Nicotinic- and muscarinic-evoked of canine adrenal catecholamines
release and peptides
S. L. CHRITTON, M. K. DOUSA, T. L. YAKSH, AND G. M. TYCE Departments of Physiology and Biophysics and of Neurosurgery, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905
S. L., M. K. DOUSA, T. L. YAKSH, AND G. M. and muscarinic-evoked release of canine adrenal catecholamines and peptides. Am. J. Physiol. 260 (Regulatory Integrative Comp. Physiol. 29): R589-R599, 1991.-The tissue content and overflow of norepinephrine (NE), epinephrine (Epi), dopamine (DA), Met-enkephalin (Met-Enk), and neuropeptide Y (NPY) from isolated, retrogradely perfused dog adrenal glands were studied. Under resting conditions, -25% of the overflow of autocoids from the glands was Ca” dependent; the cholinergic antagonists hexamethonium and atropine had no effects on basal efflux. Stimulation with the nicotinic agonist l,l-dimethyl-4-phenylpiperazinium iodide (DMPP; 3 or 50 PM) or with the muscarinic agonist pilocarpine (50 PM or 1 mM) evoked releases of autocoids. These releases were blocked or dramatically reduced by appropriate antagonists or by the removal of Ca2’ from the perfusate. Expressed as percentages of tissue stores, the rank order of overflow of autocoids was E - DA >> NE during resting conditions, DA >> E - NE during stimulation with 50 PM DMPP, and DA > Epi > NE during stimulation with 1 mM pilocarpine. These data are consistent with different mechanisms of release for the catecholamines, perhaps from different cell populations. The data support corelease of peptides and catecholamines, although clear pairing of autocoids could not be confirmed. CHRITTON,
TYCE. Nicotinic-
adrenal medulla; chromaffin cells; epinephrine; norepinephrine; dopamine; methionine-enkephalin; neuropeptide Y; pilocarpine; 1,1-dimethyl-4-phenylpiperazinium iodide
Two neuropeptides, Met-enkephalin immunoreactive material (Met-Enk) and neuropeptide Y immunoreactive material (NPY), are also released by cholinergic stimuli from isolated, perfused adrenals (1, 18, 36). There is continuing controversy regarding the localization of these peptides in chromaffin cells. Although Met-Enk has been shown to be localized mainly in bovine Epicontaining cells in histological studies (17, 19), cosecretion of Met-Enk with NE as well as with Epi has been observed with perfused bovine glands and cultured bovine chromaffin cells (1, 39). NPY has been shown to be colocalized with Epicontaining granules by biochemical .techniques (2) and with Epi-containing cells by histochemistry and in situ hybridization in bovine adrenal glands (27). The release of NPY from cat adrenals has been more closely associated with Epi release than NE release (18). However, in another report, NPY immunoreactivity in bovine adrenals was found to be restricted to NE-containing cells (19) The purpose of this study was to characterize the simultaneous efflux of the autocoids Epi, NE, DA, MetEnk, and NPY from isolated dog adrenals during basal conditions and under nicotinic or muscarinic stimulation. METHODS
secretes a variety of hormones into the blood in response to acetylcholine released by splanchnic nerves. Most abundant among these hormones are epinephrine (Epi) and norepinephrine (NE), which have been shown to be stored in two different cell populations within the adrenal (5). Release of Epi and NE from the isolated adrenal occurs in different proportions with muscarinic vs. nicotinic stimulation in both the cat (6, 26) and dog (34). This suggests that the NEand Epi-containing cells respond differently to nicotinic and muscarinic agonists. Much less studied than Epi and NE is dopamine (DA), which is also released from the adrenals on stimulation (21). It has been suggested that DA has various roles as precursor, hormone, and intra-adrenal modulator of release (9,29). DA must indeed be present in NE- and Epicontaining cells as a precursor, but it appears that there is also a separate group of small granule chromaffin cells, which may contain DA as their only catecholamine (32).
