The actions of Pasteurella multocida toxin on neuronal cells

Abstract

Pasteurella multocida toxin (PMT) activates the G-proteins Gαi(₁₋₃), Gα(q), Gα₁₁, Gα₁₂ and Gα₁₃ by deamidation of specific glutamine residues. A number of these alpha subunits have signalling roles in neurones. Hence we studied the action of this toxin on rat superior cervical ganglion (SCG) neurones and NG108-15 neuronal cells. Both Gα(q) and Gα₁₁ could be identified in SCGs with immunocytochemistry. PMT had no direct action on Kv7 or Cav2 channels in SCGs. However PMT treatment enhanced muscarinic receptor mediated inhibition of M-current (Kv7.2 + 7. 3) as measured by a 19-fold leftward shift in the oxotremorine-M concentration-inhibition curve. Agonists of other receptors, such as bradykinin or angiotensin, that inhibit M-current did not produce this effect. However the amount of PIP₂ hydrolysis could be enhanced by PMT for all three agonists. In a transduction system in SCGs that is unlikely to be affected by PMT, Go mediated inhibition of calcium current, PMT was ineffective whereas the response was blocked by pertussis toxin as expected. M1 muscarinic receptor evoked calcium mobilisation in transformed NG108-15 cells was enhanced by PMT. The calcium rises evoked by uridine triphosphate acting on endogenous P2Y₂ receptors in NG108-15 cells were enhanced by PMT. The time and concentration dependence of the PMT effect was different for the resting calcium compared to the calcium rise produced by activation of P2Y₂ receptors. PMT's action on these neuronal cells would suggest that if it got into the brain, symptoms of a hyperexcitable nature would be seen, such as seizures.

Keywords: Calcium current; G-protein; Intracellular calcium; Kv7 channels; M-current; Muscarinic receptors; NG108-15 cells; Neurones; P2Y receptors; Pasteurella multocida toxin; Superior cervical ganglion cell.

Fig. 1

Immunostaining of rat SCG cells with antibodies to rat Gαq and Gα11. A: representative immunostaining of control cells for antibodies directed against Gαq or Gα11. These images were taken at the same camera exposure of 1.0 s to compare fluorescent staining of the two G-protein subtypes. B: comparative immunostaining for Gαq (upper row) and Gα11 (lower row) in control and PMT (500 ng ml⁻¹, 18–24 h) pre-treated cells. Exposure times were 0.6 s and 2 s for Gαq and Gα11 respectively. The scale bar (25 μm) applies to all panels.

Fig. 2

Preincubation with PMT or CTX has no effect on M-current activation curves. A) Example of a current–voltage relationship. Sympathetic SCG neurons were held at −20 mV to activate the Kv7/M-current then stepped negative every 30 s for 1 s in −10 mV increments to deactivate the current. Current traces show the time-dependent current decline as M-channels close. B) Activation curves measured from the current deactivations (see Methods) for control cells (open squares), cells preincubated in CTX (500 ng ml⁻¹) for 18–24 h (open circles) or in PMT (500 ng ml⁻¹) for 18–24 h (filled circles). Solid lines are Boltzmann fits to the mean data (see Methods and Materials); parameters of the curves are given in Results.

Fig. 3

Preincubation with PMT increases the sensitivity of the M-current to inhibition by oxotremorine-M. A(i) example of a PMT-treated cell's response to oxotremorine-M. Records show current relaxations evoked by holding the cell at −20 mV and then stepping to −50 mV for 1 s. Trace cont: control; trace oxo-M: in the presence of 0.1 μM oxotremorine-M; trace wash: after washing in drug free solution. A(ii): time-course of the changes in deactivation current amplitudes illustrated in A(i). Measurements were made every 30 s. Oxotremorine-M application is shown by the solid bar beneath the points. B) Mean (SEM) concentration–inhibition curves in control cells (open circles), CTX treated cells (filled circles) and PMT treated cells (filled squares). Curves show least-squares Hill fits (solid lines) with the numerical data given in Table 1.

