Carol A Whiteis

The Ion Channel ASIC2 Is Required for Baroreceptor and Autonomic Control of the Circulation

Arterial baroreceptors provide a neural sensory input that reflexly regulates the autonomic drive of circulation. Our goal was to test the hypothesis that a member of the acid-sensing ion channel (ASIC) subfamily of the DEG/ENaC superfamily is an important determinant of the arterial baroreceptor reflex. We found that aortic baroreceptor neurons in the nodose ganglia and their terminals express ASIC2. Conscious ASIC2 null mice developed hypertension, had exaggerated sympathetic and depressed parasympathetic control of the circulation, and a decreased gain of the baroreflex, all indicative of an impaired baroreceptor reflex. Multiple measures of baroreceptor activity each suggest that mechanosensitivity is diminished in ASIC2 null mice. The results define ASIC2 as an important determinant of autonomic circulatory control and of baroreceptor sensitivity. The genetic disruption of ASIC2 recapitulates the pathological dysautonomia seen in heart failure and hypertension and defines a molecular defect that may be relevant to its development.

Chemoreceptor Hypersensitivity, Sympathetic Excitation, and Overexpression of ASIC and TASK Channels Before the Onset of Hypertension in SHR

Rationale: Increased sympathetic nerve activity has been linked to the pathogenesis of hypertension in humans and animal models. Enhanced peripheral chemoreceptor sensitivity which increases sympathetic nerve activity has been observed in established hypertension but has not been identified as a possible mechanism for initiating an increase in sympathetic nerve activity before the onset of hypertension. Objective: We tested this hypothesis by measuring the pH sensitivity of isolated carotid body glomus cells from young spontaneously hypertensive rats (SHR) before the onset of hypertension and their control normotensive Wistar–Kyoto (WKY) rats. Methods and Results: We found a significant increase in the depolarizing effect of low pH in SHR versus WKY glomus cells which was caused by overexpression of 2 acid-sensing non–voltage-gated channels. One is the amiloride-sensitive acid-sensing sodium channel (ASIC3), which is activated by low pH and the other is the 2-pore domain acid-sensing K+ channel (TASK1), which is inhibited by low pH and blocked by quinidine. Moreover, we found that the increase in sympathetic nerve activity in response to stimulation of chemoreceptors with sodium cyanide was markedly enhanced in the still normotensive young SHR compared to control WKY rats. Conclusions: Our results establish a novel molecular basis for increased chemotransduction that contributes to excessive sympathetic activity before the onset of hypertension.

Acid-sensing ion channels contribute to transduction of extracellular acidosis in rat carotid body glomus cells

Carotid body chemoreceptors sense hypoxemia, hypercapnia, and acidosis and play an important role in cardiorespiratory regulation. The molecular mechanism of pH sensing by chemoreceptors is not clear, although it has been proposed to be mediated by a drop in intracellular pH of carotid body glomus cells, which inhibits a K+ current. Recently, pH-sensitive ion channels have been described in glomus cells that respond directly to extracellular acidosis. In this study, we investigated the possible molecular mechanisms of carotid body pH sensing by recording the responses of glomus cells isolated from rat carotid body to rapid changes in extracellular pH using the whole-cell patch-clamping technique. Extracellular acidosis evoked transient inward current in glomus cells that was inhibited by the acid-sensing ion channel (ASIC) blocker amiloride, absent in Na+-free bathing solution, and enhanced by either Ca2+-free buffer or addition of lactate. In addition, ASIC1 and ASIC3 were shown to be expressed in rat carotid body by quantitative PCR and immunohistochemistry. In the current-clamp mode, extracellular acidosis evoked both a transient and sustained depolarizations. The initial transient component of depolarization was blocked by amiloride, whereas the sustained component was eliminated by removal of K+ from the pipette solution and partially blocked by the TASK (tandem-p-domain, acid-sensitive K+ channel) blockers anandamide and quinidine. The results provide the first evidence that ASICs may contribute to chemotransduction of low pH by carotid body chemoreceptors and that extracellular acidosis directly activates carotid body chemoreceptors through both ASIC and TASK channels.

