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Originally published In Press as doi:10.1074/jbc.M403819200 on June 16, 2004

J. Biol. Chem., Vol. 279, Issue 36, 37291-37297, September 3, 2004
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Modulation of L-type Calcium Channels in Drosophila via a Pituitary Adenylyl Cyclase-activating Polypeptide (PACAP)-mediated Pathway*

Anindya Bhattacharya{ddagger}, Sukhwinder S. Lakhman§, and Satpal Singh

From the Department of Pharmacology and Toxicology, State University of New York at Buffalo, Buffalo, New York 14214-3000

Received for publication, April 6, 2004 , and in revised form, June 15, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Modulation of calcium channels plays an important role in many cellular processes. Previous studies have shown that the L-type Ca2+ channels in Drosophila larval muscles are modulated via a cAMP-protein kinase A (PKA)-mediated pathway. This raises questions on the identity of the steps prior to cAMP, particularly the endogenous signal that may initiate this modulatory cascade. We now present data suggesting the possible role of a neuropeptide, pituitary adenylyl cyclase-activating polypeptide (PACAP), in this modulation. Mutations in the amnesiac (amn) gene, which encodes a polypeptide homologous to human PACAP-38, reduced the L-type current in larval muscles. Conditional expression of a wild-type copy of the amn gene rescued the current from this reduction. Bath application of human PACAP-38 also rescued the current. PACAP-38 did not rescue the mutant current in the presence of PACAP-6–38, an antagonist at type-I PACAP receptor. 2',5'-dideoxyadenosine, an inhibitor of adenylyl cyclase, prevented PACAP-38 from rescuing the amn current. In addition, 2',5'-dideoxyadenosine reduced the wild-type current to the level seen in amn, whereas it failed to further reduce the current observed in amn muscles. H-89, an inhibitor of PKA, suppressed the effect of PACAP-38 on the current. The above data suggest that PACAP, the type-I PACAP receptors, and adenylyl cyclase play a role in the modulation of L-type Ca2+ channels via cAMP-PKA pathway. The data also provide support for functional homology between human PACAP-38 and the amn gene product in Drosophila.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Ca2+ channels play an important role in many cellular functions, including excitation-contraction and excitation-secretion coupling, sensory and motor function, gating of other ion channels, and long term potentiation (1–4). The channels are regulated by many factors, such as aging, development, nutritional status, depolarization, and the action of neurotransmitters, neurotoxins, and drugs (1, 5, 6). Regulation of L-type channels is altered in a number of cardiovascular, neurological, and other human diseases (7–10). In many instances, regulation of channels is mediated via signal transduction pathways (1, 11–15). However, detailed mechanisms underlying modulation or regulation of ion channels are not well understood in many cases, and exploring these mechanisms is important from both basic and clinical points of view.

Molecular pathways involved in channel modulation can be analyzed with the help of mutations that disrupt specific pathway components. With many such mutations available, Drosophila provides an excellent model system for these studies. The role of cAMP-mediated pathway in modulating L-type Ca2+ channels in Drosophila has been suggested by the use of mutations and pharmacological agents that affect steps in this pathway (16). These observations raise a question as to what the likely steps are prior to cAMP in the pathway, particularly the likely endogenous signals that might lead to modulation of L-type Ca2+ channels in Drosophila.

To address this question, we considered the possible involvement of the peptide neurotransmitter pituitary adenylyl cyclase-activating polypeptide (PACAP)1 in cAMP-mediated modulation of L-type Ca2+ channels in Drosophila. PACAP is a member of a peptide superfamily that includes vasoactive intestinal peptide (VIP), secretin, and glucagon (17). It consists of either 38 amino acids (PACAP-38) or 27 amino acids (PACAP-27). There are three PACAP receptors, type-I (PAC1), type-II (VPAC1), and type-III (VPAC2). The PAC1 receptor has the highest affinity for PACAP-38 and is about 1000-fold more potent for PACAP-38 than for VIP (18, 19). There are several reasons for considering the possibility of PACAP involvement in modulating L-type Ca2+ channels in Drosophila. Binding of PACAP-38 to PAC1 receptors leads to the activation of adenylyl cyclase (AC) (19). Neuromuscular synapses in Drosophila larvae show immunoreactivity to PACAP (20). The amnesiac (amn) gene product in Drosophila, a polypeptide closely related to human PACAP-38, plays an important role in modulating the cAMP pathway in Drosophila (21–24). These observations, along with those on a role for AC, cAMP, and PKA in modulating L-type Ca2+ channels (16), raise the possibility that PACAP may be involved in this modulation. The current study examines this possibility with the help of mutations and drugs that act in the PACAP-mediated pathway.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Drosophila Strains and Genetics—The amnx8, amn28A, yctrl, and hs-amn+-7 strains were obtained from the laboratory of U. Heberlein at the University of California, San Francisco (23). The amnx8 mutant males were crossed to FM3/+ virgin females. FM3/amnx8 females from the progeny of this cross were mated to males carrying the hs-amn+-7 transgene. Because FM3 males die as early first instar larvae, all third instar male larvae would be of the amnx8; hs-amn+-7/+ genotype. These larvae were used for examining the effect of heat shock-induced expression of the wild-type amn gene on the L-type Ca2+ channel currents in the amnx8 background.

