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Cardiovascular Research 2003 57(1):217-224; doi:10.1016/S0008-6363(02)00648-X
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Copyright © 2003, European Society of Cardiology

Involvement of cyclic AMP-mediated pathway in neural release of noradrenaline in canine isolated mesenteric artery and vein

Violeta N. Mutafova-Yambolieva*, Lisa Smyth and Janette Bobalova

Department of Physiology and Cell Biology, Anderson Medical Building/MS 352, University of Nevada School of Medicine, Reno, NV 89557-0046, USA

* Corresponding author. Tel.: +1-775-784-4302; fax: +1-775-784-6903. vnm{at}med.unr.edu

Received 14 March 2002; accepted 20 August 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Our major hypothesis is that cyclic adenosine-3',5'-monophosphate (cAMP)-mediated modulation of neurotransmitter release plays different roles at low and high activity of the sympathetic nervous system. We further hypothesize that cAMP-mediated neuromodulation might underlie disparate neurovascular control in mesenteric arteries and veins. Methods: Electrical field stimulation (EFS)-evoked overflow of noradrenaline (NA) was evaluated in the absence or presence of activators and inhibitors of cAMP-dependent pathway at low (4 Hz) and high (16 Hz) frequencies of stimulation of endothelium-denuded secondary and tertiary branches of the canine isolated inferior mesenteric arteries and veins. The content of NA in samples of the superfusates collected before and during nerve stimulation was assayed by high-performance liquid chromatography (HPLC) technique in conjunction with electrochemical detection. Student's t-test and ANOVA analyses were applied for statistical analysis. Results: Activation of cAMP-dependent pathway with either isoproterenol (ISO, 10 µM), forskolin (1 µM), dibutyryl cAMP (100 µM) or combined site-specific activators of cAMP-dependent protein kinase (PKA) [i.e. N6-phenyl-adenosine-3',5'-cyclic monophosphate, 8-(6-aminohexyl) aminoadenosine-3',5'-cyclic monophosphate, and the Sp-isomer of 5,6-dichloro-1-D-ribofuranosylbenzimidazole-3',5'-cyclic monophosphorothioate, each 100 µM] caused an enhancement of the EFS-evoked overflow of endogenous NA at 16 Hz of stimulation but was without an effect at 4 Hz of stimulation both in artery and vein. The EFS (16 Hz)-evoked overflow of NA in vein was also increased in the presence of inhibitors of phosphodiesterase (PDE) III and PDE IV (i.e. milrinone, 0.4 µM, and roilpram, 30 µM), whereas these inhibitors did not affect the overflow of NA in the artery. The facilitating effect of activators of cAMP-dependent pathway on the EFS-evoked release of NA at 16 Hz appears to be more pronounced in the vein than in artery. The increasing effect of ISO (10 µM) was inhibited with either propranolol (1 µM) or the adenylyl cyclase (AC) inhibitor [9-(tetrahydro-2'-furyl)adenine] (SQ 22,536, 100 µM) in both blood vessels. The ISO effect was inhibited by the PKA inhibitor 14–22 amide (PKI14–22), 1 µM, in the artery but not in vein. The enhancing effect of FSK was inhibited by pretreatment of the tissue with SQ 22,536, 100 µM, or the PKA inhibitors PKI14–22, 1 µM, and 4-cyano-3-methylisoquinoline, 50 nM. However, the inhibitors alone did not significantly change the EFS-evoked overflow of NA in both blood vessels. Conclusions: Activation of AC–cAMP–PKA pathway appears to play a role in modulating NA release at higher stimulation frequencies as might be expected during stress, strenuous exercise, or hemorrhage. The AC–cAMP pathway plays a more pronounced role in the autonomic neural control of mesenteric veins than of the corresponding arteries, whereas the PKA contribution is more distinct in the arteries.

