased reactivity to NE released from the sympathetic nervous system in DOCA-salt MV that causes VSMC depolarization and increased venomotor tone. In DOCA-salt rats, in vivo ET-1 reactivity is maintained in MV, but reduced in MA. membrane potential; venomotor tone; norepinephrine; endothelin-l; hypertension doi:10.1152/ajpheart.01414.2006

Continuous amperometry with a diamond microelectrode and video microscopy were used to record (in vitro) endogenous norepinephrine release simultaneously with the evoked contractile response of a mesenteric artery from a healthy Sprague Dawley rat. Norepinephrine (NE) is a vasoconstricting neurotransmitter released from sympathetic nerves that innervate the smooth muscle cell layers surrounding arteries and veins. Using these two techniques along with several drugs, the NE released at sympathetic neuroeffector junctions nearby the microelectrode was measured as an oxidation current. Key to the amperometric measurement was the use of a diamond microelectrode because of the response sensitivity, reproducibility, and stability it provided. NE release was elicited by electrical stimulation at frequencies between 1 and 60 Hz, with a maximum response seen at 20 Hz. Confirmation that the oxidation current was, in fact, associated with endogenous NE came from the results of several drugs. Tetrodotoxin (TTX, 0.3 [micro]M), a voltage-dependent sodium channel antagonist that blocks nerve conduction, abolished both the oxidation current and the arterial constriction. The [[alpha].sub.2]-adrenergic autoreceptor antagonist, yohimbine (1.0 [micro]M), caused an increase in the oxidation current and the corresponding constriction. The addition of cocaine (10 [micro]M), an antagonist that inhibits neuronal NE reuptake, caused both the oxidation current and the contractile response to increase. These results, combined with the fact that the hydrodynamic voltammetric [E.sub.1/2] for endogenous NE was identical to that for a standard solution, confirmed that the oxidation' current was due to NE and that this compound caused, at least in part, the contractile response. The results demonstrate that continuous amperometric monitoring of NE with a diamond microelectrode and video imaging of vascular tone allow real time local measurement of the temporal relationship between nerve-stimulated NE release and arterial constriction.

Dai and colleagues (Dai X, Galligan JJ, Watts SW, Fink GD, and Kreulen DL. Hypertension 43: 1048-1054, 2004) found that endothelin (ET) stimulated [O.sup.-.sub.2] production in sympathetic ganglion neurons in vitro by activating [ET.sub.B] receptors. The objective of the present study was to determine whether activation of [ET.sub.B] receptors in vivo elevates Of levels in sympathetic ganglia. Because [ET.sub.B] receptor activation increases blood pressure, we also sought to determine whether alteration in [O.sup.-.sub.2] levels was a direct effect of [ET.sub.B] receptor activation on sympathetic ganglia or an indirect consequence of hypertension. Male Sprague-Dawley rats received intravenous infusions of either the specific [ET.sub.B] receptor agonist sarafotoxin 6c (S6c; 5 pmol x [kg.sup.-1] x [min.sup.-1]) or isotonic saline at 0.01 ml/min (control) for 120 min. To measure [O.sup.-.sub.2] levels, we removed the inferior mesenteric ganglion immediately after infusion and stained it with dihydroethidine (DHE). Mean arterial pressure increased 26.6 [+ or -] 1.7 mmHg in the S6c-treated rats and 3.65 [+ or -] 6 mmHg in control rats. Measurements of average pixel intensity revealed that the DHE fluorescence in ganglionic neurons and surrounding glial cells were 96.7% and 160% greater in S6c-treated than in control rats, respectively. To evaluate the effect of elevated blood pressure on [O.sup.-.sub.2] production, a separate group of rats received phenylephrine (PE; 10 [micro]g x [kg.sup.-1] x [min.sup.-1] iv) for 2 h. MAP increased 31 [+ or -] 1.2 mmHg in PE-infused rats. The DHE fluorescence intensity in ganglia of PE-infused rats was significantly greater than that of control rats, 137.7% in neurons and 104.6% in glia but significantly lower than in ganglia from S6c rats. We conclude that ETB receptor activation in vivo significantly enhances [O.sup.-.sub.2] levels in sympathetic ganglia, due to both pressor effects and direct stimulation of [ET.sub.B] receptors in ganglion cells. hypertension; sarafotoxin 6c; sympathetic nervous activity; oxidative stress; reactive oxygen species

