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Am J Physiol Heart Circ Physiol 280: H1058-H1065, 2001;
0363-6135/01 $5.00
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Vol. 280, Issue 3, H1058-H1065, March 2001

Pituitary adenylate cyclase-activating polypeptide activates KATP current in rat atrial myocytes

Anne Baron, Dominique Monnier, Angela Roatti, and Alex J. Baertschi

Department of Physiology, Centre Médical Universitaire, CH-1211 Geneva 4, Switzerland


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Because the electrophysiological effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on the heart are little known, we studied the regulation of the atrial ATP-sensitive K+ (KATP) current by PACAP on primary cultured neonatal rat atrial myocytes. PACAP-38 stimulates cAMP production with EC50 = 0.28 nmol/l (r = 0.92, P < 0.02). PACAP-38 and PACAP-27 (10 nmol/l) have similar maximal effects, whereas 100 nmol/l vasoactive intestinal polypeptide (VIP) is 2.7 times less effective (P < 0.05). RT-PCR shows the presence of cloned PACAP receptors PAC1 (>= 2 isoforms), VPAC1, and VPAC2. PACAP-38 dose dependently activates the whole cell atrial KATP current with EC50 = 1-3 nmol/l (n = 44). Maximal effects occur at 10 nmol/l (91 ± 15 pA/pF, n = 18). Diazoxide further increases the PACAP-activated current by 78% (P < 0.05; n = 6). H89 (500 nmol/l), a protein kinase A (PKA) inhibitor, reduces the PACAP-activated KATP current to 17.8 ± 9.6% (n = 5) of the maximal diazoxide-induced current and totally inhibits the cAMP-induced KATP current. A protein kinase C (PKC) inhibitor peptide (50 µmol/l) in the pipette reduces the PACAP-38-induced KATP current to 33 ± 17 pA/pF (P < 0.05, n = 6) without significantly affecting the currents induced by cAMP or VIP. The results suggest that: 1) PAC1, VPAC1, and VPAC2 are present in atrial myocytes; and 2) PACAP-38 activates the atrial KATP channels through both PKA and PKC pathways.

cAMP; patch-clamping; protein kinase C; RT-PCR; vasoactive intestinal polypeptide


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PITUITARY ADENYLATE CYCLASE-ACTIVATING POLYPEPTIDE (PACAP) is a neuropeptide originally isolated from the ovine hypothalamus (28), exhibiting 68% homology with vasoactive intestinal polypeptide (VIP) (1). It was discovered on the basis of its ability to increase adenylate cyclase activity in the rat pituitary (28). VIP and PACAP belong to the same superfamily (the glucagon/secretin superfamily) and act on G protein-coupled receptors with seven conserved transmembrane domains (2, 29, 34). In the central nervous system, PACAP and VIP have been involved in various biological functions, including neurotransmission, neuroprotection, neuronal growth and differentiation, pain transmission, and hypophysial secretion (2, 11, 27, 29). In peripheral tissues, PACAP exhibits a variety of biological activities involving activation of adenylate cyclase and protein kinase A (PKA), an increase in intracellular Ca2+ concentration, and/or the activation of phospholipase C and protein kinase C (PKC) (2, 29, 34). In pancreatic beta -cells, PACAP appears to be by far the most potent insulinotropic peptide known, stimulating insulin release at 0.1 pmol/l (23, 39).

PACAP-related peptides also strongly affect cardiac function (4, 10, 35), but few studies on their effects on cardiac excitability exist. We hypothesized that PACAP peptides modulate cardiac ATP-sensitive K+ (KATP) channels for three reasons. First, KATP channels play a fundamental role in cardiac excitability (3). Second, PACAP peptides are known to induce an endothelium-independent relaxation of the human and porcine coronary artery as well as the pulmonary artery, partly via activation of KATP channels (6, 7, 21). Third, the PACAP-related peptide VIP has been detected in intracardiac nerve endings (12), raising the possibility that PACAP-related peptides may affect both vascular smooth muscle and striated myocytes of the heart. The atrial myocytes were the main focus of our study, because atrial KATP channels potently modulate the stimulated atrial natriuretic peptide (ANP) secretion (20, 38), and PACAP peptides are known to affect various endocrine systems (4, 23, 39). The atrial KATP channel is also interesting because of its high sensitivity to various KATP channel modulators (3).