Mechanosensory transduction of vagal and baroreceptor afferents revealed by study of isolated nodose neurons in culture

Changes in arterial pressure and blood volume are sensed by baroreceptor and vagal afferent nerves innervating aorta and heart with soma in nodose ganglia. The inability to measure membrane potential at the nerve terminals has limited our understanding of mechanosensory transduction. Goals of the present study were to: (1) Characterize membrane potential and action potential responses to mechanical stimulation of isolated nodose sensory neurons in culture; and (2) Determine whether the degenerin/epithelial sodium channel (DEG/ENaC) blocker amiloride selectively blocks mechanically induced depolarization without suppressing membrane excitability. Membrane potential of isolated rat nodose neurons was measured with sharp microelectrodes. Mechanical stimulation with buffer ejected from a micropipette (5, 10, 20 psi) depolarized 6 of 10 nodose neurons (60%) in an intensity-dependent manner. The depolarization evoked action potentials in 4 of the 6 neurons. Amiloride (1 microM) essentially abolished mechanically induced depolarization (15 +/- 4 mV during control vs. 1 +/- 2 mV during amiloride with 20-psi stimulation, n = 6) and action potential discharge. In contrast, amiloride did not inhibit the frequency of action potential discharge in response to depolarizing current injection (n = 6). In summary, mechanical stimulation depolarizes and triggers action potentials in a subpopulation of nodose sensory neurons in culture. The DEG/ENaC blocker amiloride at a concentration of 1 microM inhibits responses to mechanical stimulation without suppressing membrane excitability. The results support the hypothesis that DEG/ENaC subunits are components of mechanosensitive ion channels on vagal afferent and baroreceptor neurons.

Nitric oxide enhances slow inactivation of voltage-dependent sodium currents in rat nodose neurons.

Nitric oxide (NO) can alter neuronal excitability by decreasing the current through voltage-sensitive sodium channels. We hypothesized that NO inhibits sodium currents in part by promoting slow inactivation. We performed whole-cell voltage clamp experiments on sensory neurons from the nodose ganglion. The voltage-dependence of inactivation was determined after stepping the neurons to various potentials between -100 and 30 mV for 200 ms (fast inactivation) and 3 min (slow inactivation) prior to depolarization to 10 mV. NO shifted the voltage of half-inactivation for fast and slow inactivation to more hyperpolarized potentials by 7 and 12 mV, respectively. Sodium currents exhibited a more profound closed state and slow inactivation after exposure to NO. These results demonstrate for the fist time that the slow inactivation of sodium currents is subject to modulation. Due to its effects on fast and slow inactivation, NO may cause a prolonged decrease in neuronal excitability.

TACROLIMUS (FK506) MODULATES CALCIUM RELEASE AND CONTRACTILITY OF INTESTINAL SMOOTH MUSCLE

Several proteins have been identified that associate with calcium release channels and potentially regulate their function. Using tacrolimus as a pharmacological tool, we investigated whether the immunophilin FKBP12 modulates ryanodine receptor channels in intestinal smooth muscle. Results with PCR demonstrated the presence of type-3 ryanodine receptor and FKBP12 in this tissue. Tacrolimus caused an irreversible increase of the intracellular calcium concentration, which was abolished by pretreatment with caffeine. The calcium channel blocker verapamil did not affect the response to tacrolimus. Tacrolimus decreased the calcium concentration in the sarcoplasmic reticulum. Caffeine, but not inositol 1,4,5-trisphosphate or heparin, abolished this effect. Finally, tacrolimus significantly and irreversibly decreased the tension generated by intestinal muscle strips. These data support our hypothesis that the immunophilin FKBP12 modulates ryanodine receptor function in smooth muscle. Interactions between such regulatory proteins and calcium release channels may play an important role in excitation-contraction coupling and other intracellular signaling processes.

Adenovirus-mediated gene transfer to cultured nodose sensory neurons.

Recent advances have enabled transfer of genes to various types of cells and tissues. The goals of the present study were to transfer genes to nodose sensory neurons using replication-deficient adenovirus vectors and to define the conditions needed to optimize the gene transfer. Neurons were dissociated from rat nodose ganglia and maintained in culture. Cultures were exposed for 30 min to vectors containing the beta-galactosidase gene lacZ driven by either the Rous sarcoma virus (RSV) or the cytomegalovirus (CMV) promoter. Cultures were fixed and treated with X-gal to evaluate lacZ expression 1-7 days after exposure to virus. Increasing concentrations of virus led to dose-related increases in the number of neurons expressing lacZ. LacZ was expressed in 8 +/- 2, 39 +/- 6, and 82 +/- 3% of neurons 1 day after exposure to 10(7), 10(8), and 10(9) pfu/ml of AdRSVlacZ, respectively (P < 0.05). The same doses of AdCMVlacZ led to expression in 41 +/- 9, 60 +/- 10, and 86 +/- 4% of neurons. Expression driven by the CMV promoter was essentially maximal within 1 day and remained stable for at least 7 days. In contrast, expression driven by the RSV promoter was less on day 1 but increased over time (1-7 days). There was no lacZ expression in vehicle-treated cultures and exposure to the adenovirus vectors did not adversely influence cell viability. Exposure of the neuronal cultures to an adenovirus vector containing the gene for green fluorescent protein (AdRSVgfp, 10(9) pfu/ml) enabled visualization of successful gene transfer in living neurons. The results indicate that gene transfer to cultured nodose neurons can be accomplished using adenovirus vectors. The expression of the transferred gene persists for at least 7 days, occurs more rapidly when expression is driven by the CMV compared with the RSV promoter, and occurs without adversely affecting cell viability.