KEYWORDS Arteries; Autonomic nervous system; Neurotransmitters; Second messengers; Veins


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
The resistive and capacitative vessels in the splanchnic circulation differ in their mechanical, electrical, and neurochemical responses to sympathetic nerve stimulation [1–5]. However, the mechanisms by which neuroeffector processes differ in arteries and veins remain unknown. It is likely that the two vascular networks demonstrate distinct activities of signal transduction pathways that mediate neurotransmitter release. There is evidence that raised intracellular levels of cyclic adenosine-3',5'-monophosphate (cAMP) can increase noradrenaline (NA) release from both central and peripheral neurons suggesting a role for this nucleotide in the regulation of sympathetic neurotransmission [6]. The formation of cAMP from adenosine-5'-triphosphate (ATP) is caused by virtue of activating of a family of adenylyl cyclases (AC) [7]. The major target molecule of cAMP is cAMP-dependent protein kinase (PKA) that mediates its effects through phosphorylation of specific substrates including components of neurotransmitter release machinery [7–9]. Thus, differential activity of cAMP-mediated mechanisms might underlie differential neurovascular control in arteries and veins.

Prejunctional {alpha}2-adrenoceptors that are preferentially coupled with Gi-group G-proteins and downregulate AC [10] appear to more prominently modulate neurotransmitter release in the mesenteric veins than in the corresponding arteries [5] indirectly suggesting that distinct AC-associated mechanisms of neurotransmitter release modulation might contribute to the differences between arteries and veins. Furthermore, {alpha}2-adrenoceptor activators reduce NA release at low but not at high frequency of electrical field stimulation (EFS) of postganglionic sympathetic nerve terminals [5]. Thus, one might assume that cAMP-mediated neuromodulation would be more prominent at low frequency of stimulation if cAMP-mediated mechanisms underlie automodulation mediated by the prejunctional {alpha}2-adrenoceptors. In some instances prejunctional {alpha}2-adrenoceptors are indeed negatively coupled to AC [11]. The general validity of this assumption has been questioned however, by findings which imply that cAMP is not always involved in the {alpha}2-adrenoceptor modulation of NA release [12,13]. Therefore, controversy still remains regarding the contribution of this pathway to the function of the prejunctional {alpha}2-adrenoceptors. Consequently, both cAMP- and prejunctional {alpha}2-adrenoceptor-mediated modulation of neurotransmitter release might have different patterns and/or frequency dependence in various systems. The role of cAMP-mediated mechanisms in neurotransmitter release in arteries and veins from the same vascular bed has not been previously addressed.

The present study was carried out therefore, to examine and compare the effects of activators and inhibitors of AC–cAMP–PKA pathway on NA release in response to EFS of postganglionic nerve terminals in canine mesenteric arteries and the corresponding veins at low and high frequency of stimulation. These experiments provide a basis for understanding how cyclic nucleotide systems might affect neurotransmitter release in blood vessels as well as how cyclic nucleotide systems might contribute to differential autonomic control of the arterial and venous sides of the splanchnic circulation.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Tissue preparation
Sixty-one mongrel dogs of either sex (averaging 15 kg) were obtained from vendors licensed by the United States Department of Agriculture. The use of dogs for these experiments was approved by the University of Nevada's Animal Care and Use Committee. The animals were sacrificed with an overdose of pentobarbitone sodium (100 mg/kg intraperitoneally) which is consistent with the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no. 85-23, revised 1996). The abdomen was opened and segments of second and third order branches of the inferior mesenteric artery (0.7–1 mm in diameter) and vein (0.9–1.2 mm in diameter) were dissected out and bathed in regular Krebs solution of the following composition: 150 mM NaCl, 4.6 mM KCl, 1.2 mM MgCl2, 2.5 mM CaCl2, 24.8 mM NaHCO3, 1.2 mM KH2PO4 and 5.6 mM dextrose, while perfusing them with distilled water for 30 min to remove endothelium. This procedure has been shown to successfully remove endothelium while the smooth muscle contractility remains intact [14].