THE ADRENAL
MEDULLA
0363-6119/91
$1.50 Copyright
AdrenaL perfusion. Left and right adrenals were removed from mongrel dogs of either sex. The dogs were “pool” dogs, in that different tissues were used by a number of investigators. The dogs were anesthetized with 30 mg/kg iv pentobarbital sodium and then bled by cutting both carotid arteries. The adrenals were removed within 5 min of exsanguination and placed in ice-cold modified Krebs-Ringer (KR) solution (118.3 mM NaCl, 5.7 mM KCl, 2.5 mM CaC12, 1.2 mM KH2P04, 1.2 mM MgSOd, 25 mM NaHC03, 10 mg/lOO ml sodium metabisulfite, 1.5 mg/lOO ml EDTA, 200 mg/lOO ml dextrose), which had been bubbled with 95% oxygen-5% carbon dioxide gas. In a few experiments, 50 mg/lOO ml bovine serum albumin (BSA) was present in the KR solution. This concentration did not increase values of Met-Enk or NPY when 49 initial basal perfusate samples without BSA were compared with 34 initial basal samples with BSA. One adrenal, usually the left gland because of its more amenable anatomy, was perfused. Perfusion was started within 15 min of the adrenal harvest. The adrenal
(c> 1991 the American
Physiological
Society
R589
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R590
NICOTINIC-
AND
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was retrogradely perfused via a cannula secured within the adrenolumbar vein (24). Slits were made in the end of each lobe of the adrenal through the capsule and cortex to allow perfusate to exit. Perfusions were carried out in a chamber with a controlled temperature of 37°C and a humidity of X0%. The KR solution was warmed and equilibrated with 95% oxygen-5% carbon dioxide gas (final pH 7.4 t 0.1) before being pumped at l-3 ml/min through the adrenal. The pump exerted a constant pressure and was adjusted to provide flow around 1.5 ml/ min; in a few experiments, a constant-flow peristaltic pump was used. Perfusate was collected in graduated tubes on ice, and an aliquot was removed and frozen for peptide analysis; 10 ~1 of 5% sodium metabisulfite were added per milliliter of the remaining perfusate, which was reserved for catecholamine analysis. After a 65-min collection of perfusate, an initial lomin basal sample was collected. The tubing leading to the adrenal was temporarily clamped off while the stimulation solution was injected through a narrow-gauge needle inserted into tubing proximal to the adrenal. The stimulation solutions were either 3 or 50 PM l,l-dimethyl-4-phenylpiperazinium iodide (DMPP), or 50 PM or 1 mM pilocarpine, and were prepared by diluting a concentrated stock with warmed, oxygenated KR solution from the perfusion reservoir just before injection. The volume injected (-3 ml) was calculated to match the previous flow through the gland. After a 2min collection while the stimulation solution was injected, the original perfusion was resumed, and an 8min poststimulation sample was collected. This was followed by a ZOmin sample and a final IO-min basal perfusion sample. Efflux of autocoids was calculated in terms of picomoles per minute. In some experiments, stimulations were done in the presence of the nicotinic antagonist hexamethonium (100 PM), the muscarinic antagonist atropine (50 PM), or in calcium-free KR solution containing 1 mM ethylene glycol-bis(@aminoethyl ether)N,N,N’,N’-tetraacetic acid (EGTA). When using calcium-free KR, additional NaCl was added to maintain isotonicity with normal KR solution. During these experiments, additional samples were taken to evaluate the effects of the KR solution containing antagonists or calcium-free KR on basal adrenal efflux. Extraction of autocoids from adrenal tissues. The unperfused glands were kept in ice-cold KR solution until they were cleaned of surrounding tissue and frozen on dry ice. The unperfused glands were frozen within 1 h of harvest. At the end of the perfusion, the perfused glands were also cleaned, frozen on dry ice, and then both glands were stored at -20°C. The glands were homogenized in 0.1 N HCl, and an aliquot was heated in a water bath at 100°C in preparation for peptide analysis. Another aliquot was rapidly deproteinized using 0.4 M perchloric acid before catecholamine analysis. Catecholamine analysis. For catecholamine extraction, 5 ml or less of perfusate was combined with 500 mg activated alumina and 1 ml of 2 M tris(hydroxymethyl)aminomethane. HCI buffer containing 5% EDTA at pH 8.6. The mixture was adjusted to pH 8.4-8.6 with Na2COg, shaken for 10 min, and then poured into 5-mm
ADRENAL
AUTOCOID
RELEASE