Heat Shock—The heat shock protocol used was as described by Moore et al. (23). The amnx8; hs-amn+-7/+ larvae obtained in the cross described above were subjected to heat shock at 37 °C for 1 h, twice daily, starting early in their development. Non-heat-shocked male siblings were used as control for this experiment.

Drugs and Solutions—Human PACAP-38 (HSDGIFTDSYSRYRKQMAVKKYLAAVLGKRYKQRVKNK) and PACAP-6–38 (FTDSYSRYRKQMAVKKYLAAVLGKRYKQRVKNK) were purchased from Peninsula Laboratories, San Carlos, CA. N-[2-((p-Bromocinnamyl) amino)ethyl]-5-isoquinolinesulfonamide (H-89) and 2',5'-dideoxyadenosine (ddA) were obtained from Calbiochem. PN-200–110 was a generous gift from Dr. D. J. Triggle (School of Pharmacy and Pharmaceutical Sciences, SUNY at Buffalo, NY). All final concentrations of drugs were made fresh every day. Drugs were applied at the final concentration indicated for each experiment to the bath solution. The dissection solution contained (in mM): NaCl (77.5), KCl (5), MgCl2 (4), NaHCO3 (2.5), trehalose (5), sucrose (115), and HEPES (5). Recording saline contained (in mM): NaCl (77.5), KCl (5), MgCl2 (4), NaHCO3 (2.5), trehalose (5), sucrose (115), HEPES (5), tetraethylammonium (20), 4-AP (1), quinidine (0.1), and BaCl2 (10) (25, 26). The pH was adjusted to 7.1.

Preparation—Ca2+ channel currents of the larval body-wall muscles were recorded using two-microelectrode voltage-clamp techniques (26–29). Wandering third instar larvae were used throughout the study (30). Flies were grown on a standard cornmeal medium at 21 °C (31). Larvae were pinned dorsal side up on a dissection dish. The cuticle was cut along the dorsal midline and pinned back. All internal organs were removed (28). Recordings were made from muscle fiber 12 (32) and were completed within 30 min from the start of dissection (16, 29).

Electrophysiology—Electrodes were pulled from thin walled, 1.0-mm (outside diameter) borosilicate glass capillaries (World Precision Instruments, Sarasota, FL) using a David Kopf Instruments puller, model 750. Voltage electrode was filled with 2.5 M KCl and the current electrode with a 3:1 mixture of 2.5 M KCl:2 M potassium citrate (28, 30). Resistance of electrodes was in the range of 10–20 Mohms. Currents were elicited by 500-ms voltage steps from a holding potential of –40 mV to potentials between –60 and +40 mV in 10-mV increments. Interpulse duration was 10 s. Potassium currents were blocked by tetraethylammonium, quinidine, and 4-aminopyridine (28–30, 33). Control recordings were performed independently for each set of experiments, and percentage effects on currents under different recording conditions were calculated with respect to controls performed specifically for those recording conditions. Recording temperature was maintained at 21 °C with a Peltier junction and measured for each larva with a thermocouple probe after the experiment (26).

Equipment and Software—A Macintosh IISi computer provided the voltage-clamp command pulses through a 12-bit digital to analog converter using a MacADIOS II/16 board from GW Instruments (Somerville, MA). A TEC 01C/02/03 amplifier (NPI Electronic GmbH, Haeldenstrasse, Germany) was used for recordings. Data were acquired after 16-bit analog to digital conversion. Further analysis was performed by a program written in Think-C (Symantec Corporation, Cupertino, CA).