2.2 Transmitter overflow experiments
Isolated segments of endothelium-denuded mesenteric arteries (73.42±2.1 mg wet weight, n=74) or mesenteric veins (46.88±1.24 mg wet weight, n=74) were placed in 200-µl BRANDEL superfusion chambers as previously described [4,5,15,16]. After 45 min equilibration, the tissues were subjected to a 30-s ‘conditioning’ stimulation with a train of square wave pulses of 0.3 ms duration and a frequency of 4 Hz. Thirty minutes later the blood vessels were subjected to EFS for 2 min with a train of suprathreshold pulses of 0.3 ms duration at 4 Hz followed by EFS at 16 Hz 30 min later. Samples of the superfusion solution were collected before the electrical stimulation (resting overflow) and during the electrical stimulation (electrically-evoked overflow) in ice-cold test tubes. Samples were analyzed for NA content by high-performance liquid chromatography (HPLC) technique in conjunction with electrochemical detection. In some experiments, tissues were superfused with either activators [i.e. isoproterenol, ISO, 10 µM; forskolin, FSK, 1 µM; 1,9-dideoxy-FSK, 1 µM; dibutyryl cAMP, 100 µM; milrinone, 0.4 µM and rolipram, 30 µM; combination of PKA activators N6-phenyl-adenosine-3',5'-cyclic monophosphate (6-Phe-cAMP), 8-(6-aminohexyl) aminoadenosine-3',5'-cyclic monophosphate (8-AHA-cAMP), and the Sp-isomer of 5,6-dichloro-1-D-ribofuranosylbenzimidazole-3',5'-cyclic monophosphorothioate (Sp-5, 6-DCl-cBIMPS), each 100 µM], or inhibitors [(9-(tetrahydro-2'-furyl)adenine, SQ 22,536, 100 µM; protein kinase A inhibitor 14–22 amide, PKI14–22, 1 µM and 4-cyano-3-methylisoquinoline, CMI, 50 nM] of the AC–cAMP–PKA pathway. In some experiments tissues were perfused with either PKI14–22 (1 µM) or propranolol (1 µM) or SQ 22,536 (100 µM) for 20 min before addition of ISO (10 µM) or FSK (1 µM) to the superfusion solution. Perfusion with drugs started immediately after the conditioning stimulation and continued throughout the experiment. Only one drug or combination of drugs was tested in each tissue.

2.3 HPLC assay of NA
To measure the overflow of NA, 115-µl aliquots from the samples were acidified with 3 µl 1 M perchloric acid to pH 2.6, filtered twice through a 0.22 µm Cameo 3N syringe filter and injected (70 µl) into an isocratic HP1100 HPLC system equipped with a HP1049A electrochemical detector (Agilent Technologies, Wilmington, DE, USA) and an MD-150 column (ESA, Chelmsford, MA, USA). The mobile phase for separation consisted of the following (mM): Na2PO4 (50); EDTA (0.2); 1-heptanesulfonic acid (3.0), LiCl (10.0), and methanol 3% v/v in deionized water. The pH was adjusted to 2.6 with o-phosphoric acid. The electrochemical detector was equipped with a glassy carbon electrode and NA was detected using amperometry mode at a potential of 0.5 V. The HPLC system was controlled by and data collected by a HP Kayak XA computer equipped with HP ChemStation (A.06.03) software from Hewlett Packard (Agilent Technologies).

The amounts of NA in each sample were calculated from calibration curves of NA standards run simultaneously with every set of unknown samples. Results were normalized for sample volume and tissue weight and the overflow of NA was expressed in pmol/mg tissue.

2.4 Drugs
Forskolin, isoproterenol, propranolol, [9-(tetrahydro-2'-furyl)adenine] (SQ 22,536), 1,9-dideoxyforskolin, ethylene-diamine tetraethylacetic acid (EDTA), 1-heptanesulfonic acid and lithium chloride were from Sigma (St. Louis, MO). N6-Phenyl-adenosine-3',5'-cyclic monophosphate (6-Phe-cAMP), 8-(6-aminohexyl) aminoadenosine-3',5'-cyclic monophosphate (8-AHA-cAMP), and the Sp-isomer of 5,6-dichloro-1-D-ribofuranosylbenzimidazole-3',5'-cyclic monophosphorothioate (Sp-5, 6-DCl-cBIMPS) were from BioLog (La Jolla, CA). Dibutyryl cyclic AMP, milrinone, rolipram, myristoylated PKA inhibitor 14–22 amide (PKI14–22), 4-cyano-3-methylisoquinoline (CMI) were from Calbiochem (La Jolla, CA). FSK, 1,9-dideoxy-FSK, milrinone, rolipram, and CMI were dissolved in 50% dimethylsulfoxide (DMSO) and further diluted in double distilled water or Krebs solution. The final concentration of DMSO was less than 0.1% DMSO. All other drugs were initially dissolved in redistilled water and further diluted in Krebs solution. Methanol and disodium phosphate were HPLC grade (Fisher Scientific, USA) and the other chemicals were reagent grade. Water was deionized in a MilliQ water purification system (Millipore, Bedford, MA).

