Mechanisms mediating pituitary adenylate cyclase-activating polypeptide depolarization of rat sympathetic neurons

Abstract

The direct effects of pituitary adenylate cyclase-activating polypeptides (PACAP) on sympathetic neurons were investigated using rat superior cervical ganglion neurons. Electrophysiological and pharmacological analyses were used to evaluate PACAP modulation of sympathetic neuron membrane potentials and to investigate potential ionic and intracellular signaling mechanisms mediating the responses. More than 90% of the sympathetic neurons were depolarized by the PACAP peptides even when stimulated release was blocked, indicating that the PACAP peptides elicited primary responses in the postganglionic neurons. The response profile was consistent for activation of PACAP-selective PAC(1) receptors: nanomolar concentrations of PACAP27 and PACAP38 were required to stimulate depolarization, whereas vasoactive intestinal peptide failed to evoke any response. Furthermore, depolarizations elicited by PACAP27 were reduced by the PAC(1) receptor antagonist PACAP(6-38). Both sodium influx and inhibition of a potassium current contributed to the peptide-induced depolarizations. Activation of neither pertussis toxin- nor cholera toxin-sensitive G-proteins was required for generation of the depolarizations. cAMP and diacylglycerol production and activation of protein kinase A or protein kinase C also were not requisite for the responses. By contrast, phospholipase C (PLC)-dependent inositol 1,4,5-triphosphate (IP(3)) synthesis was crucial to the PACAP-mediated depolarizations. Although calcium release from IP(3)-sensitive stores was not required for the PACAP-induced responses, inhibition of IP(3) receptors reduced the depolarizations. Thus, among the many signal transduction pathways coupled to the PAC(1) receptor, the PACAP-induced depolarization of sympathetic neurons appears to require activation of PLC and subsequent generation of IP(3).

Fig. 1.

PACAP27 induces membrane depolarizations and inward currents in sympathetic neurons. A, PACAP-induced depolarization of a sympathetic neuron in response to 1 sec pressure ejection of 50 μm PACAP27 (arrow).B, In the presence of 300 nm TTX, pressure application of 50 μm PACAP27 to sympathetic neurons voltage-clamped to −50 mV revealed the current underlying the PACAP-induced depolarizations.

Fig. 2.

Current–voltage studies support PACAP modulation of multiple conductances. A,I–V curves generated by voltage ramps from −120 to −30 mV at 25 mV/sec were measured before (Control) and during the peak (PACAP27) of PACAP-induced inward currents.B, Data denote differences in ramp currents before and after PACAP application. Representative data from seven separate experimental recordings.

Fig. 3.

PACAP-induced SCG sympathetic neuron depolarizations are concentration-dependent. SCG neurons were superfused (30 sec) with the indicated concentrations of PACAP27 (●), PACAP38 (▴), or VIP (▪), and neuronal depolarization was measured by intracellular recording. Maximal depolarizations of ∼10 mV were attained with 32–100 nm PACAP peptides; PACAP27 and PACAP38 demonstrated identical half-maximal responses at 5 nm peptide. The data represent the mean depolarization (mV) ± SEM (n = 3–7 neurons per concentration).

Fig. 4.

Schematic representation of PAC₁receptor intracellular signaling. PAC₁ receptors are coupled to SCG sympathetic neurons adenylyl cyclase and PLC, resulting in diverse intracellular signaling responses. Second messenger pathway activators and inhibitors used to elucidate the signaling mechanisms underlying the PACAP-induced depolarization are indicated inboxes. The data suggested that PACAP-induced stimulation of IP₃ production results in IP₃ receptor activation; activated IP₃ receptors may directly gate a nonselective cationic conductance, postulated to be a mammalian Trp family channel. PAC₁R, PACAP-selective, PAC₁ receptor; CTX, cholera toxin; FSK, forskolin; dBcAMP, dibutyryl cAMP; PTX, pertussis toxin; NEM,N-ethylmaleimide; BimI, bisindolylmaleimide I; PMA, phorbol myristate acetate;CPA, cyclopiazonic acid; XeD, xestospongin D; XeC, xestospongin C;IP₃R, IP₃ receptor;NSCC, nonselective cationic conductance;AC, adenylyl cyclase; PKA, protein kinase A; PLC, phospholipase C; DAG, diacylglycerol; PKC, protein kinase C.

Fig. 5.

SCG neurons express multiple Trp channel transcripts. Total RNA from rat brain and SCG and primary rat sympathetic neurons in vitro was reverse-transcribed, and the cDNA was amplified using each of the seven oligonucleotide primer sets specific for the Trp channel transcripts (Table 1). The amplified products were resolved on 2% agarose–GelTwin II gels, stained with ethidium bromide, and visualized by UV illumination. The predicted sizes of the seven products are as follows:Trp1, 373 bp; Trp2, 413 bp;Trp3, 363 bp; Trp4, 343 bp;Trp5, 431 bp; Trp6, 465 bp; andTrp7, 558 bp. Trp6 may exhibit multiple transcript isoforms. Among Trp channels, Trp3 Xe sensitivity and store independence have been best studied to date.