Vascular capacitance is reduced by endothelin-1 (ET-1) in deoxycorticosterone (DOCA)-salt hypertensive rats. This may contribute to hypertension development. Because the splanchnic blood vessels (especially veins) are important in determining vascular capacitance, we tested the hypothesis that ET-1 levels in the splanchnic vasculature are elevated in hypertensive DOCA-salt compared with normotensive rats. Tissue ET-1 content was measured by ELISA in aorta, vena cava, superior mesenteric artery and vein, and small mesenteric arteries and veins from normotensive sham-operated (sham) and 4-wk DOCA-salt rats. We also determined ET-1 concentration in aortic and portal venous blood (draining the nonhepatic splanchnic organs) in anesthetized and conscious sham and DOCA-salt rats before and after acute blockade of E[T.sub.B] receptor-mediated plasma clearance of ET-1. Results showed a higher ET-1 content in veins than in arteries of similar size. However, ET-1 content was similar in vessels from sham and DOCA-salt rats, except in aorta and superior mesenteric artery, where ET-1 content was greater in DOCA-salt rats. ET-1 concentration was significantly higher in portal venous than in aortic blood, indicating net nonhepatic splanchnic release (nNHSR) of ET-1. However, nNHSR of ET-1 was similar in sham and DOCA-salt rats. Although nNHSR of ET-1 increased significantly after E[T.sub.B] receptor blockade in sham rats, it was completely unchanged in DOCA-salt rats. These data suggest that, despite the absence of E[T.sub.B] receptor-mediated plasma clearance of ET-1, neither the venous peptide content nor the net release of ET-1 is increased in the splanchnic vasculature of DOCA-salt rats. These results argue against the hypothesis that increased venomotor tone in DOCA-salt hypertension is caused by increased ET-1 concentration around splanchnic venous smooth muscle cells. endothelin-1; splanchnic vasculature; vascular capacitance; veins; deoxycorticosterone

Currents carried by L-, N-, and P/Q-type calcium channels do not account for the total calcium current in myenteric neurons. This study identified all calcium channels expressed by guinea pig small intestinal myenteric neurons maintained in primary culture. Calcium currents were recorded using whole cell techniques. Depolarizations (holding potential = -70 mV) elicited inward currents that were blocked by Cd[Cl.sub.2] (100 [micro]M). Combined application of nifedipine (blocks L-type channels), [OMEGA]-conotoxin GVIA (blocks N-type channels), and [OMEGA]-agatoxin IVA (blocks P/Q-type channels) inhibited calcium currents by 56%. Subsequent addition of the R-type calcium channel antagonists, Ni[Cl.sub.2] (50 [micro]M) or SNX-482 (0. 1 [micro]M), abolished the residual calcium current. Ni[Cl.sub.2] or SNX-482 alone inhibited calcium currents by 46%. The activation threshold for R-type calcium currents was -30 mV, the half-activation voltage was -5.2 [+ or -] 5 mV, and the voltage sensitivity was 17 [+ or -] 3 mV. R-type currents activated fully in 10 ms at 10 mV. R-type calcium currents inactivated in 1 s at l0 mV, and they inactivated (voltage sensitivity of 16 [+ or -] 1 mV) with a half-inactivation voltage of -76 [+ or -] 5 mV. These studies have accounted for all of the calcium channels in myenteric neurons. The data indicate that R-type calcium channels make the largest contribution to the total calcium current in myenteric neurons. The relatively positive half-activation voltage and rapid activation kinetics suggest that R-type channels could contribute to calcium entry during somal action potentials or during action potential-induced neurotransmitter release. ion channels; enteric nervous system; electrophysiology