diameter glass columns plugged with glass wool. The alumina was washed with 5 ml distilled water, and catecholamines were eluted with 6 ml of 0.05 N perchloric acid. Quantitation of catecholamines was by high-performance liquid chromatography (HPLC). Catecholamines were measured in diluted eluates or tissue homogenates after a reversed-phase separation using a C-18 solid phase on 5-pm diameter particles (Ultrasphere, Beckman Instruments) packed in a 4.6 mm X 25 cm column. Samples were autoinjected (Waters Intelligent Sample Processor, Millipore) into a 1 ml/min flow of mobile phase through the HPLC column. The mobile phase contained 70 mM NaH2P04, 2 mM heptane sulfonate, 0.2 mM EDTA, and 5% acetonitrile at a pH of 3.4. Amperometric detection of catecholamines was accomplished using a Bioanalytical Systems detector at a potential of 0.65 V relative to a silver-silver chloride electrode. 3,4-Dihydroxybenzylamine (DHBA) was used as an internal standard. Mean recoveries of Epi, NE, DA, and DHBA standards were 82, 83, 76, and 81% (for all, SE = 2%) from the alumina; values reported have been corrected for recovery. Peptide analysis. Peptide immunoreactivity was measured by radioimmunoassay using antibodies directed at Met-Enk (antibody no. N302) and NPY (antibody no. 221) (13, 40). The limits of detection for Met-Enk and NPY were 400 t 30 and 8 t 2 (SE) fmol/min. HPLC of peptides was performed on some samples using an LKB model 2150 with a reverse-phase C-18 column (Rainin Short One). Samples were lyophilized and reconstituted in 0.6 ml of the initial mobile phase. The mobile phase was 0.1 M sodium acetate, pH 4, with acetonitrile gradients of lo-60% for Met-Enk or 25-45% for NPY. After the sample was injected and the gradient was started, I-min fractions of 0.5 ml were collected from the column and lyophilized. Met-Enk fractions separated by HPLC were enzymatically cleaved to reveal encrypted Met-Enk immunoreactivity. Fractions were incubated at 37°C for 1 h with 25 pg trypsin followed by 1 h with 0.5 pg carboxypeptidase B and then were lyophilized and assayed for Met-Enk. NPY immunoreactivity of HPLC fractions was performed with no further processing. Calibration standards of synthetic human NPY and synthetic human NPY oxidized with 3% hydrogen peroxide were run separately. Calculations and statistics. Release was measured in the lo-min period immediately after the first basal sample; 20 min after the release period a second basal sample was measured. To calculate net evoked release of an autocoid, a calculated underlying basal efflux of the autocoid was subtracted from the total release evoked by the stimulation. To model the behavior of basal efflux, unstimulated efflux was studied. Unstimulated efflux after 60 min of perfusion declined as a single exponential curve; on a plot of logarithmic values of efflux agains t the time of perfusion, the efflux descended in a straigh t line. The slope of such a line would be ln(B2) - In(B1) tB2
-
tB1
where Bl and B2 are effluxes of the autocoid into basa .l
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NICOTINIC-
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samples before and after stimulation, respectively, and tgl and tBZ are the midpoints in time of the basal samples. The slope of the same line can also be calculated as - ln(B1)
ln(BR) tR
-
tB1
where BR is the predicted basal efflux during evoked release and tR is the midpoint in time of the evoked release. Setting the two expressions equal and solving for BR, one finds ln(B1)+
BR = e{
[ln(B2)--ln(Bl)]
[ht tB2
-
tB1
-
tBl] 1
where net release = total release - BR. One-way analysis of variance (ANOVA) and repeatedmeasures ANOVA were performed on the fractional release data. Other statistical methods used in this study were the sign test, Wilcoxon’s signed-rank test, MannWhitney U test, paired Student’s t test, and correlations to determine Pearson’s and Spearman’s correlation coefficients. Drugs. l,l-Dimethyl-4-phenylpiperazinium iodide, pilocarpine nitrate, hexamethonium chloride, atropine sulfate, trypsin, and carboxypeptidase B were obtained from Sigma Chemical (St. Louis, MO). Peptide standards were obtained from Peninsula Labs (Belmont, CA). RESULTS
Catecholamines and peptides in perfusates under basal conditions. The unstimulated efflux of each autocoid, as