Data Handling—Currents were digitally sampled every 500 µs except during examination of capacitative transients (28), which were sampled every 100 µs. Currents were digitally corrected for linear leakage with respect to currents from –60 mV. Current densities, expressed as nanoamperes/nanofarad (nA/nF) were calculated by dividing the absolute currents by cell capacitance to avoid differences due to fiber size. Mutations used in the study did not appear to affect membrane capacitance. All traces depict average data from a number of fibers as mentioned. Current amplitude for each pulse was measured at the peak value to generate a current-voltage relationship. Currents recorded under different conditions were compared for their peak values at –10 mV. All data are expressed as mean values ± S.E. of means.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Reduction of the L-type Ca2+ Channel Current in amn Mutants—The amn gene in Drosophila encodes a polypeptide closely related to human PACAP-38 (21). The L-type currents recorded from the larval muscles of the wild-type (CS) and the amn28A mutant are shown in Fig. 1A. Currents were elicited by voltage steps from a holding potential of –40 mV to potentials between –60 and +40 mV in increments of 10 mV (29). Fig. 1B shows the relationship between membrane potential and peak current (current-voltage relationship). Maximum peak current was obtained for pulses to –10 mV. Percentage changes in currents were calculated for voltage pulses to this potential. The peak current density at –10 mV was 47.1 ± 1.5 nA/nF for the wild-type muscles. The amn28A mutant showed a reduction of about 21% in the current to 37.4 ± 3.4 nA/nF. To check for any effects arising from differences in the genetic background between the CS and the amn28A strain, the amn28A current was compared with that from yctrl, the strain used to generate the amn28A mutation by a P-element insertion (23). The yctrl larvae showed similar currents as the CS larvae, giving a peak current of 45.5 ± 1.8 nA/nF at –10 mV (data not shown), thus supporting the idea that amn28A reduces the L-type current in Drosophila larvae.



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  		FIG. 1.
Effect of the amn mutations on the L-type Ca2+ channel current in Drosophila larval muscles. Currents shown here and in all subsequent figures were elicited with 500-ms voltage pulses. A, average current traces from the wild-type (CS), amnx8, amn28A, and amn1 larvae as labeled. B, current voltage relationships for these strains. The amn mutations reduced the current. In this and the other figures, data are shown as mean ± S.E. Data shown were collected from a number of fibers (F) taken from a number of larvae (L) as specified. For CS, L = 11, F = 22; for amnx8, L = 9, F = 18; for amn28A, L = 4, F = 7; for amn1, L = 5, F = 10.

 
Muscles from male larvae of a null mutant allele, amnx8, generated by imprecise excision of the P-element in amn28A (23), showed a current of 38.7 ± 0.8 nA/nF (Fig. 1). Females in the amnx8 strain used in our experiments carry an attached X chromosome (34) homozygous for the wild-type amn gene. It has been shown previously that the L-type Ca2+ channel currents recorded from larval muscles are similar between males and females (26). Thus, wild-type female siblings from the amnx8 strain provide a control for the mutant males. The female larvae showed similar currents as the CS larvae, giving a current of 47.3 ± 2.3 nA/nF at –10 mV (data not shown).

The above experiments still leave open a small likelihood that the current reduction may be caused by an additional mutation, different from amn28A, present in the mutant strain. A good way to address this possibility is to record currents from another, independently identified, mutant allele in the amn gene. The probability of having the same additional mutation present in different, independently identified, amn strains is extremely low. Recordings from amn1, the original amn allele identified for disruption in learning and memory (35), showed a current of 37.2 ± 0.7 nA/nF (Fig. 1). Thus, disruption of the amn gene seems to result in the reduction of L-type current in Drosophila larvae.

Rescue from the Mutational Effect by the Wild-type amn Gene—The above data indicate that the amn mutations are responsible for reduction in the L-type current. If this is so, it may be possible to rescue the current by expressing the wild-type amn transgene in a fly strain mutant for amn. This possibility was examined with the help of the hs-amn+-7 strain, a strain containing the wild-type amn gene linked to the heat shock protein-70 promoter (23).

