Pituitary adenylate cyclase-activating polypeptide may function as a neuromodulator in guinea-pig adrenal medulla

Abstract

The role of pituitary adenylate cyclase-activating polypeptide (PACAP) in catecholamine secretion from dissociated adrenal chromaffin cells of the guinea-pig was investigated using amperometry, the patch clamp technique and immunochemistry. Pretreatment of adrenal chromaffin cells with 0.3-10 nM PACAP for 2 min resulted in enhancement of nicotine- and muscarine-induced secretions in either the presence of external Ca2+ ions or nominally Ca2+-free solution, with no change in basal secretion or the holding current at -60 mV in most of the cells tested. Pretreatment with PACAP augmented the muscarine-induced non-selective cation current, but did not affect the muscarine-induced outward current or nicotine-induced current. PACAP-induced enhancement of nicotine- and muscarine-induced secretions was suppressed by the simultaneous application of PACAP and the protein kinase inhibitors 100 microM HA1004 or 2 microM H89. Application of forskolin enhanced both muscarine- and nicotine-induced secretions, whereas application of a phorbol ester augmented the nicotine-induced secretion, but suppressed the muscarine-induced secretion in a reversible manner. Immunohistochemical analysis of adrenal medullae revealed that PACAP-like immunoreactivity was present in nerve fibres surrounding putative chromaffin cells. PAC1R-like immunoreactivity was distributed diffusely in the plasma membrane, whereas nicotinic ACh receptor-like immunoreactivity was concentrated at the plasma membrane near the nucleus, where the synapses were mainly localized. These observations suggest that PACAP in the guinea-pig adrenal medulla functions as a neuromodulator to facilitate ACh-induced secretion through a cAMP-protein kinase A-dependent pathway.

Figure 1. Application of PACAP38 does not induce secretion but does enhance the subsequent catecholamine secretion evoked by nicotine

A, amperometric recordings of catecholamine secretion in response to 10 μm nicotine. Nicotine (N) and 1 nm PACAP38 were added to the perfusate for the periods indicated (bars for N and double line for PACAP38). Chart records were interrupted for the indicated times. B, cumulative curves of secretion; a-c shown in A correspond to a-c in B. Zero on the abscissa represents the time when nicotine was applied.

Figure 2. Dose- and time-dependent enhancement of nicotine-induced secretion by PACAP pretreatment

A, amperometric recordings of catecholamine secretion in response to 10 μm nicotine. Nicotine was bath applied during the periods indicated (bars) before (con) and after treatment with 10 nm (upper traces) and 0·3 nm (lower traces) PACAP27 (PACAP; P, double lines). PACAP was applied for about 2 min in this and the following figures. Some of the recordings during PACAP pretreatment are not shown. Upper and lower records are from the same cell. B, amount of nicotine-induced secretion, expressed as a fraction of that just before PACAP pretreatment (see Methods), plotted against time after PACAP pretreatment; a-e shown in A correspond to a-e in B. Squares represent nicotine-induced secretion following 0·1 nm PACAP pretreatment. C, relative amount of nicotine-induced secretion plotted against the concentration of PACAP applied for pretreatment. Means ±s.e.m. of 4–15 cells for each concentration of PACAP (○) and of 4–8 cells for PACAP38 (▵). The line represents y = 2·65(C/(C + 2·06)) + 1, where y is the relative amount of secretion and C is the concentration of PACAP.

Figure 3. Enhancement of muscarine-induced secretion in the presence of external Ca²⁺ ions and Ca²⁺-deficient solution by PACAP pretreatment

A–C, amperometric recordings of secretion in response to 3 μm muscarine (M), 10 μm muscarine in Ca²⁺-deficient solution, and 10 μm nicotine (N). Chemicals were bath applied during the periods indicated (bars for m and N, double line for PACAP (P)) and Ca²⁺-deficient solution was substituted for standard saline during the periods indicated by the dotted line. Some of the recordings during exposure to 1 nm PACAP are not shown. Numbers represent approximate times (min) after PACAP pretreatment. B and C were from the same cell (different from that in A). D, relative amount of secretion in response to 10 μm nicotine, 3 μm muscarine and 10 μm muscarine in Ca²⁺-deficient solution (M Ca(−)). The amount of secretion is expressed as a fraction of that before 1 nm PACAP pretreatment. Means and s.e.m. of 15 cells for N, 7 cells for m and 6 cells for m Ca(−). *P = 0·023, **P = 0·101 (Mann-Whitney test).

Figure 4. Enhancement of nicotine-induced secretion by pretreatment with phorbol 12,13-dibutyrate and forskolin

A, amperometric recordings of secretion in response to 10 μm nicotine. Nicotine (N, bar), 0·1 μm phorbol 12,13-dibutyrate (PB, double line) or 10 μm forskolin (F, double line) was bath applied during the indicated periods. Some of the recordings during 2 min exposure to forskolin are not shown. B, amount of secretion relative to that evoked by the first application of nicotine, plotted against recording time; a-f shown in A correspond to a-f in B. Bars indicate exposure to PB or F.