For these reasons, the goals of this study on primary cultured neonatal rat atrial myocytes were the following: 1) identify and characterize the subtypes of PACAP receptors (2, 14, 16, 27, 29, 34, 36), 2) identify and characterize a PACAP-stimulated KATP current with the whole cell patch-clamp technique, and 3) determine whether the signaling pathway involved PKA, PKC, or both.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Atrial myocyte cultures. Atrial appendage myocytes from 2- to 3-day-old rats were cultured as described previously (3, 20, 24). Briefly, the atrial appendages were minced, dispersed enzymatically with 0.2% trypsin (Worthington; Allschwil, Switzerland), and dispersed mechanically by repeated pipetting in Ca2+- and Mg2+-free medium. Cells were grown in 10% FCS-containing culture medium, a 1:1 mixture of Dulbecco's modified Eagle's medium, and Ham's F-12 (GIBCO-BRL; Basel, Switzerland) for 2-4 days in a 5% CO2 incubator. Cultured atrial myocytes were shown to express ANP mRNA by RT-PCR (Fig. 1C) and ANP by immunostaining (20), with at least 70% of all cells being myocytes on days 2 and 3 of culture. Between 10 and 50% of the myocytes contracted spontaneously at a rate of 0.1-1 Hz, and noncontracting myocytes could be induced to contract by mechanical stimulation, indicating that the myocytes were normally polarized.


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  		Fig. 1.   Characterization of atrial pituitary adenylate cyclase-activating polypeptide (PACAP) receptors by cAMP responses and RT-PCR. A: cellular accumulation of cAMP in response to PACAP-38 (*significantly different from baseline; a, significantly different from 0.1 nmol/l PACAP-38). B: cellular accumulation of cAMP in response to PACAP-related peptides normalized with respect to the stimulatory effect of 10 nmol/l PACAP-38 [*significantly different from baseline; b, significantly different from 10-100 nmol/l vasoactive intestinal polypeptide (VIP)]. C: RT-PCR of RNA extracts from atrial appendages, ventricles, and atrial myocyte cultures. Ladder (in bp) on left. Control without RT but with beta -actin primers. Expected sizes for PCR fragments numbers were the following (in bp): 595 for beta -actin, 535 for atrial natriuretic factor (ANF), 301 for short PAC1, 385 for PAC1-Hip or PAC1-Hop-1, 382 for PAC1-Hop-2, 469 for PAC1-HipHop-1, 298 for VPAC1, and 297 for VPAC2.

Patch-clamp recording of KATP current. KATP currents were recorded on culture days 2-4 at room temperature from initially beating atrial myocytes with the use of the whole cell configuration of the patch-clamp technique (3, 13, 20). Room temperature was chosen to aid the performance of long-term recordings. The cells were observed with an inverted microscope (Diaphot TMD, Nikon; Tokyo, Japan). The currents were recorded by an Axopatch 200B patch-clamp amplifier, digitized by a TL-1 dimethyl amiloride interface, and sampled with the use of a personal computer running pCLAMP software (Axon Instruments). Borosilicate glass patch pipettes were pulled with a BB-CH puller (Mecanex; Nyon, Switzerland) to a resistance of 2-4 MOmega . The standard pipette solution contained (in mmol/l) 121 KCl, 1.5 CaCl2, 10 EGTA, 1.3 MgCl2, 5 ATP, 10 glucose, 34 KOH, and 10 HEPES, pH 7.45 with KOH. The bathing solution contained (in mmol/l) 5 KCl, 1 CaCl2, 1 MgCl2, 118 NaCl, 10 glucose, and 10 HEPES, pH 7.5 with NaOH. The osmolality was adjusted with sucrose to 290 mosmol/kg H2O. The currents were filtered at 2 kHz and sampled at the frequency of 0.8 kHz.