2.5 Statistics
Data are presented as means±S.E.M. Means were compared by analysis of variance (one-way and two-way ANOVA) (GraphPadPrizm v. 3, GraphPad Software). A probability value of less than 0.05 was considered significant. The results were obtained from 150 tissue segments (n) of 61 animals.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 General characteristics of EFS-evoked overflow of NA in canine isolated inferior mesenteric artery and vein
There were no detectable amounts of NA in samples collected for 2 min before stimulation of endothelium-free canine mesenteric arteries or veins suggesting that spontaneous efflux of NA does not occur under our experimental conditions. EFS of both the canine mesenteric artery and vein evokes overflow of NA at 4 Hz and 16 Hz of stimulation. The EFS-evoked NA overflow at 4 Hz was 0.0045±0.0008 pmol/mg tissue (n=12) in the artery and 0.012±0.002 pmol/mg tissue (n=13) in vein, whereas at 16 Hz of stimulation the overflow of NA was 0.032±0.0026 (n=14) and 0.111±0.022 (n=12) in the artery and vein, respectively. Thus, the overflow of NA in vein exceeded the EFS-evoked overflow of NA in artery both at 4 and 16 Hz of stimulation (Fig. 1, controls). Previous study in our laboratory [5], using procedures and EFS parameters virtually identical to those employed in the present study, indicates that pretreatment of the tissue with either tetrodotoxin (1 µM) or guanethidine (10 µM) inhibits the EFS (4 and 16 Hz)-evoked NA overflow in both blood vessels suggesting that NA originates exclusively from the postganglionic sympathetic nerve terminals under these experimental conditions.


Figure 1
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  		Fig. 1 The effect of isoproterenol (ISO, 10 µM), forskolin (FSK, 1 µM), 1,9-dideoxy forskolin (1,9-dideoxy-FSK, 1 µM), dibutyryl cAMP (db-cAMP, 100 µM), milrinone (M, 0.4 µM) and rolipram (R, 30 µM) and the PKA activators (PKA act) N6-phenyladenosine-3',5'-monophosphate (6-Phe-cAMP), 8-(6-aminohexyl)aminoadenosine-3',5'-cyclic monophosphate (8-AHA-cAMP) and Sp-isomer of 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole-3',5'-cyclic monophosphorothioate (Sp-5,6-DCl-cBIMPS) (PKA act, each at 100 µM) on the overflow of NA evoked by EFS (0.3 ms, 15 V, 2 min) at 4 Hz (panel A) and 16 Hz (panel B) in canine isolated mesenteric artery and vein. Values are expressed as means±S.E. from 3–6 experiments. Asterisks denote P<0.05 between control and treated preparations. Panel C and panel D represent chromatographic peaks of NA from original chromatograms of superfusate samples collected during EFS at 16 Hz of the canine isolated mesenteric artery (C) and vein (D) in the absence (control) or presence of the aforementioned activators of the AC–cAMP–PKA pathway. Each trace represents a chromatographic peak of NA in an individual experiment carried out on a separate tissue preparation. The tissue preparations in the examples shown ranged from 64 to 66 mg tissue wet weight for arteries and 53 to 55 mg wet weight for veins.

 
3.2 Raising cAMP levels facilitates the EFS-evoked NA release
Fig. 1 shows the results of numerous maneuvers for stimulating AC–cAMP–PKA pathway. Original traces of chromatograms obtained by assaying the superfusate samples collected during EFS at 16 Hz of artery (Fig. 1C) and vein (Fig. 1D) are shown, as well.