Amitriptyline Inhibits Voltage-Sensitive Sodium Currents in Rat Gastric Sensory Neurons

Recent studies indicate that peripheral mechanisms contribute to the analgesic effect of amitriptyline. We hypothesized that amitriptyline inhibits voltage-dependent sodium currents in gastric sensory neurons. To label gastric neurons, the stomach was exposed in male Sprague Dawley rats through a midline incision to inject the retrograde tracer DiI into the gastric wall. Seven days after surgery, nodose ganglia were harvested. Neurons were dissociated and cultured for 4–24 hr to record whole cell sodium currents with the patch-clamp technique. Amitriptyline reversibly inhibited voltage-sensitive sodium currents with an IC50 of 20 μM. At clinically relevant concentrations, the peak sodium current decreased by about 15%. This was associated with a slowed recovery from inactivation, leading to a significantly enhanced cumulative inhibition during brief repetitive depolarizations. These findings are consistent with a use-dependent block of voltage-dependent sodium channels by amitriptyline. This effect may contribute to the analgesic properties of tricyclic antidepressants.

Mechano-and chemosensitivity of rat nodose neurones - : selective excitatory effects of prostacyclin

Nodose ganglion sensory neurones exert a significant reflex autonomic influence. We contrasted their mechanosensitivity, excitability and chemosensitivity in response to the stable prostacyclin (PGI2) analogue carbacyclin (cPGI) in culture. Under current clamp conditions we measured changes in membrane potential (ΔmV) and action potential (AP) responses to mechanically induced depolarizations and depolarizing current injections before and after superfusion of cPGI (1 μm and 10 μm). Chemosensitivity was indicated by augmentation of AP firing frequency and increased maximum gain of AP frequency (max. dAP/dΔmV), during superfusion with cPGI. Results indicate that two groups of neurones, A and B, are mechanosensitive (MS) and one group, C, is mechanoinsensitive (MI). Group A shows modest depolarization without AP generation during mechanical stimulation, and no increase in max. dAP/dΔmV, despite a marked increase in electrical depolarization with cPGI. Group B shows pronounced mechanical depolarization accompanied by enhanced AP discharge with cPGI, and an increase in max. dAP/dΔmV. Group C remains MI after cPGI but is more excitable and markedly chemosensitive (CS) with a pronounced enhancement of max. dAP/dΔmV with cPGI. The effect of cPGI on ionic conductances indicates that it does not sensitize the mechanically gated depolarizing degenerin/epithelial Na+ channels (DEG/ENaC), but it inhibits two voltage‐gated K+ currents, Maxi‐K and M‐current, causing enhanced AP firing frequency and depolarization, respectively. We conclude that MS nodose neurones may be unimodal MS or bimodal MS/CS, and that MI neurones are unimodal CS, and much more CS to cPGI than MS/CS neurones. We suggest that the known excitatory effect of PGI2 on baroreceptor and vagal afferent fibres is mediated by inhibition of voltage‐gated K+ channels (Maxi‐K and M‐current) and not by an effect on mechanically gated DEG/ENaC channels.

Slow inactivation of sodium currents in the rat nodose neurons.

Nodose neurons express sodium currents that can be differentiated based on their sensitivity to tetrodotoxin. Several studies have demonstrated significant differences in voltage-dependence and kinetics of activation and inactivation between tetrodotoxin-sensitive and tetrodotoxin-resistant currents. However, little is known about the slow inactivation. Using whole cell patch-clamp technique fast and slow inactivation of sodium currents were studied in cultured rat nodose neurons. Tetrodotoxin-resistant currents recovered much more rapidly after a 15-ms depolarization than tetrodotoxin-sensitive currents. However, repeated 5-ms depolarizations at 10 Hz induced a cumulative inhibition that was more prolonged in tetrodotoxin-resistant compared to tetrodotoxin-sensitive currents. Consistent with these findings, slow inactivation proceeded more rapidly and was more complete for the tetrodotoxin-resistant than for tetrodotoxin-sensitive currents. While the voltage-dependence of fast inactivation differed significantly between the pharmacologically distinct currents, the voltage-dependence of slow inactivation was similar for both sodium currents. We conclude that slow inactivation of sodium currents can be triggered by trains of brief depolarizations. The resulting prolonged decrease in membrane excitability may contribute to the different patterns of action potential generation observed in primary afferent neurons.