The wild-type amn gene in the hs-amn+-7 strain is present on an autosome. Larvae carrying the null amnx8 mutation and the wild-type amn gene (hs-amn+-7) (23) were obtained by the cross shown in Fig. 2A. The wild-type amn gene was induced by giving a heat shock to larvae as described under "Experimental Procedures." Fig. 2, B and C, represents the currents recorded from these larvae without and with heat shock. The peak current density from non-heat-shocked larvae was 34.5 ± 1.1 nA/nF. This value is comparable with currents recorded from amnx8 larvae as would be expected in the absence of expression of the wild-type amn gene. With heat shock-induced expression, the current increased to 47.0 ± 2.1 nA/nF, a value comparable with the control peak current. These data strongly support the idea that the amn gene product is involved in modulation of L-type Ca2+ channels in Drosophila.



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  		FIG. 2.
Rescue from the mutational effect by the wild-type amn gene. A, the genetic cross to obtain males carrying a wild-type amn gene (hs-amn+-7) in the background of the amnx8 mutation. B, current traces obtained without and with heat shock. C, the corresponding current-voltage relations. Induction of the wild-type amn gene by heat shock rescued the current to normal levels. In the absence of heat shock, the current remained at the levels seen in the amn mutants. Data shown were collected from a number of fibers (F) taken from a number of larvae (L) as specified. Without heat shock, L = 7, F = 20; with heat shock, L = 5, F = 8.

 
Rescue from the Mutational Effect by PACAP-38 —Because the amn gene encodes a protein that is homologous to human PACAP-38, the above data predict that application of PACAP-38 to the muscle preparation may rescue the current from the reduction caused by the amn mutation. Effect of bath-applied human PACAP-38 was examined on muscles in amnx8.

Fig. 3 shows the effect of various concentrations of PACAP-38 on the L-type current in amnx8. Current density recorded from amnx8 in the absence of exogenous PACAP-38 was 37.5 ± 1.2 nA/nF. Addition of PACAP-38 in the bath solution, ~10 min before the recordings, increased the current in a concentration-dependent manner. Concentration of PACAP-38 as low as 10 nM was able to increase the current significantly. At 100 nM PACAP-38, the current increased to 45.3 ± 1.2 nA/nF, bringing it close to the wild-type current. Thus, addition of PACAP-38 rescued the amnx8 current defect, lending support to the argument that disruption of PACAP activity in the amn mutants may be responsible for the reduction of L-type current in larval muscles.



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  		FIG. 3.
Rescue from the mutational effect by PACAP-38. The amn gene codes for a protein that is homologous to PACAP-38. Application of human PACAP-38 rescued the L-type current in the amnx8 mutant larvae in a concentration-dependent manner. A concentration of 100 nM PACAP-38, which brought the current in the amnx8 mutant close to the wild-type level, was used in subsequent experiments involving PACAP-38. Current-voltage relationships for various concentrations of PACAP-38 are shown (A). B, the amplitude of the peak current obtained at –10 mV in the presence of different concentrations of PACAP-38. Data shown were collected from a number of fibers (F) taken from a number of larvae (L) as specified. CS (wild-type), L = 11, F = 22; amnx8 without PACAP-38, L = 7, F = 14;1nM PACAP-38, L = 3, F = 11; 10 nM PACAP-38, L = 4, F = 7; 100 nM PACAP-38, L = 3, F = 7; 1000 nM PACAP-38, L = 5, F = 11.

 
The effect of PACAP-38 seen above may also be explained by PACAP-38 inducing a new, non-L-type Ca2+ current and thus increasing the total inward current seen in recordings. Such a possibility can be examined by using a pharmacological agent, such as PN200–110, that specifically blocks the L-type Ca2+ current (29, 36). Fig. 4 shows the sensitivity of the amnx8 current to 10 µM PN-200–110 in the absence or presence of PACAP-38. The current observed in the presence of PACAP-38, 45.3 ± 1.2 nA/nF (Fig. 3), was reduced by about 83% to 7.8 ± 1.8 nA/nF by PN200–110. Current in the absence of PACAP-38 was reduced from 38.9 ± 1.2 nA/nF to 4.9 ± 1.0 nA/nF, showing a reduction of about 87%. The total current observed in the presence of PACAP-38 thus showed a similar sensitivity to blockade by PN200–110 as the current in the absence of PACAP-38. Therefore, the neuropeptide is likely to have rescued the residual L-type current in amnx8 to the normal levels rather than having induced a novel non-L-type current.