Isoproterenol (ISO, 10 µM), an agent that is known to activate AC via activation of Gs-protein coupled β-adrenoceptors [11], had no effect on the EFS-evoked overflow of NA at 4 Hz of stimulation in both blood vessels (Fig. 1A). At 16 Hz however, the NA overflow was enhanced to 0.065±0.005 pmol/mg tissue (n=4) in the artery, and to 0.206±0.026 pmol/mg tissue (n=4) in the vein (P<0.05 as compared to controls) (Fig. 1B–D). The results with ISO suggest that stimulation of AC and production of cAMP might lead to facilitation of the EFS-evoked NA release at high frequency of stimulation but not at low frequency of stimulation. Other maneuvers that also increase cAMP levels in the cells should then potentiate the EFS-evoked NA overflow at 16 Hz but not at 4 Hz of stimulation. To determine whether this is the case, three additional methods of cAMP level enhancement were tested including (1) direct activation of AC with FSK (1 µM), (2) inhibition of phosphordiesterase (PDE) III with milrinone (0.4 µM) and PDE IV with rolipram (30 µM), and (3) exogenous application of the cell membrane permeant analogue of cAMP, dibutyryl cAMP (100 µM). Indeed, FSK increased the EFS (16 Hz)-evoked NA overflow to 0.053±0.008 pmol/mg tissue (n=6) and 0.233±0.03 pmol/mg tissue (n=6) in artery and vein, respectively (Fig. 1B–D). Likewise, combined milrinone and rolipram increased the NA overflow at 16 Hz to 0.174±0.017 pmol/mg tissue (n=6) in the vein (Fig. 1B and D) although it did not affect significantly the EFS-evoked overflow of NE in the artery (Fig. 1B and C). Finally, dibutyryl cAMP enhanced the NA overflow in response to EFS at 16 Hz to 0.051±0.009 pmol/mg tissue in artery and to 0.216±0.01 pmol/mg tissue in vein (Fig. 1B). However, none of these interventions significantly affected the NA overflow at 4 Hz in both blood vessels (Fig. 1A). 1,9-Dideoxy-FSK, a structural analog of FSK that lacks AC stimulating activity [17] was without an effect in both blood vessels (Fig. 1A and B).

The EFS-evoked overflow of NA at 16 Hz can also be facilitated by direct activation of PKA. Combined administration of the site-selective PKA activators 6-Phe-cAMP, 8-AHA-cAMP, and Sp-5,6-DCl-cBIMPS (each at 100 µM) enhanced the EFS (16 Hz)-evoked overflow of NA to 0.097±0.01 pmol/mg tissue (n=3) and to 0.140±0.013 pmol/mg tissue (n=3) in the artery and vein, respectively (Fig. 1B–D).

3.3 Inhibitors of AC/PKA do not affect EFS-evoked release of NA
Both SQ 22,536, an inhibitor of AC [18,19], and PKI14–22 amide, a membrane permeant (myristoylated) protein kinase A inhibitor [20], significantly reduced the enhancing effect of FSK on the EFS-evoked overflow of NE at 16 Hz (Fig. 2) suggesting that the effect of FSK on NA release is mediated by AC/PKA activation. Interestingly, in the presence of PKI14–22 the effect of FSK in the vein was even inverted; that is, FSK reduced the EFS-evoked NA overflow at 16 Hz in the presence of PKI14–22 (Fig. 2). The enhancing effect of ISO on the NA release at 16 Hz was inhibited by pretreatment with the nonselective β-adrenoceptor blocker propranolol (not shown) and the AC inhibitor SQ 22,536 in both artery and vein (Fig. 3) suggesting that the effect of ISO is indeed mediated by β-adrenoceptors and activation of AC. Interestingly, PKI14–22 abolished the enhancing effect of ISO only in arteries, whereas in the veins this effect of ISO remained intact (Fig. 3) suggesting that PKA is more involved in mediating the effects of β-adrenoceptor activation in the arteries than in veins. Neither SQ 22,536 nor PKI14–22 alone or CMI, another cell permeant PKA inhibitor [21], significantly affected the EFS-evoked NA overflow at all experimental conditions (Fig. 4) suggesting that inhibition of the resting activity of either AC or PKA might not be sufficient to modulate neurotransmitter release.


Figure 2
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  		Fig. 2 The effect of FSK (1 µM) on the EFS-evoked overflow of NA in the presence of the adenylyl cyclase inhibitor SQ 22,536 (100 µM) and the PKA inhibitor PKI14–22 (1 µM) at 16 Hz of EFS (0.3 ms, 15 V, 2 min) in canine mesenteric artery and vein. Asterisks denote P<0.05 compared to control group; open circles denote P<0.05 compared to FSK group.

 

Figure 3
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  		Fig. 3 The effect of ISO (10 µM) on the EFS-evoked overflow of NA in the presence of the adenylyl cyclase inhibitor SQ 22,536 (100 µM) and the PKA inhibitor PKI14–22 (1 µM) at 16 Hz of EFS (0.3 ms, 15 V, 2 min) in canine mesenteric artery and vein. Asterisks denote P<0.05 compared to control group; open circles denote P<0.05 compared to ISO group.