Whole cell KATP currents were recorded on initially beating atrial cardiomyocytes during a 10-s voltage ramp from -80 to +90 mV. A holding potential of -40 mV and the slow ramp protocol prevented the activation of voltage-dependent channels. The protocol also allowed for the recording of current voltage (I-V) relationships, indicating the typically low inward rectification of the KATP channels. The EGTA-buffered low Ca2+ concentration in the patch pipette (10.5 nmol/l free Ca2+), as calculated by the WinMAX chelator software (version 1, 1995, Stanford University), prevented the activation of Ca2+-dependent currents. The presence of 5 mmol/l ATP in the pipette prevented the spontaneous activation of KATP current even during prolonged recordings (30-50 min), but did not prevent the activation by KATP channel openers (3). Recordings with cell membranes that were stretched by drifting patch pipettes were discarded. The initial leak current was subtracted. The KATP current amplitude was measured at +50 mV and plotted as a function of time of whole cell recording or expressed as current density (pA/pF) to calculate mean values. The mean membrane capacitance of atrial cardiomyocytes was 13.0 ± 0.4 (SE) pF (n = 134). The KATP current was compared, whenever possible, with the maximal current activated by 100 µmol/l diazoxide on the same cell. The membrane hyperpolarization triggered by the activation of the KATP current was measured in current clamp mode at 0 pA current. Inhibition by <1 µmol/l glyburide further identified the activated K+ current as a KATP current, because concentrations >= 1 µmol/l are required to significantly modulate voltage-dependent channel activity.

Chemicals and drugs. EGTA, ATP, glyburide, diazoxide, 8-(4-chlorophenyl-thio)-cAMP (8-CPT-cAMP) were all purchased from Sigma (St. Louis, MO). 8-CPT-cAMP was dissolved in water, whereas glyburide and diazoxide were dissolved in DMSO at the stock concentration of 10 and 100 mmol/l, respectively. H-89 dihydrochloride (H89) and 1-oleoyl-2-acetyl-sn-glycerol (OAG) were purchased from Calbiochem (La Jolla, CA) and dissolved in DMSO at the stock concentration of 1 and 50 mmol/l, respectively. The PKC inhibitor (PKC-I) peptide (19-31) was purchased from Upstate Biotechnology (Lake Placid, NY) and added to the pipette solution at 50 µmol/l. VIP, PACAP-27, and PACAP-38 were purchased from Novabiochem (Laufelfingen, Switzerland) and dissolved in 5% ethanol at the stock concentration of 200, 20, and 20 µmol/l, respectively. The two molecular forms of PACAP occur in the brain as well as in peripheral tissues (22, 31). Because PACAP-38 is the predominant form (90%) in tissues (2), most experiments presented here used PACAP-38.

cAMP assays. Atrial myocytes were cultured for 3-4 days in 24-well culture plates at a density of 250,000 cells/well, equilibrated for 80 min in an extracellular HEPES buffer (see the description above) at 37°C, and stimulated for 40 min in the absence of phosphodiesterase inhibitors with PACAP-38, PACAP-27, or VIP at concentrations indicated in Fig. 1. Cells were extracted with 65% ethanol, and the samples were frozen at -30°C, pending radioimmunoassay with the use of a kit (model RPA509, Amersham; Buckinghamshire, UK). On the day of the assay, the samples were evaporated at 65°C under a stream of nitrogen, reconstituted in 500 µl assay buffer, and assayed in each tube by using a sample volume of 50 µl and 25 µl (2,500-3,000 counts/min) of iodinated tracer and 25 µl of diluted antibody. Standard curves were similar to manufacturer specifications, with a mean correlation coefficient of 0.958 ± 0.016 (n = 4 assay series).

RT-PCR analysis of PACAP receptors. The nomenclature follows that of Ref. 14. The three cloned PACAP/VIP receptors (2, 26, 29, 34) are called PAC1, showing a 1,000-fold greater affinity for PACAPs than VIP (16), and VPAC1 and VPAC2, showing similar binding properties for PACAPs and VIP (19, 26). Eight splice variants of PAC1 have been reported (2, 29). VPAC2 is broadly distributed throughout the peripheral tissues, including the pancreas and skeletal muscle, whereas all three PACAP receptor types are found together only in heart, brain, and adipose tissue (2, 14, 27, 36). Atrium alone was not examined before.