Luo, Min, Gregory D. Fink, Keith J. Lookingland, John A. Morris, and James J. Galligan. Impaired function of [[alpha].sub.2]-adrenergic autoreceptors on sympathetic nerves associated with mesenteric arteries and veins in DOCA-salt hypertension. Am J Physiol Heart Circ Physiol 286: H1558-H1564, 2004. First published December 11, 2003; 10.1152/ajpheart.00592.2003.--The present study tested the hypothesis that there is impaired function of [[alpha].sub.2]-adrenergic autoreceptors and increased transmitter release from sympathetic nerves associated with mesenteric arteries and veins from DOCA-salt rats. High-performance liquid chromatography was used to measure the overflow of ATP and norepinephrine (NE) from electrically stimulated mesenteric artery and vein preparations in vitro. In sham arteries, nerve stimulation evoked a 1.5-fold increase in NE release, whereas in DOCA-salt arteries there was a 3.9-fold increase in NE release over basal levels (P < 0.05). In contrast, stimulated ATP release was not different in DOCA-salt arteries compared with sham arteries. In sham veins, nerve stimulation evoked a 2.9-fold increase in NE release, whereas in DOCA-salt veins there was a 8.4-fold increase in NE release over basal levels (P < 0.05). In sham rats NE release, normalized to basal levels, was greater in veins than in arteries (P < 0.05). The [[alpha].sub.2]-adrenergic receptor antagonist yohimbine (1 [micro]M) increased ATP and NE release in sham but not DOCA-salt arteries. The [[alpha].sub.2]-adrenergic receptor agonist UK-14,304 (10 [micro]M) decreased ATP release in sham but not DOCA-salt arteries. In sham veins, UK-14,304 decreased, but yohimbine increased, NE release; effects that were not observed in DOCA-salt veins. These data show that nerve stimulation causes a greater increase in NE release from nerves associated with veins compared with arteries. In addition, impairment of [[alpha].sub.2]-adrenergic autoreceptor function in sympathetic nerves associated with arteries and veins from DOCA-salt rats results in increased NE release. norepinephrine; ATP; vasoconstriction; splanchnic circulation

Signaling mechanisms coupled to activation of different neurotransmitter receptors interact in the enteric nervous system. ACh excites myenteric neurons by activating nicotinic ACh receptors (nAChRs) and muscarinic receptors expressed by the same neurons. These studies tested the hypothesis that muscarinic receptor activation alters the functional properties of nAChRs in guinea pig small intestinal myenteric neurons maintained in primary culture. Whole cell patch-clamp techniques were used to measure inward currents caused by ACh (1 mM) or nicotine (1 mM). Currents caused by ACh and nicotine were blocked by hexamethonium (100 [micro]M) and showed complete cross desensitization. The rate and extent of nAChR desensitization was greater when recordings were obtained with ATP/GTP-containing compared with ATP/GTP-free pipette solutions. These data suggest that ATP/GTP-dependent mechanisms increase nAChR desensitization. The muscarinic receptor antagonist scopolamine (1 [micro]M) decreased desensitization caused by ACh but not by nicotine, which does not activate muscarinic receptors. Phorbol 12,13-dibutyrate (10-100 nM), an activator of protein kinase C (PKC), but not 4-[alpha]-phorbol 12-myristate 13-acetate (a PKC inactive phorbol ester), increased nAChR desensitization caused by ACh and nicotine. Forskolin (1 [micro]M), an activator of adenylate cyclase, increased nAChR desensitization, but this effect was mimicked by dideoxyforskolin, an adenylate cyclase inactive forskolin analog. These data indicate that simultaneous activation of nAChRs and muscarinic receptors increases nAChR desensitization. This effect may involve activation of a PKC-dependent pathway. These data also suggest that nAChRs and muscarinic receptors are coupled functionally through an intracellular signaling pathway in myenteric neurons. enteric nervous system; electrophysiology; intracellular signaling