 

Figure 4
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  		Fig. 4 The effect of the adenylyl cyclase inhibitor SQ 22,536 (100 µM) or PKA inhibitors PKI14–22 (1 µM) and CMI (0.05 µM) on the EFS (0.3 ms, 15 V, 2 min)-evoked overflow of NA at 4 Hz (A) and 16 Hz (B) in canine mesenteric artery and vein.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The arteries and veins from the splanchnic circulation are densely innervated [3,22,23] and the two vascular networks differ significantly in the mechanisms of neurovascular control [1–5,24] which might contribute to serving distinct functions (i.e. resistance vs. capacitance) in the peripheral circulation. Besides, distinct mechanisms of neurovascular control might be involved at low and high activities of the autonomic nervous system. For example, the prejunctional {alpha}2-adrenoceptor mediated modulation of neurotransmitter release generally appears to be an important feedback mechanism at lower frequencies of stimulation [5,25–27]. Likewise, the role of N-type voltage-operated Ca2+ channels appears to be more prominent at lower frequencies of nerve stimulation. On the other hand, the role of P/Q-type neuronal Ca2+ channels increases with stimulation frequency [28]. Therefore, the role of intracellular signal transduction pathways in neurotransmitter release might also vary at low and high levels of autonomic nervous system activity.

A large number of hormones, neurotransmitters and other signal substances utilize cAMP as an intracellular second messenger. Cyclic AMP has generally been implicated as an important mediator of neuroeffector transmission in both the central and peripheral nervous systems [6,29] although a direct correlation between cAMP-mediated pathways and release of neurotransmitters is sometimes difficult to identify [13,30]. The role of cAMP-mediated mechanisms in neurotransmitter release in arteries and veins from the same circulatory bed has not been previously addressed.

In the present study we have directly measured the EFS-evoked overflow of endogenous NA upon activation or inhibition of the AC–cAMP–PKA pathway in isolated strips of the canine inferior mesenteric artery and vein. Several lines of evidence suggest that cAMP may be an important intracellular messenger in regulating neurotransmitter release in the mesenteric vasculature primarily at high frequency of stimulation. For example, activation of AC elicited with β-adrenoceptor stimulation and consequent activation of the guanine nucleotide-binding protein Gs with ISO led to a facilitated overflow of NA at 16 Hz but not at 4 Hz of nerve stimulation. The enhancing effect of ISO was reduced by the AC inhibitor SQ 22,536. Further, direct activation of AC with FSK enhanced the NA overflow at 16 Hz but not at 4 Hz of stimulation both in artery and vein. The effect of FSK was reduced by the AC inhibitor SQ 22,536. This observation together with the finding that the inactive analog of FSK, 1,9-dideoxy-FSK, had no effect on neurotransmitter release, provide functional evidence that the FSK-induced enhancement of EFS-evoked overflow of NA is due primarily to increased AC activity. Similar effects of FSK and ISO have previously been reported in the rat tail artery [31] and in frog neuromuscular junction preparations [32]. FSK has also been shown to cause coordinated up-regulation of gene expression and enzyme activities of three catecholamine-synthesizing enzymes (tyrosin hydroxylase, dopamine beta-hydroxylase, and phenyethanolamine N-methyltransferase) resulting in a large increase in NA content and dramatic release in primary cultured bovine adrenomedullary chromaffin cells [33]. This large enhancement of catecholamine release implicates an important physiological role for cAMP in the regulation of in vivo sympathetic activities. Exogenous application of cAMP (i.e. membrane permeant dibutyryl cAMP) in the present study caused a similar enhancement of the EFS-evoked NA overflow at 16 Hz of stimulation both in artery and vein, whereas at 4 Hz of stimulation dibutyryl cAMP produced no effect.

The levels of cAMP in the tissue are modulated by the activity of various PDEs. Of particular significance for cAMP are PDE III and PDE IV [34]. Blockade of the PDE III and PDE IV activity in the mesenteric vein also led to an increase of the EFS-evoked overflow of NA at 16 Hz but not at 4 Hz of stimulation. This observation is particularly intriguing since it suggests that the basal production of cAMP in vein, unaltered by metabolism, is sufficient to lead to activation of neurotransmitter release at high frequency of nerve stimulation. This result also raises the interesting possibility that an additional manner in which neurotransmitter release may be regulated is through modulation of PDE activity.