Total RNA from 2-day-old cultured neonatal atrial appendage myocytes, or from 4-day-old atrial appendage or ventricular tissue, was extracted by TRIzol Reagent (GIBCO-BRL) and subjected to RQ1 DNase (Promega; Wallisellen, Switzerland) digestion. Total RNA (1 µg) was used for RT experiments. cDNAs were synthesized with random hexaprimers (Boehringer) and Superscript II RT (200 units; GIBCO-BRL) at 37°C during 2 h. PCR was carried out in a HYBAID personal cycler in a final volume of 50 µl containing 1 µl of the RT reaction, 1 unit of Taq polymerase (Appligene Oncor), 1.5 mmol/l MgCl2, 250 µmol/l of each dNTP, and 20 pmol of each primer. The primers used for detecting PAC1 were the following: forward, 5'-CTTGTACAGAAGCTGCAGTC-3' (nucleotide position 987-1006); and reverse, 5'-GGTGCTTGAAGTCCATAG-3' (position 1288-1266). The spanned region covers the splice sites for a short form (301 bp) and two long forms (Hip, 385 bp; Hop-1, 385 bp; and Hop-2, 382 bp). For VPAC1, the primers were the following: forward, 5'-GCCCCCATCCTCCTCTCCATC-3' (position 959-978); and reverse: 5'-TCCGCCTGCACCTCACCATTG-3' (position 1258-1236, product size 298 bp). For VPAC2, the primers were the following: forward, 5'-GTCAACTTTGCCCTCTTCATCA-3' (position 944-966); and reverse, 5'-GCCTCTCCACCTTCTTTTCAG-3' (position 1241-1220, product size 297 bp). The PCR conditions were 40 cycles at 95°C/30 s, 60°C/30 s, and 72°C/30 s. The PCR products were separated by electrophoresis on a 2.5% agarose gel and visualized with ethidium bromide under ultraviolet fluorescence. RT was omitted in controls.

Statistical analysis. cAMP assay and electrophysiology results are presented as means ± SE and analyzed for statistically significant differences by ANOVA and Duncan's multiple-range test from the SAS Institute (Cary, NC).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cultured atrial cells contain functional PACAP receptors. PACAP-38 (0.01-100 nmol/l) dose dependently stimulates the production of cAMP in atrial appendage cultures (Fig. 1A), with an EC50 = 0.28 nmol/l (r = 0.92, P < 0.02). A similar EC50 (0.49 nmol/l) was established in a repeat experiment (r = 0.94, P < 0.02). PACAP-27 at 10-100 nmol/l has the same effects as PACAP-38 (Fig. 1B), but the effects of 100 nmol/l VIP on cAMP are 1.9 times (Fig. 1B) to 3.5 times less (repeat experiment, not shown, P < 0.05). The EC50 for VIP cannot be accurately estimated but is between 1 and 10 nmol/l.

Characterization of PACAP receptors by RT-PCR clearly shows the presence of mRNA coding for at least two isoforms of PAC1 and for VPAC1 and VPAC2 in atrial appendage cell culture (Fig. 1C, right). The expected sizes of the PCR products correspond to the position of the bands in the gel. A similar expression pattern is observed for mRNA extracts of atrial appendages (Fig. 1C, left) and ventricular tissue (Fig. 1C, middle). Tissue and culture extracts show a strong band for both beta -actin and atrial natriuretic factor. Controls without RT and with beta -actin primers are negative.

PACAP-38 activates a KATP current in atrial appendage myocytes. No spontaneous activation of the KATP channel occurs in the presence of 5 mmol/l ATP in the patch pipette. However, the extracellular application of 100 nmol/l PACAP-38 strongly activates the KATP current (Fig. 2, A and B, trace b), although there is a variable delay between drug application and increase in current. The current shows a weak inward-rectifying I-V relationship with a reversal potential near -80 mV (Fig. 2A). Diazoxide can stimulate this current further (Fig. 2, A and B, trace c). In 44 myocytes, the effect of PACAP-38 is dose dependent, with an EC50 close to 3 nmol/l and a maximal effect at 10 nmol/l (Fig. 2C). The maximal current density reached with 10 nmol/l PACAP-38 (91 ± 15 pA/pF, n = 18) represents 91 ± 15% of the current density reached in 22 myocytes with 100 µmol/l diazoxide. A better estimate of the relative effect of PACAP-38 is obtained in six experiments, where both drugs could be tested on the same cell, and where diazoxide increases the PACAP-38-induced current by 78% (P < 0.05). This indicates that PACAP-38 does not elicit maximal effects on the KATP current. With only 2 mmol/l ATP in the pipette solution, 1 nmol/l PACAP activates a KATP current of 85 ± 35 pA/pF (n = 4) compared with 26 ± 15 pA/pF (n = 9) with 5 mmol/l ATP, suggesting that low internal ATP increases the effectiveness of PACAP-38.