Fig. 4

PMT alters M-current sensitivity to muscarinic receptor but not angiotensin or bradykinin receptor mediated inhibition. Comparison of the effect of 1 nM bradykinin (filled bars) and 100 nM oxotremorine-M (open bars) on M-current amplitude in control (A(i): open symbols) and PMT-pretreated (A(ii): filled symbols) SCG neurons. Note that the inhibition by oxotremorine-M is enhanced whereas inhibition by bradykinin was unaffected by PMT. M-currents were measured every 30 s as deactivation tail amplitudes using a voltage command to −50 mV from a holding potential of −20 mV for 1 s. B: % inhibition of M-current induced by angiotensin II (ATII, 10 and 100 nM) and bradykinin (BK, 1 nM) in control (light bars) and PMT pre-treated cells (dark bars). Numbers of cells in each group are indicated in brackets.

Fig. 5

Effects of PMT on GFP-PLCδ-PH translocation. (A). Example of the membrane-to-cytosolic translocation of the GFP-PLCδ-PH construct (measured as increase in cytosolic fluorescence over basal fluorescence (F/F₀) in a defined region of interest) in a control cell in response to 0.1 μM and 10 μM oxotremorine-M (open and light filled bar respectively) and to 1 nM bradykinin II (dark filled bar). Note: inset shows expanded trace of oxotremorine-M (0.1 μM) application. (B). Similar experiment to (A) but in a PMT (500 ng ml⁻¹, 18–24 h) pre-treated SGC neuron. Drug applications and inset as in (A). (C). Pooled data showing % increase (mean ± SEM) over basal cytosolic fluorescence for two concentration of oxotremorine-M (0.1 and 10 μM), 0.1 μM angiotensin II and 1 nM (0.001 μM) bradykinin. Open bars: control cells; filled bars: PMT pre-treated cells. Numbers in brackets indicate number of cells tested and * indicates significant change (P < 0.05).

Fig. 6

PMT does not alter Go mediated calcium channel current inhibition. (A). Example traces of calcium currents evoked by a twin-pulse protocol (c). Cells were voltage clamped at −70 mV and stepped to 0 mV for 100 ms before (P1) and after (P2) a 50 ms command pulse to +90 mV. In a control cell inhibition produced by norepinephrine (n, 10 μM) shows a strong block during P1 which is very much reduced after the +90 mV depolarising pulse at P2. The pooled data (B) shows that there was no difference in the inhibition between the control cells (filled bars) and those pretreated with PMT (500 ng ml⁻¹ 18–14 h; shaded bars). On the other hand cells pretreated with Pertussis toxin (PTX, 500 ng ml⁻¹, 18–24 h) showed almost complete abolition of the response, suggesting that the inhibition recorded under these conditions (whole cell and high calcium buffering) was mostly mediated by Go activation. The number of cells tested for each condition is given in brackets.

Fig. 7

M1 muscarinic receptor mediated [Ca]_i increases in transformed NG108-15 cells are enhanced by PMT. Concentration response curves for oxotemorine-M evoked calcium increases in NG108-15 cells. Control cell (open circles, N = 10), PMT (500 ng ml⁻¹, 18–24 h) pre-treated cells (filled circles, N = 6) and PMT C1165S (500 ng ml⁻¹, 18–24 h) pre-treated cells (open triangles, N = 5). The points where fit with Hill curves as described in text.

Fig. 8

UTP concentration response curves showing responses in PMT treated cells compared to control cells. A) Example traces where bars indicate UTP application and B) concentration response curves of control (open circles) and 500 ng ml⁻¹ (128-24 h) PMT treated (filled squares) NG108-15 cells. N = 5 for both groups of cells and * indicates P < 0.05 (t-test).

Fig. 9

Concentration and time dependence of PMT on UTP induced calcium responses in NG108-15 cells. A) PMT concentration- and B) time-dependent changes in resting [Ca]_i (filled circles), maximal UTP evoked calcium response (filled squares) and UTP EC₅₀ (filled triangles). The concentration dependence was done at 18–24 h and the symbols on the vertical axis of A indicate control values, in the absence of PMT. The time dependence was done at 500 ng ml⁻¹. N = 5 for all points and * indicates P < 0.05 by ANOVA, compared to controls in A and time zero in B.