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  		FIG. 4.
Pharmacological sensitivity of the current observed in the presence of PACAP-38. The increased current seen in the presence of PACAP-38 shows the same sensitivity to blockade by PN200–110 as the current seen in the absence of PACAP-38. Current traces and current-voltage relationships are shown, respectively, in panels A and B. Data shown were collected from a number of fibers (F) taken from a number of larvae (L) as specified. amnx8, L = 4, F = 6; amnx8 + PN200–110, L = 5, F = 20; amnx8 + PN200–110 + PACAP-38, L = 7, F = 20.

 
Role of the PAC1 Receptor in Mediating the PACAP Effect— PACAP-38 acts selectively at the PAC1 receptor (19). PACAP-6–38, a PACAP-38 peptide missing the first 5 amino acids, acts as an antagonist at PAC1. If PAC1 is involved in rescue of the current in the amn mutant by PACAP-38, then PACAP-6–38 is expected to interfere with this rescue. Fig. 5 shows the effect of PACAP-6–38 on the ability of PACAP-38 to rescue the current in amnx8. In the absence of PACAP-6–38, 100 nM PACAP-38 increased the amnx8 current from 37.1 ± 0.9 nA/nF to 44.2 ± 0.6 nA/nF. This increase in the amnx8 current failed to occur with the addition of 1 µM PACAP-6–38 to the recording solution about 5 min before adding 100 nM PACAP-38. In this case, the current measured in the presence of 100 nM PACAP-38 was 35.3 ± 1.3 nA/nF. Thus, PACAP-38 seems to mediate its action via the PAC1 receptor.



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  		FIG. 5.
Role of the PAC1 receptors in mediating the PACAP effect. PACAP-6–38, which antagonizes the interaction of PACAP-38 with the PAC1 receptors, compromises the ability of PACAP-38 to rescue the current from the effect of the amnx8 mutation. A, currents recorded from amnx8, control larvae, amnx8 with PACAP-38, and amnx8 with PACAP-38 as well as PACAP-6–38, as indicated. The control recordings were made from female larvae from the same strain as amnx8 but carrying attached X chromosomes with wild-type copies of the amn gene. B, current-voltage relationships. Data shown were collected from a number of fibers (F) taken from a number of larvae (L) as specified. amnx8, L = 7, F = 20; amnx8 + PACAP-38, L = 9, F = 22, amnx8 + PACAP-38 + PACAP-6–38, L = 9, F = 22.

 
Role of Adenylyl Cyclase in Mediating the Effect of PACAP— Action of PACAP-38 at PAC1 leads to activation of AC (19). This raises the possibility that the action of PACAP-38 on L-type Ca2+ channels may be mediated by AC and the likely resulting increase in the cAMP concentration. If AC is involved in the modulatory pathway, then inhibition of this enzyme is expected to mimic the amn effect by reducing the L-type current in the wild-type muscles. Fig. 6A shows this to be the case. 50 µM ddA, an AC inhibitor, reduced the current in the wild-type from 47.1 ± 1.5 nA/nF to 33.5 ± 2.8 nA/nF.



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  		FIG. 6.
Role of adenylyl cyclase in mediating the effect of PACAP. The effect of the amn mutations was mimicked by 2',5'-dideoxyadenosine (ddA), which inhibits adenylyl cyclase. A, the effect of 50 µM ddA on the L-type current in the wild-type (CS). B, currents recorded from the amn1 mutant were not reduced much further by ddA. C, with the inhibition of adenylyl cyclase by ddA, PACAP-38 did not rescue the current observed in amn1. The dotted line in the current-voltage curves shows the amn1 current from panel B for comparison. Data shown were collected from a number of fibers (F) taken from a number of larvae (L) as specified. CS, L = 11, F = 22; CS + ddA, L = 5, F = 14; amn1, L = 5, F = 10; amn1 + ddA, L = 4, F = 11; amn1 + PACAP-38, L = 4, F = 12; amn1 + ddA + PACAP-38, L = 5, F = 12.

 
If the level of activation of AC is already low in the amn mutants due to disruption of PACAP, then application of ddA may not lead to much further reduction in the amn current. This is shown in Fig. 6B, with the amn1 muscles showing a current of 37.2 ± 0.7 nA/nF in the absence of ddA and 34.1 ± 1.0 nA/nF in the presence of ddA.

In the light of the observations above, which indicate that the effect of PACAP-38 is likely to be mediated via AC, inhibition of AC by ddA is expected to suppress the ability of PACAP-38 to rescue the current in amn. Recordings from amn muscles in the presence of ddA and PACAP-38 are shown in Fig. 6C. With ddA present in the recording solution, the current without and with PACAP-38 was, respectively, 37.2 ± 0.7 nA/nF and 37.2 ± 1.7 nA/nF. These observations provide strong support for the argument that AC is involved in mediating the effect of PACAP-38 on L-type Ca2+ channels in Drosophila.