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  		Fig. 2.   Activation of atrial ATP-sensitive K+ (KATP) current by PACAP-38. A: original current traces for activation of atrial KATP current from rest (trace a) by 100 nmol/l PACAP-38 (PACAP, trace b) with further activation by 100 µmol/l diazoxide (Dia, trace c) by 5 mmol/l ATP in patch pipette. The voltage ramp protocol is shown above. The activation of the KATP current at -40 mV is represented by the resting current amplitude, as also shown in subsequent figures. B: time course of KATP current measured at +50 mV corresponding to the experiment shown in A. C: mean PACAP-38-induced KATP current density at +50 mV as a function of PACAP-38 concentration (*significantly different from baseline; d, significantly different from 1 nmol/l; no. of myocytes shown above bars).

Permeant analog of cAMP activates the atrial KATP current. To determine whether cAMP was involved in the activation of atrial KATP current by PACAP-38, we studied the effects of a permeant analog of cAMP, 8-CPT-cAMP. The bath application of 100 µmol/l 8-CPT-cAMP activates a KATP current (Fig. 3, A and B, trace b), which can be further activated by 100 µmol/l diazoxide (Fig. 3, A and B, trace c). The 8-CPT-cAMP (100 µM) activates a mean maximal current of 31.8 ± 6.2 pA/pF (n = 16) compared with the mean maximal current of 100.3 ± 10.3 pA/pF (n = 22) activated by 100 µmol/l diazoxide. However, the 8-CPT-cAMP-activated KATP current induces a near-maximal membrane polarization, reaching -63 ± 4 mV (n = 16) compared with -69 ± 2 mV (n = 22) for 100 µmol/l diazoxide and -68 ± 2 mV (n = 18) for 10 nmol/l PACAP-38. Considering the eight experiments where diazoxide and 8-CPT-cAMP could be applied on the same cell, the 8-CPT-cAMP-induced current is 30 ± 11% of the maximal diazoxide-induced current.


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  		Fig. 3.   Activation of atrial KATP current by 8-(4-chlorophenyl-thio)cAMP (8-CPT-cAMP). A: original current traces for activation of the atrial KATP current from rest (trace a) by 100 µmol/l 8-CPT-cAMP (cAMP, trace b) with further activation by 100 µmol/l Dia (trace c). B: time course of KATP current corresponding to the experiment shown in A. C: mean KATP current density measured at +50 mV, activated by 100 µmol/l 8-CPT-cAMP and 100 µmol/l Dia. (*significantly different from baseline; d, significantly different from 100 µmol/l 8-CPT-cAMP; no. of myocytes shown above in italics).

Effects of the PKA inhibitor H89 on the PACAP-activated atrial KATP current. The PKA inhibitor H89 was used to block the effects of cAMP because it is described as a rather selective inhibitor of PKA, with inhibitory constant (Ki) = 48 nmol/l, compared with PKC, where Ki = 31.7 µmol/l (8). Used externally at 1 µmol/l, H89 strongly inhibits the diazoxide-activated KATP current to 7.5 ± 1.9 pA/pF (n = 3) from a control amplitude of 100.3 ± 10.3 pA/pF (n = 22). At 0.5 µmol/l, H89 much more strongly reduces PACAP-38 than the diazoxide-induced current (Fig. 4, A and B). The diazoxide-induced KATP current is reduced to 30.5 ± 25.2 pA/pF (n = 6) (Fig. 4C, open bar), corresponding to 30.4% of the control current. This concentration of H89 totally inhibits the KATP current induced by 100 µmol/l 8-CPT-cAMP (Fig. 3C) and reduces the 10 nmol/l PACAP-38-induced KATP current to 5.0 ± 2.7 pA/pF (n = 5) (Fig. 4C, hatched bar), corresponding to 8 ± 4% (n = 5) of the current induced by diazoxide on the same cell. Thus, although H89 exerts a probably nonspecific partial inhibition of the KATP current, it strongly affects the 8-CPT-cAMP- and PACAP-38-induced currents. Figure 4, A and B, also shows that the PACAP plus diazoxide-activated current increases after the wash out of H89, illustrating the reversible inhibition of the atrial KATP current by H89. Furthermore, low concentrations of glyburide strongly inhibit the KATP current as expected (3).


