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. 2015 May 27;10(5):e0125566.
doi: 10.1371/journal.pone.0125566. eCollection 2015.

Modulation of voltage-gated Ca2+ channels by G protein-coupled receptors in celiac-mesenteric ganglion neurons of septic rats

Mohamed Farrag  1 , Lacee J Laufenberg  2 , Jennifer L Steiner  3 , Gregory E Weller  1 , Charles H Lang  3 , Victor Ruiz-Velasco  1
Affiliations

Modulation of voltage-gated Ca2+ channels by G protein-coupled receptors in celiac-mesenteric ganglion neurons of septic rats

Mohamed Farrag et al. PLoS One. .

Abstract

Septic shock, the most severe complication associated with sepsis, is manifested by tissue hypoperfusion due, in part, to cardiovascular and autonomic dysfunction. In many cases, the splanchnic circulation becomes vasoplegic. The celiac-superior mesenteric ganglion (CSMG) sympathetic neurons provide the main autonomic input to these vessels. We used the cecal ligation puncture (CLP) model, which closely mimics the hemodynamic and metabolic disturbances observed in septic patients, to examine the properties and modulation of Ca2+ channels by G protein-coupled receptors in acutely dissociated rat CSMG neurons. Voltage-clamp studies 48 hr post-sepsis revealed that the Ca2+ current density in CMSG neurons from septic rats was significantly lower than those isolated from sham control rats. This reduction coincided with a significant increase in membrane surface area and a negligible increase in Ca2+ current amplitude. Possible explanations for these findings include either cell swelling or neurite outgrowth enhancement of CSMG neurons from septic rats. Additionally, a significant rightward shift of the concentration-response relationship for the norepinephrine (NE)-mediated Ca2+ current inhibition was observed in CSMG neurons from septic rats. Testing for the presence of opioid receptor subtypes in CSMG neurons, showed that mu opioid receptors were present in ~70% of CSMG, while NOP opioid receptors were found in all CSMG neurons tested. The pharmacological profile for both opioid receptor subtypes was not significantly affected by sepsis. Further, the Ca2+ current modulation by propionate, an agonist for the free fatty acid receptors GPR41 and GPR43, was not altered by sepsis. Overall, our findings suggest that CSMG function is affected by sepsis via changes in cell size and α2-adrenergic receptor-mediated Ca2+ channel modulation.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Normalized current-voltage (I-V) relationships of acutely isolated CSMG neurons from sham control (A) and septic (B) rats 48 hr post-sepsis induction by CLP.
The I-V curves represent the mean Ca2+ current amplitude for each test potential. Ca2+ currents were evoked every 3 s with a 70 ms pulse from a holding potential of -80 mV to test potential between -60 and +60 mV. The current amplitude was measured 10 ms following the onset of the test pulse and normalized to +15 mV. The superimposed Ca2+ current traces shown to the left were evoked to potentials from -25 to +40 mV. The number of neurons tested for each group is shown in parenthesis.
Fig 2
Fig 2. Biophysical properties of CSMG neurons from sham control and septic rats.
Measurement of the mean absolute Ca2+ current amplitude (A) at the depolarizing potential to +10 mV. Ca2+ current density (B) was calculated from the peak Ca2+ current amplitude at the test pulse of +10 mV and normalized to membrane capacitance. The cell membrane capacitance (C) was determined from the numerical integration of a transient elicited with a depolarizing pulse from -80 mV to -70 mV prior to electronic compensation. Summary graph of the basal facilitation ratio (D), calculated as the ratio of Ca2+ current amplitude determined from the test pulse (+10 mV) occurring after (postpulse) and before (prepulse) the +80 mV conditioning pulse (see Fig 3A, top right). The current amplitude was measured isochronally 10 ms after initiation of the test pulse. The numbers in parenthesis indicate the number of neurons tested; * indicates P < 0.05, unpaired t-test, NS indicates not significantly different, P > 0.05.
Fig 3
Fig 3. Time courses of Ca2+ current amplitude for prepulse (●) and postpulse (○) acquired from the application of NE in acutely isolated CSMG neurons from sham control (A) and septic (B) rats 48 hr post-sepsis induction by CLP.
Ca2+ channel currents were evoked every 10 sec with the ‘double-pulse’ voltage paradigm (A, top right). The numbered current traces in A and B are shown to the right. C, summary bar graph illustrating the mean (± SE) prepulse inhibition (first test pulse to +10 mV) and postpulse inhibition (second test pulse to +10 mV) mediated by 10 μM NE. The numbers in parenthesis indicate the number of neurons tested. D, NE concentration-response relationship of CSMG neurons from sham control (▲) and septic (●) rats. Each data point represents the mean (± SE) NE-mediated prepulse current inhibition from 4 to 16 neurons, except for 1 and 30 nM where n = 1. The smooth curves were obtained by fitting the points to the Hill equation and the EC50 (nM) values, shown in the legend, were significantly (P < 0.001) different from each other.
Fig 4
Fig 4. Time courses of Ca2+ current amplitude for prepulse (●) and postpulse (○) acquired from the application of Noc in acutely isolated CSMG neurons from sham control (A) and septic (B) rats 48 hr post-sepsis induction by CLP.
Ca2+ channel currents were evoked as described for Fig 3. C, Noc concentration-response relationship of CSMG neurons from sham control (▲) and septic (●) rats. Each data point represents the mean (± SE) Noc-mediated prepulse current inhibition from 4 to 12 neurons. Both smooth curves were obtained by fitting the points with the Hill equation and the fits were not significantly different (P = 0.95). The legend indicates the EC50 values (nM). D, effect of overnight pretreatment with PTX (500 ng/ml) on the NE (10 μM)- and Noc (3 μM)—mediated Ca2+ current inhibition in both sham control and septic rats. Plot indicates the mean (± SE) current inhibition produced by NE and Noc. Numbers in parenthesis indicate the number of neurons tested. * indicates P < 0.05 compared to each respective control, unpaired t-test.
Fig 5
Fig 5. CSMG neurons express mu opioid (A) and free fatty acid (B) receptors.
A, left, superimposed Ca2+ current traces (evoked as described for Fig 3) in the absence (lower traces, black) and presence (upper traces, gray) of 10 μM DAMGO for sham control and septic rats. A, right, DAMGO concentration-response relationship of CSMG neurons from sham control (▲) and septic (●) rats. Each data point represents the mean (± SE) DAMGO-mediated prepulse current inhibition from 3 to 9 neurons, (except for 0.1 nM DAMGO for sham group where n = 2). Both smooth curves were obtained by fitting the points with the Hill equation and the fits were not significantly different (P > 0.05). The legend indicates the EC50 values (μM). B, left, propionate—mediated Ca2+ current inhibition in sham control and septic rats. B, right, summary plot indicates the mean (± SE) current inhibition produced by 1 and 3 mM propionate. Numbers in parenthesis indicate the number of neurons tested.
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