examined in five unstimulated glands, displayed two phases (data not shown). During the first 60 min of perfusion, efflux was high but dropped off rapidly at a rate of -3%/min. After 60 min of perfusion, both basal efflux and its subsequent rate of decline were low; catecholamine effluxes decreased at -0.5%/min. There were no differences in the rates of decline between catecholamines and peptides during the second phase. Basal effluxes of adrenal autocoids after 60 min of perfusion were (in pmol/min t SE; n = 23) 4,600 t 560 Epi; 520 t 81 NE; 46 t 7.7 DA; 2.7 t 0.36 Met-Enk. NPY was detected in basal samples in 13 of 23 perfusions, at a level of 8.6 t 1.7 fmol/min. When glands perfused with KR containing 2.5 mM calcium were switched to calcium-free KR, basal effluxes of catecholamines tended to decrease (Table 1). However, the decreases were not significantly different from those seen in washout control experiments with no removal of calcium. When perfusion with calcium-free KR continued for 45 min after a 2-min stimulation, then a return to KR containing 2.5 mM calcium significantly increased the effluxes of Epi, NE, and DA (Table 1). These increases in Epi, NE, and DA effluxes were significantly different from those in washout control experiments. Basal effluxes of Met-Enk and NPY did not show any clearly calcium-dependent components. Catecholamines and peptides in perfusates with nicotinic stimulation. When 3 PM DMPP was infused into
the adrenal, it significantly enhanced the effluxes of Epi, NE, DA, and Met-Enk (Fig. 1). The mean net releases
ADRENAL
AUTOCOID
RELEASE
R591
evoked by 3 PM DMPP of Epi, NE, DA, Met-Enk, and NPY were 33, 57, 63, 67, and 20%, respectively, above the predicted baselines. At a concentration of 50 PM, DMPP evoked a brisk release of autocoids from the adrenal (Fig. 2). Mean net releases of Epi, NE, DA, and Met-Enk were 230, 520, 500, and 580%) respectively, above predicted baselines. The highest level of release measured for each autocoid occurred during the 2 min of actual infusion, although during the 8 min after infusion autocoids remained substantially above baseline. NPY was not consistently detected except during the lo-min release period in these experiments. Releases with 50 PM DMPP were blocked by the addition of 100 PM hexamethonium to the perfusate (Fig. 3). Introduction of hexamethonium did not affect the basal overflows of autocoids. Releases of Epi, NE, and DA with 50 PM DMPP were dependent on the presence of calcium in the medium (Table 2). Release of Met-Enk with no calcium was reduced to 4% of the mean release with calcium, which was NE (Fig. 6). Thus, during basal conditions, Epi and DA preferentially overflowed from stores in relation to NE. Met-Enk and NPY overflowed from stores at levels between NE and the other catecholamines.
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NICOTINIC-
AND
MUSCARINIC-EVOKED
ADRENAL
100,Wo
AUTOCOID
R593
RELEASE
lW,Wo
r_L,,
10,Wo
T
EPI 1
1
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1,~
1
EPI NE
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1
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MET-ENK 1
1
1
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*
1
1
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34 3 k
~\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\~~~
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100
MET-ENK 1
1
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HEXAMETHONIUM 0.1
0.1
1 0.01
I
0.01
0.001
I
60
IH
I
6ola DMPP MINUTES
I
I
100
I
0.001
I
I
60
120
N
I
I
Rho PILOCARPINE
NPY 1
I
I
loo
I
I
120
OF PERFUSION
FIG. 3. Mean k SE rate of efflux of autocoids in perfusates after introduction of 100 PM hexamethonium and with infusion of 50 PM DMPP for 2 min (n = 2). DMPP-evoked release was antagonized by hexamethonium.
FIG. 4. Mean t SE rate of efflux of autocoids in perfusates with infusion of pilocarpine (1 mM) for 2 min (n = 5). Peak release occurred during 8 min after infusion. *Levels of autocoid were significantly increased during IO-min release period (P < 0.05, Student’s t test after logarithmic transformation).
2. Calcium dependency of DMPPand pilocarpine-evoked releases
TABLE
Basal
lo-Min Release
Net Release
50 PM DMPP stimulation (n = 2, except for NPY where n = 1) No calcium + 1 mM EGTA Epi, nmol/min NE, pmol/min DA, pmol/min Met-Enk, pmol/min NPY, fmol/min
2.63 470 31 1.85 5.9
2.36 430 26.48 2.11 6.2
-0.27 -30 -5 0.26 0.3
#E
K
EPI t
t
NE -1
loo
DA
E
P
f
1
I’
v
MET-ENK I0
m
5
1 mM pilocarpine No calcium Epi, nmol/min NE, pmol/min DA, pmol/min Met-Enk, pmol/min NPY, fmol/min
stimulation (n = 1) + 1 mM EGTA 3.1 430 20 1.16 6.8
3.1 450 24 1.15 5.3
1
i
kl
-0.02 20 4 -0.01 -1.5
Perfusion was carried out for -70 min with KR containing 2.5 mM calcium, then for 35 min in KR containing no calcium and 1 mM EGTA. Glands were then stimulated for 2 min with 50 PM DMPP or 1 mM pilocarpine. Perfusion with no calcium and 1 mM EGTA continued until 45 min after stimulation. Basal efflux in calcium-free medium was calculated from logarithmic slope between samples taken 5 min before and 40 min after stimulation.