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  		Fig. 4.   Effects of the protein kinase A (PKA) inhibitor H-89 dihydrochloride (H89) on PACAP-38-activated atrial KATP current. A: original current traces demonstrating a weak activation of KATP current from rest (trace a) by 10 nmol/l PACAP-38 in the presence of 0.5 µmol/l H89 (PACAP, trace b), with further activation by 100 µmol/l Dia (trace c). Incubation with H89 began 10 min before the beginning of the recording. Further increase of KATP current on washout of H89 is shown in trace d. B: time course of KATP current corresponding to the experiment shown in A. Note strong inhibition at the end of the experiment by 10 and 100 nmol/l glyburide (Glyb). C: mean KATP current densities measured at +50 mV, activated by 100 µmol/l 8-CPT-cAMP (solid bars), 10 nmol/l PACAP-38 (hatched bars), and 100 µmol/l Dia. Controls are shown on left and effects of 0.5 µmol/l H89 on right (*significantly different from baseline; e, significantly different from 8-CPT-cAMP; f, significantly different from control; g, significantly different from 8-CPT-cAMP/H89; no. of myocytes shown above bars).

Effect of PKC-I peptide on the PACAP-38-activated atrial KATP current. To test the involvement of PKC in PACAP-38-activation of the atrial KATP current, we used the specific PKC-I peptide (19-31) (IC50 = 0.2 µM; see Ref. 17), a synthetic peptide corresponding to the pseudosubstrate region of PKC (33). With 50 µmol/l PKC-I in the patch pipette and after 10 min of whole cell recording to allow the diffusion of the inhibitor into the cytoplasm, 10 nmol/l PACAP induces a significantly reduced KATP current of 33.2 ± 17.8 pA/pF (n = 6) (Fig. 5, A and B, trace b). This result compares with 91 ± 15 pA/pF (n = 18) in the absence of PKC-I (Fig. 5C, hatched bars, P < 0.05). The presence of PKC-I does not affect the cAMP-activated KATP current (Fig. 5C, solid bars), whereas the OAG-activated KATP current amplitude is reduced from 50.9 ± 16.5 pA/pF (n = 7) in control condition to 15.3 ± 10.7 pA/pF (n = 5) in the presence of 50 µM of PKC-I (not shown).


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  		Fig. 5.   Effects of the PKC inhibitor peptide (PKC-I) on PACAP-38- and VIP-activated atrial KATP current. A: original current traces illustrating a moderate activation of KATP current from rest (trace a) by 10 nmol/l PACAP-38 in the presence of 50 µmol/l PKC-I in the pipette solution (PACAP, trace b) and further activation by 10 nmol/l VIP (trace c). B: time course of KATP current corresponding to the experiment shown in B. C: mean KATP current density measured at +50 mV, activated by 10 nmol/l PACAP-38, 10 nmol/l VIP, or 100 µmol/l 8-CPT cAMP (cAMP). Controls are shown on left and responses in the presence of 50 µmol/l PKC-I on right (*significantly different from baseline; d, significantly different from corresponding PKC-I; number of myocytes shown above bars).

Interestingly, VIP is able to elicit a KATP current in the presence of PKC-I, as shown in the example of Fig. 5, A and B (trace c), and statistically in Fig. 5C (open columns). The responses to 10 nmol/l VIP are variable, however, because some myocytes fail to respond to VIP, whereas they respond to 10 nmol/l PACAP-38 and 10 µmol/l diazoxide (example, Fig. 6).