Figure 5. Abolition of muscarine-evoked secretion by pretreatment with a phorbol ester

A–C, amperometric recordings of secretion in response to 3 μm muscarine. Muscarine (M) and 10 nm PACAP, 1 μm phorbol 12,13-dibutyrate (Phorbol) or 10 μm forskolin were bath applied during the periods indicated (M, bar; PACAP, phorbol or forskolin, double line). A and B were obtained from the same cell, but C was from a different cell. Approximate times after the pretreatment are indicated. D, relative amount of nicotine (N)- or muscarine (M)-induced secretion after pretreatment with 10 μm forskolin or 1 or 0·1 μm phorbol 12,13-dibutyrate. The amount of nicotine- or muscarine-evoked secretion after the pretreatment is expressed relative to that before pretreatment. Means and s.e.m. of 3 cells for N and 4 cells for m with forskolin, and 5 cells for N and 4 cells for m with phorbol.

Figure 6. Lack of augmentation of PACAP-induced enhancement of secretion by simultaneous application of PACAP and IBMX

A, amperometric recording of secretion in response to 10 μm nicotine. Nicotine (N) and 0·1 mm IBMX, 1 nm PACAP (P) or IBMX + PACAP were bath applied during the periods indicated (N, bar; IBMX, PACAP or PACAP and IBMX, double line). Since addition of IBMX induced an upward shift of the basal current level, IBMX was washed out in standard saline prior to the application of nicotine. B, increase in nicotine-induced secretion after pretreatment with IBMX, PACAP, and PACAP and IBMX. IBMX solutions were sonicated, using an ultrasonic homogenizer. Means and s.e.m. of 4 cells.

Figure 7. Abolition of PACAP-induced enhancement of secretion by protein kinase inhibitors

A, amperometric recordings of 10 μm nicotine (N; a)- and 3 μm muscarine (M; b)-induced secretions. Upper and lower traces, pretreatment with 1 nm PACAP (P) and with PACAP and 100 μm HA1004 (HA; a) or 2 μm H89 (b), respectively. a and b were from different cells. Chemicals were bath applied during the periods indicated (N and M, bar; PACAP or PACAP and HA or H89, double line). Some of the recordings during pretreatment are not shown. B, relative amount of nicotine- and muscarine-induced secretion after pretreatment with 1 nm PACAP, 1 nm PACAP and 100 μm HA1004, and 1 nm PACAP and 2 μm H89. Means and s.e.m. of 3 cells for HA and 4 cells for H89.

Figure 8. Effects of PACAP on resting whole-cell current and muscarine- and nicotine-induced currents

A and C, traces of whole-cell current recorded at −60 mV with the perforated patch method. Chemicals (10 μm muscarine in Ca²⁺-deficient solution, m Ca(−); 3 μm muscarine, M; 10 μm nicotine, N; 1 nm PACAP) were bath applied during the indicated periods. B, half-decay times (T_½) and amplitudes (I_O) of 10 μm muscarine-induced outward currents in Ca²⁺-deficient solution after exposure to PACAP, expressed as fractions of those before exposure. Means and s.e.m. of 5 cells. D, amplitudes of muscarine- and nicotine-induced inward currents (I_M and I_N, respectively) after exposure to PACAP, expressed as fractions of those prior to exposure. Means and s.e.m. of 11 cells for I_M and 4 cells for I_N. The relative amplitudes of currents induced by 3 μm muscarine (1·39 ± 0·21, n = 6) and 10 μm muscarine in Ca²⁺-deficient solution (1·84 ± 0·11, n = 5) were pooled.

Figure 9. Immunochemistry of PACAP and PAC1R in adrenal medullary cells

A, section of adrenal medulla immunostained for PACAP. PACAP-like immunoreactive nerve fibres (arrows) with varicosities (arrowhead) were distributed in close proximity to chromaffin cells. PACAP-positive nerve fibres were also visible around capillaries. B, confocal microscopic images of cells immunostained for PAC1R. Images represent superimposition of whole-cell images and PAC1R-like immunoreactivity (yellow) (see Methods). Z-axis analysis was performed with a full width at half-maximal intensity of ca 0·7 μm. Arrows indicate sites for XZ (a) and YZ (b) images. Asterisks in this and the following figures indicate the location of the nucleus. C, histogram of the distribution of PAC1R-like immunoreactivity. N and Non indicate the presence of PAC1R-like immunoreactivity in the plasma membrane of the nuclear and non-nuclear portions of the cell, respectively, and m indicates its presence in both compartments.

Figure 10. PAC1R in Golgi complex and plasma membrane

Immunostainings for PAC1R (upper image) and βCOP (lower image). PAC1R-like immunoreactivity and βCOP-like immunoreactivity were visualized using FITC- and rhodamine-conjugated secondary antibodies, respectively (see Methods). The area adjacent to the nucleus (arrowhead) was immunoreactive for both PAC1R and βCOP, whereas part of the plasma membrane was immunoreactive only for PAC1R (arrow). The strongest fluorescence is displayed in red.

Figure 11. Localization of nAChR-like immunoreactivity and synapse

A, confocal microscopic images of nAChR-like immunoreactivity. Upper and lower images correspond to YZ and XY images. Z-axis analysis was similar to that in Fig. 9. Arrowheads represent spots of nAChR-like immunoreactivity. Arrow indicates the site of the YZ image. B, histogram of nAChR-like immunoreactive spots with various distances between spots and the nearest rim of the nucleus. Distances are expressed as fractions of the longest diameter of the cell (see text). C, electron micrograph of chromaffin cells. Arrow indicates an asymmetrical synapse; the inset shows this area at a higher magnification.