Role of Protein Kinase A in Mediating the Effect of PACAP— Previous studies in our laboratory have shown that cAMP is likely to modulate L-type Ca2+ channels in Drosophila via PKA (16). In the context of these studies, the experiments discussed above suggest that PACAP-38-mediated activation of AC may modulate L-type Ca2+ channels via increased cAMP levels and the resultant increase in PKA activity. Possible involvement of PKA in mediating the action of PACAP-38 was examined with the help of H-89, an inhibitor of PKA. In the absence of H-89, 100 nM PACAP-38 increased the amnx8 current from 34.3 ± 2.2 nA/nF to 44.4 ± 1.7 nA/nF. However, with 10 µM H-89 in the recording solution, the current in amnx8 was 32.1 ± 1.5 in the presence of 100 nM PACAP-38 (Fig. 7). Thus, PACAP-38 could not rescue the amnx8 current in the presence of H-89. This suggests that PKA is likely to be involved in mediating the modulatory effect of PACAP-38 on Ca2+ channels. However, it may be noted that in addition to PKA, H-89 inhibits mitogen- and stress-activated protein kinase 1 (MSK1), p70 ribosomal protein S6 kinase (S6K1), and rho-dependent protein kinase-II (ROCK-II) with comparable potency (37).



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  		FIG. 7.
Role of protein kinase A in mediating the effect of PACAP. Current observed in the amnx8 mutant could not be rescued by PACAP-38 in the presence of H-89, an inhibitor of protein kinase A. Voltage-clamp traces and current-voltage relationships are shown in panels A and B, respectively. Data shown were collected from a number of fibers (F) taken from a number of larvae (L) as specified. amnx8, L = 5, F = 12; amnx8 + PACAP-38, L = 4, F = 11; amnx8 + PACAP-38 + H-89, L = 6, F = 13.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The L-type Ca2+ channels in Drosophila larval body-wall muscles are modulated by the cAMP-mediated pathway (16). This raises questions about the likely initial physiological signals that may play a role in starting the modulatory cascade in vivo. The present study suggests that the initial steps in this modulatory pathway may involve the action of PACAP on the PAC1 receptors. Thus, the following components are likely to play a role in modulating L-type Ca2+ channels in Drosophila: 1) a polypeptide encoded by the amn gene and homologous to human PACAP-38, 2) type-1 PACAP receptor PAC1, 3) AC, 4) cAMP, and 5) PKA. The involvement of these steps is shown by reduction of the L-type channel current by amn mutations; by rescue of this current reduction by wild-type amn transgene under the control of a heat shock promoter, with rescue occurring only if larvae are subjected to heat shock; by rescue of the current by PACAP-38; by inability of PACAP-38 to rescue the current in the presence of PACAP-6–38; by inhibition of the wild-type current by an AC inhibitor, with the inhibitor not showing any effect in the amn mutants; by inability of PACAP-38 to increase the current in the presence of an AC inhibitor; and by inability of PACAP-38 to rescue the current if PKA is inhibited by H-89. Thus, the observations presented here provide in vivo physiological significance to the cAMP-mediated modulation of the L-type Ca2+ channels in Drosophila larval muscles (16).

PACAP mediates a number of physiological functions (19, 38), such as somatodendritic vasopressin release (39), catecholamine secretion (40, 41), growth hormone secretion (42), secretin release (43), gene expression (44, 45), neuronal protection from apoptosis (46), effects on muscle excitability (47, 48), circadian rhythms (44, 49), and behavioral and psychomotor phenomena (50, 51). Many of these processes are mediated via increase in intracellular Ca2+ concentration. This PACAP-mediated increase in intracellular Ca2+ concentration occurs by influx of Ca2+ through voltage-dependent Ca2+ channels, including L-type Ca2+ channels and/or by release of Ca2+ from intracellular stores via a phospholipase C-{beta} (PLC)-mediated pathway (42, 49, 52–54). The data presented here delineate steps in the pathway involved in modulation of L-type Ca2+ channels by PACAP and subsequent increase in Ca2+ influx through these channels. Thus, the studies presented here reveal steps in the pathway linking actions of the PACAP neurotransmitter with the physiological functions influenced by it.