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  		Fig. 6.   Lack of responsiveness to VIP in a PACAP-38-responsive myocyte. A: original current traces showing an activation of atrial myocyte KATP current from rest (trace a) by 10 nmol/l PACAP-38 (PACAP, trace c) and lack of responsiveness to VIP (trace b). Current is further activated by 10 µmol/l Dia at the end of the experiment (trace d) (note superposition of traces a and b). B: time course of KATP current corresponding to the experiment shown in A.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study we show the following: 1) PACAP-related peptides potently stimulate cAMP production in primary cultured atrial myocytes, with an order of potency PACAP-38 = PACAP-27 > VIP; 2) atrial myocytes in culture have at least two splice variants of PAC1 and also contain VPAC1 and VPAC2; 3) PACAP- 38 activates an atrial myocyte KATP current in a dose-dependent manner, PACAP-38 being significantly more potent than VIP; and 4) pharmacological blockade of the PKA or PKC pathway attenuates the KATP current response to PACAP-38.

Multiple types of PACAP receptors in atrial myocytes. Functional and molecular evidence supports the notion that atrial myocytes contain multiple types of PACAP receptors. First, cAMP responses are much more pronounced for PACAP-38 than VIP (Fig. 1B), as also shown by others on cultured cardiocytes (4), suggesting the presence of PAC1 (= PVR1) in combination with VPAC1 (= PVR2) and/or VPAC2 (= PVR3) (compare with Table 1 in Ref. 29). Second, PACAP-38 much more potently and consistently stimulates the atrial KATP current than VIP (Fig. 5 and 6), either because it more potently stimulates PKA or because it stimulates both PKA and PKC. In either case, one would infer from Rawlings and Hezareh (29) that PACAP-38 stimulates PAC1 in combination with VPAC1 and/or VPAC2. Third, RT-PCR indicates the existence of three main types of PACAP-38 receptors in atrial myocytes, PAC1, VPAC1, and VPAC2, with at least two splice forms for PAC1.

The PCR product of 301 bp corresponds to the short form of PAC1 (34), whereas the 385-bp product contains an 84-bp insert represented by Hip or Hop-1. It cannot be distinguished here from a 382-bp insert corresponding to Hop-2. It is known that Hip strongly diminishes the cAMP responses (34), and as Fig. 1A shows that cAMP is strongly activated by PACAP-38 (EC50 0.28 nmol/l), this suggests that the insert most likely is not Hip. Further experiments, such as Southern blot with specific probes for each form, would be needed to confirm this. The PAC1-Hip-Hop forms are not detectable (Fig. 1C). These results on atrial myocytes differ from those of Braas et al. (5), who found that PAC1 receptors in cardiac ganglia mostly do not contain an insert in the third cytoplasmic loop as shown here.

The RT-PCR results of Fig. 1C also show that the distribution of different PACAP receptors is quite similar in atrial tissue, ventricular tissue, and primary atrial cell culture. Although Northern blots fail to demonstrate cardiac VPAC receptors (18, 19), the presence of the three main PACAP receptor types in the whole heart has previously been shown by RNAse protection assay (36). The great majority of cells in the atrial cell cultures are myocytes (20), but it is not possible to conclude that all myocytes contain all isoforms shown. Indeed, the heterogeneity in responsiveness of the KATP current to VIP (Fig. 5 and 6) would suggest that some myocytes do not contain VPAC1 and VPAC2. Single cell RT-PCR, as shown by Rawlings and Hezareh (29) on pituitary cells, may resolve this issue in future studies.

PACAP-38 activates the atrial KATP current via PKA and PKC. Consistent with the presence of multiple PACAP receptor types, PACAP-38 potently and dose dependently increases the KATP current in atrial myocytes. Indeed, the results strongly suggest the involvement of both the PKA and PKC signaling pathways.

The involvement of PKA is indicated by the inhibition by H89 of the KATP current induced by 10 nmol/l PACAP-38 (Fig. 4). This inhibition is total for stimulation by 8-CPT-cAMP, as expected. However, H89 also partially inhibits the stimulation of the KATP current by diazoxide, either because of a basal tone of PKA, or because diazoxide may boost cAMP via inhibition of phosphodiesterase, or because of unknown nonspecific effects. Because the inhibition of stimulation by PACAP-38 is far greater than the inhibition of stimulation by diazoxide (Fig. 4C), it is still possible to conclude that H89 exerts a specific inhibition of the PACAP-38-stimulated PKA pathway. This result is supported by the strong stimulation by PACAP-38 of cAMP (Fig. 1A) and by the significant stimulation of the atrial KATP current by 8-CPT-cAMP (Fig. 3C).