The principal intracellular target for cAMP in mammalian cells is the PKA [35] although some effects of this cyclic nucleotide might also be mediated by direct action of cAMP on substrate molecules [36]. To provide direct evidence for the involvement of this kinase in facilitating the EFS-evoked neurotransmitter release, we applied three different site-selective activators of PKA (i.e. 6-Phe-cAMP, 8-AHA-cAMP, Sp-5, 6-DCl-cBIMPS). This combination of drugs together activate both the A- and B-sites of both type I and type II PKA [37,38]. This combination of drugs led to an increase of the EFS-evoked overflow of NA at 16 Hz but not at 4 Hz, providing further evidence that facilitation of neurotransmitter release at higher frequency of stimulation is mediated by PKA. Therefore, direct activation of endogenous PKA enhances EFS-evoked transmitter release at 16 Hz of stimulation. Interestingly, direct PKA activation had more pronounced effect in the artery than in vein, although AC–cAMP pathway seems to generally play a greater role in the vein. Along these lines, it is particularly intriguing that the enhancing effect of ISO was abolished by the PKA inhibitor PKI14–22 in the artery, whereas the ISO enhancing effect in the vein remained untouched. The latter finding suggests that in the artery a large portion of the ISO effect is mediated by PKA, whereas in the vein the enhancing effect of ISO on NA release might be directly mediated by cAMP or be PKA-independent.

Interestingly, while activation of AC–cAMP–PKA pathway leads to enhancement of the EFS-evoked NA release at high frequency of stimulation, inhibitors of this pathway (i.e. SQ22,536, PKI14–22 and CMI) generally appear not to affect the EFS-evoked overflow of neurotransmitter neither at high nor at low frequency of stimulation.

As discussed in the Introduction, one might anticipate the AC–cAMP–PKA pathway-mediated neuromodulation be more pronounced at lower frequency of stimulation if this pathway underlies the {alpha}2-adrenoceptor-mediated neuromodulation, since the autoregulation mediated by these receptors appear to be more prominent at low stimulation frequency. However, although all maneuvers for cAMP/AC/PKA pathway activation tend to influence the NA overflow at 4 Hz and 16 Hz of stimulation in a similar pattern, the present study clearly indicates that in the canine mesenteric circulation the AC–cAMP–PKA pathway plays a more important role in the facilitation of neurotransmitter release at higher frequency of stimulation.

These observations differ significantly from what we have observed previously with regard to {alpha}2-adrenoceptor-mediated modulation of NA release in the same vascular networks [5] indirectly suggesting that cAMP-mediated mechanisms might not underlie the {alpha}2-adrenoceptor-mediated neuromodulation in this system. The present findings agree with previous studies that report lack of a direct link between {alpha}2-adrenoceptor-mediated neuromodulation and AC pathway [12,13,39]. In contrast, in the rabbit iris-ciliary body, the prejunctional {alpha}2-adrenoceptors do appear to be negatively coupled to adenylyl cyclase, which contributes to autofeedback regulation of NA release [11]. These findings may help to explain the disparate nature of the mechanisms underlying prejunctional receptor-mediated neuromodulation.

Another interesting feature of the present study is that although AC–cAMP pathway appears to be involved in the neurotransmitter release at high frequency of stimulation both in artery and vein, this pathway seems to play a greater role in the vein. Intense activation of sympathetic nervous system as initiated by hemorrhage, intense exercise or stress results in marked constriction primarily of veins [40], which results in increases in venous return and central venous pressure, factors important in increasing cardiac efficiency and output. Therefore, activation of AC–cAMP-dependent pathway might participate in the prolonged venoconstriction during sympathetic nervous system hyperactivity.

In conclusion, activation of the AC–cAMP–PKA pathway does not play a significant role in modulating NA release in canine mesenteric arteries and veins at low stimulation frequencies. In contrast, AC–cAMP–PKA pathway activation appears to be more important for neuromodulation at higher stimulation frequencies and hence during intense activation of the sympathetic nervous system. In addition, the AC–cAMP-mediated neuromodulation seems to be more prominent in veins than in arteries of the splanchnic circulation, whereas the contribution of PKA is more prominent in the arteries.

Time for primary review 22 days.


   Acknowledgements
 
This work was supported by US Public Health Service Grant HL 60031 to V.M-Y.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

  1. Kreulen D.L. Activation of mesenteric arteries and veins by preganglionic and postganglionic nerves. Am J Physiol (1986) 251:H1267–H1275.[Web of Science][Medline]
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