Sensitization of enteric neurons to morphine by HIV-1 Tat protein

Abstract

Background: Gastrointestinal (GI) dysfunction is a major cause of morbidity in acquired immunodeficiency syndrome (AIDS). HIV-1-induced neuropathogenesis is significantly enhanced by opiate abuse, which increases proinflammatory chemokine/cytokine release, the production of reactive species, glial reactivity, and neuronal injury in the central nervous system. Despite marked interactions in the gut, little is known about the effects of HIV-1 in combination with opiate use on the enteric nervous system.

Methods: To explore HIV-opiate interactions in myenteric neurons, the effects of Tat ± morphine (0.03, 0.3, and 3 μM) were examined in isolated neurons from doxycycline- (DOX-) inducible HIV-1 Tat(1-86) transgenic mice or following in vitro Tat 100 nM exposure (>6 h).

Key results: Current clamp recordings demonstrated increased neuronal excitability in neurons of inducible Tat(+) mice (Tat+/DOX) compared to control Tat-/DOX mice. In neurons from Tat+/DOX, but not from Tat-/DOX mice, 0.03 μM morphine significantly reduced neuronal excitability, fast transient and late long-lasting sodium currents. There was a significant leftward shift in V(0.5) of inactivation following exposure to 0.03 μM morphine, with a 50% decrease in availability of sodium channels at -100 mV. Similar effects were noted with in vitro Tat exposure in the presence of 0.3 μM morphine. Additionally, GI motility was significantly more sensitive to morphine in Tat(+) mice than Tat(-) mice.

Conclusions & inferences: Overall, these data suggest that the sensitivity of enteric neurons to morphine is enhanced in the presence of Tat. Opiates and HIV-1 may uniquely interact to exacerbate the deleterious effects of HIV-1-infection and opiate exposure on GI function.

Keywords: HIV-1 Tat; MOR-1 expression; enteric neurons; excitability; gut; ileum; opioid drug abuse; sodium currents.

Figure 1. Effect of morphine on enteric neuronal excitability is enhanced in neurons from Tat+/DOX but not Tat−/DOX mice

Neuronal excitability of enteric neurons isolated from adult mice ileum was assessed by whole-cell patch-clamp studies in current clamp mode in neurons from Tat−/DOX (A & C) and Tat+/DOX mice (B & D). (A & C) Neurons isolated from Tat−/DOX elicit single action potentials at a rheobase of 10 pA, with (A) no effect of a low 0.03 μM concentration of morphine on excitability, but (C) a significant reduction in neuronal excitability with a high 0.3 μM concentration of morphine. (B & D) In contrast, neurons from Tat+/DOX elicit multiple action potentials at the same rheobase, with (B) the low 0.03 μM concentration and (D) the high 0.3 μM concentration of morphine significantly reducing neuronal excitability. Thus, neurons from Tat+/DOX demonstrate an increased neuronal sensitivity to morphine.

Figure 2. Effect of morphine on sodium current densities is significantly enhanced in neurons from Tat+/DOX mice

Sodium current density was assessed by voltage clamp recordings with Cs⁺ in the internal solution. (A & B) Raw traces of sodium channel currents of neurons from (A) Tat−/DOX mice show no effect for a low 0.03 μM concentration of morphine on sodium currents, whereas (B) Tat+/DOX show a significant reduction in the presence of 0.03 μM morphine. (C & D) A current density-voltage relationship shows that for the fast transient inward sodium currents (C) in neurons from Tat−/DOX, 0.3 μM inhibits sodium flow but not 0.03 μM morphine, whereas (D) in neurons from Tat+/DOX a significant reduction is noticed for 0.3 μM and 0.03 μM morphine. Insets indicate inhibition at peak responses with morphine that was significantly enhanced for morphine 0.03 μM in Tat+/DOX compared to Tat−/DOX. (E & F) A current density-voltage relationship shows that for the late component of the inward sodium currents (E) in Tat−/DOX morphine reduces the long-lasting inward sodium current density, specifically for the high 0.3 μM morphine concentration, whereas (F) in Tat+/DOX a significant reduction is noticed similarly for 0.3 μM and 0.03 μM morphine. Data are expressed as mean ± SEM. Bar graphs: one-sample t-test, *p < 0.05 vs 0 % inhibition, ^†p < 0.05 vs. Tat−/DOX + Morphine 0.03 μM, M = morphine.