The involvement of the PKC pathway is indicated by the strong inhibition, by cell dialysis with the PKC-I peptide, of the KATP current induced by 10 nmol/l PACAP-38 (Fig. 5) or by OAG (see RESULTS). This result is supported by another study showing that stimulation of PKC activates ventricular KATP channels (25). Interestingly, the KATP current induced by VIP is not affected by PKC-I, suggesting that a putative stimulation of PKC by VPAC2 (2, 18) is unlikely in atrial myocytes. It is also noted that stimulation by 8-CPT-cAMP is not affected by PKC-I, as expected.

The relative contribution of the PKA and PKC pathways to the total response of the KATP current to PACAP-38 cannot easily be inferred. The large inhibitory effect of H89 in Fig. 4 is partly nonspecific (see above) and cannot be attributed to inhibition of PKA alone. From Fig. 4 and 5, we estimate that one-third of the inhibitory effect is attributable to PKA and two-thirds are due to PKC. Such double signaling by PACAP-38 through PKA and PKC is not unprecedented, because it has also been observed for PACAP modulation of the L-type calcium channel in vascular smooth muscle (9). It is suggested that the stimulation by PKA and PKC would be even more pronounced at 37°C and in intact cells, because the enzymatic activity would be greater at 37°C and second messengers would not be diluted by the patch pipette.

Possible roles of PACAP-related peptides in cardiac function. Recent studies (5, 15) show that PACAP-related peptides are contained in nervous structures surrounding the heart, in particular in cardiac ganglia and nerve fibers, and may modulate cardiac output via stimulation of the parasympathetic system. The results of our study show that atrial myocytes could also be a target of PACAP-containing fibers surrounding the cardiac myocytes (5). The activation of the atrial KATP current by PACAP-38 released from nerve fibers would be expected to cause a shortening of the action potential, a reduction of calcium influx, and a reduction of atrial contraction. By analogy, the demonstration of PACAP receptors in ventricular tissue (Fig. 1C) suggests that PACAP-38 may reduce the ventricular contraction, thus overall causing a reduction in cardiac output. These primary cardiac effects of PACAP-related peptides are probably overshadowed in vivo by their vasodilation actions on the microcirculation and by the reflex activation of the cardiac sympathetic outflow (9, 37).

In addition to modulating cardiac pumping, PACAP-related peptides may also modulate cardiac secretion of ANP. PACAP-38 stimulates cAMP accumulation and ANP secretion from cultured cardiomyocytes (4). Because the cardiomyocytes in that study were derived mostly from ventricular tissue, and ventricular myocytes release ANP in response to cAMP (30), some of the action may be attributed to activation of the cAMP pathway. A significant response is probably also mediated by activation of PKC (4). In contrast, in atrial myocytes, cAMP mostly inhibits or has no effect on ANP secretion (32), and activation of KATP channels by diazoxide inhibits the stimulated ANP secretion in hearts (38) and in atrial myocyte culture (20). Future experiments should show whether PACAP-38 inhibits or stimulates ANP secretion from atrial tissue or cells.

In conclusion, we show that PACAP-38 potently activates a KATP current in atrial myocytes. Multiple PACAP receptors and both PKA and PKC pathways are implicated in this response. Together with other studies, the results imply that PACAP-38 would reduce cardiac output, but the initiating stimulus for PACAP release from cardiac nerve fibers is still unknown.


    ACKNOWLEDGEMENTS

This study was supported by Swiss National Science Foundation Grants 31-49798.96 and 31-059551.99, the de Reuter Foundation, the Horten Foundation, the Novartis Stiftung für Biologisch-Medizinische Forschung, the Roche Research Foundation, and the Société Académique de Genève.


    FOOTNOTES

Address for reprint requests and other correspondence: A. J. Baertschi, Dept. of Physiology, Centre Médical Universitaire, 1 rue Michel Servet, CH-1211 Geneva 4, Switzerland (E-mail: alex.baertschi{at}medecine.unige.ch).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 5 July 2000; accepted in final form 20 October 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Heart Circ Physiol 280(3):H1058-H1065
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