Modulation of L-type Ca2+ channels in the larval body-wall muscles has been previously shown to be mediated by two signal transduction pathways, one involving cAMP and PKA and the other involving PLC, diacylglycerol, and protein kinase C. A model of the pathways involved in modulating L-type Ca2+ channels in larval muscles is presented in Fig. 8. Modulation of L-type Ca2+ channels via cAMP- and PLC-mediated pathways raises some interesting questions, e.g. whether there is any cross-talk between the two modulatory pathways or whether the two pathways even converge at some point subsequent to the involvement of PKA and protein kinase C. Depending on the particular splice variant subtype of the PAC1 receptor involved, PACAP-38 can induce the activation of both AC and PLC (42, 52). It will be interesting to see whether PACAP modulates L-type channels in Drosophila not only via a cAMP-mediated pathway but also through the pathway involving PLC. Thus, involvement of PACAP in modulating these channels opens up the possibility that the two pathways may converge not only downstream of the involvement of cAMP and diacylglycerol but also upstream of these steps.



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  		FIG. 8.
A schematic representation of the pathways involved in modulation of the L-type Ca2+ channels in Drosophila, showing the likely components involved in modulation. Components examined in the present study, as well as those studied in two previous reports from this laboratory (16, 26), are shown boxed. Underlined text refers to the pharmacological effects studied on these components. The text in italics refers to the mutations used in the studies.

 
The data presented here raise several questions. For example, what regulates amn gene expression as well as release of amn-encoded polypeptide at neuromuscular synapses? How does that in turn regulate muscle contractility? What is the in vivo relevance of PACAP-mediated modulation of L-type calcium channels and the expected resultant regulation of muscle contractility? Further experiments are needed to address most of these questions. There is little information available on the initial steps of the pathway to offer a sound hypothesis at this stage. Some observations that have been reported previously are relevant to these questions. For example, PACAP-38-like immunoreactivity has been demonstrated at the larval neuromuscular junction (20). It is also interesting that some of the mutations affecting the cAMP cascade have been shown to affect neuromuscular plasticity (55). The role of the amn gene product in regulating the cAMP cascade is of particular interest. The cAMP cascade has been shown to play an important role in regulating the contraction of insect muscles (56, 57). Regenerative membrane potentials in the muscles of arthropods arise entirely through Ca2+ channels (1, 58). The data presented here provide a likely pathway by which cAMP can play a role in insect muscle contraction. Further experiments are needed to delineate the functional significance of modulation of calcium channels and of contractility by a PACAP-mediated pathway.

Analysis of intracellular pathways in model organisms such as Drosophila greatly helps in understanding the pathways in other organisms, including humans. There is a considerable similarity between Drosophila and other organisms. For example, about 61% of the genes related to human diseases appear to have orthologs in Drosophila (59). On the other hand, analysis of the Drosophila and the human genomes provides several instances of clear physiological differences between the two organisms (59). Because the PACAP-mediated pathways are involved in several important physiological phenomena such as neuronal plasticity, learning and memory, and ethanol intoxication (21–23,60), it is important to compare the extent of similarity in action at the physiological level. Data provided in the present study provide support for functional homology between the human PACAP-38 and the amnesiac gene product of Drosophila, because the human polypeptide could rescue the physiological effect caused by the absence of the peptide in Drosophila.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant GM-50779 and National Science Foundation Grant MCB-0094477. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Present address: Roche Pharmaceuticals, 3431 Hillview Ave., R2-101, Palo Alto, CA 94304. Back

§ Present address: Dept. of Pharmaceutical Sciences, 527 Cooke Hall, State University of New York at Buffalo, NY 14260-1200. Back

To whom correspondence should be addressed: Dept. of Pharmacology and Toxicology, 102 Farber Hall, State University of New York at Buffalo, Buffalo, NY 14214-3000. Tel.: 716-829-2453; Fax: 716-829-2801; E-mail: singhs{at}buffalo.edu.

1 The abbreviations used are: PACAP, pituitary adenylyl cyclase-activating polypeptide; AC, adenylyl cyclase; ddA, 2',5'-dideoxyadenosine; H-89, N-[2-((p-Bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide; PAC1, type-1 PACAP receptor; PKA, protein kinase A; amn, amnesiac; nA/nF, nanoamperes/nanofarad; CS, larval muscles of the wild-type; PLC, phospholipase C-{beta}. Back



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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