Figure 3. Effect of morphine on sodium current densities is significantly enhanced in the presence of in vitro Tat 100 nM

As seen for neurons of Tat transgenic mice, similar effects were noted for in vitro Tat exposure on sodium current density. (A & B) Raw traces of sodium channel currents of (A) control neurons show no effect of a low 0.03 μM concentration of morphine on sodium currents, whereas (B) with in vitro Tat exposure a significant reduction in the presence of 0.03 μM morphine is noted. (C & D) A current density-voltage relationship shows that for the fast inward Na⁺ currents (C) in control neurons 0.3 μM inhibits sodium flow but not 0.03 μM morphine, whereas (D) in the presence of Tat a significant reduction is noticed for 0.3 μM and 0.03 μM morphine, thus demonstrating an enhanced inhibition to 0.3 μM morphine in the presence of Tat. (E & F) A current density-voltage relationship shows that for the late component of the inward Na⁺ currents (E) in control neurons no effect was noted for either concentration of morphine, whereas (F) in the presence of Tat morphine significantly decreases the late inward sodium current density similarly for both morphine concentrations. Data are expressed as mean ± SEM. Bar graphs: one-sample t-test, *p < 0.05 vs 0 % inhibition, M = morphine.

Figure 4. Neurons from Tat+/DOX mice show increased sensitivity to morphine on the inactivation kinetics of sodium channels

Neurons from Tat transgenic mice in combination with morphine 0.03 μM were tested on the voltage-dependence of steady-state activation/inactivation of sodium channels. (A-C) Boltzmann curve analyses of inactivation kinetic of sodium channels. (A, B) In contrast to neurons from Tat−/DOX, Tat+/DOX indicate a significant downward shift of the inactivation curve for 0.03 μM morphine with the fraction of available channels being approximately 55 % decreased. (C) For both, neurons of Tat−/DOX and Tat+/DOX, a leftward shift for the inactivation curve in response to morphine is noted, indicated by significant differences in V_0.5 inactivation values. (D-F) No effects are noted on the activation kinetics of sodium channels. Data are expressed as mean ± SEM. *p < 0.05.

Figure 5. In vitro Tat 100 nM increases the sensitivity to morphine on the inactivation kinetics of sodium channels

Similarly, effects of Tat (100nM) in combination with morphine 0.3 μM were tested on the voltage-dependence of steady-state activation/inactivation of sodium channels. (A-C) Boltzmann curve analyses of inactivation kinetic of sodium channels. (A, B) In contrast to control neurons, neurons exposed to 100 nM Tat indicate a significant downward shift of the inactivation curve for 0.3 μM morphine concentration, similar to the neurons of Tat+/DOX, with the fraction of available channels being approximately 40 % decreased. (C) For both, control neurons and neurons exposed to 100 nM Tat, a leftward shift for the inactivation curve in response to morphine is noted, indicated by a significant difference in V_0.5 inactivation values. (D-F) No effects are noted on the activation kinetics of sodium channels. Data are expressed as mean ± SEM. *p < 0.05.

Figure 6. MOR-1 mRNA expression is not significantly different in ileum tissue of Tat+/DOX compared to Tat−/DOX mice

Real time PCR with a unique primer for the splice variant MOR-1 shows no significant difference in the relative abundance of MOR-1 in ileal tissue of Tat+/DOX and Tat−/DOX mice. The mean ΔC_t value from ileal tissue preparations was calculated and plotted as a histogram (3 independent experiments).

Figure 7. Effects of morphine on GI motility are significantly altered in Tat(+) mice

Analysis of the fecal pellet output in Tat(−) mice (upper panel) and Tat(+) mice (bottom panel). Fecal pellets were counted for 1hr following saline/morphine (i.p.) at doses indicated. Morphine treatment increased stool output at 0.1 mg/kg, whereas it reduced 0.3 mg/kg in Tat(+) mice but not Tat(−). Data are expressed as mean ± SEM. *p < 0.05 (n=5 for each group).