Stimulation with DMPP resulted in a different pattern: DA > Epi - NE (Fig. 6). Epi did not increase as much as the other autocoids, so that DA was preferentially released from stores in relation to Epi and NE. The peptides increased, but their variability did not allow them to be differentiated from the catecholamines. During stimulation with 1 mM pilocarpine the preferential fractional overflow of DA over NE seen during basal efflux remained (Fig. 6). The fractional efflux of
0.1
0.001
1
60
I
I
60 PILOCARPINE MINUTES
I
1
loo
I
I
120
OF PERFUSION
FIG. 5. Mean t SE rate of efflux of autocoids in perfusates after introduction of 50 PM atropine and with infusion of 1 mM pilocarpine for 2 min (n = 2). Pilocarpine-evoked release was antagonized by atropine.
Epi was lower than that of DA but did not fall to the level of NE as seen with DMPP stimulation, even though releases of catecholamines with 1 mM pilocarpine were larger than those evoked by 50 PM DMPP. The peptides, Met-Enk and NPY, increased along with the catecholamines, but their variability did not allow them to be differentiated from the fractional releases of individual
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R594
NICOTINIC-
AND
MUSCARINIC-EVOKED
ADRENAL 0.5%
3. Tissue concentrations of catecholamines, Met-Enk, and NPY with nicotinic stimulation TABLE
Unperfused Unstimulated EPi
wwg
NE, wWg DA, nmol/g Met-Enk, nmol/g NPY, pmol/g
and
Stimulated With 50 ,uM DMPP
1.99kO.50 55.35t10.84 3.31kO.68
12.5t4.9
Met-Enk,
and NPY with muscarinic stimulation Unperfused Unstimulated
EPi,
wok
NE, wok DA, nmol/g Met-Enk, nmol/g NPY, pmol/g
and With
5.50-r-0.63 1.21t0.12 51.98t4.83
Stimulated Pilocarpine
4.09kO.43 0.89kO.06" 74.91k6.50
6.32t1.15
4.91kO.88
22.4t4.4
16.0&4.2*
Values are means t SE; n = 6 dogs. One of a pair right glands, 1 left gland) was not perfused or stimulated. was perfused for 125-260 min and stimulated l-4 times pine. In correlation studies of these glands, it was found number of stimulations or a longer time of perfusion was with greater depletion of adrenal autocoids. * Concentration cantly different in stimulated gland as compared with fused gland (P < 0.05, Wilcoxon and Student’s t test).
of adrenals (5 Other gland with pilocarthat a larger not associated is signifipaired unper-
5. Tissue concentrations of catecholamines, Met-Enk, and NPY with perfusion but no stimulation TABLE
Unperfused Unstimulated W,
wol/g
NE, wok DA, nmol/g Met-Enk, nmol/g NPY, pmol/g
8 8
Right gland of a pair of adrenals gland was perfused for 195-205 PM DMPP. * Concentration is gland as compared with paired t test).
4. Tissue concentrations of catecholamines,
TABLE
and
7.24kO.75 1.55kO.11 61.92t5.97 5.78-e0.28 13.2k2.3
-
RELEASE
gg EPINEPHRINE Ed NOREPINEPHRINE DOPAMINE MET-ENKEPHALIN cl NEUROPEPTIDE
Y
8.9Ok 1.85 1.89k0.36 83.5Ok10.67" 3.84k1.34 16.4k1.7
10.10cL95
Values are means t SE; n = 5 dogs. was not perfused or stimulated. Left lnin and stimulated 2 times with 50 significantly different in stimulated unperfused gland (P < 0.001, Student’s
AUTOCOID
Perfused and Unstimulated
6.90t0.82 1.62k0.12 81.78t8.26 6.16kO.80 26.Ok6.4
Values are means & SE; n = 5 animals. Autocoid tissue levels were measured in adrenal glands after an abbreviated perfusion. Left glands of 5 pairs of adrenals were perfused for 75 min with no stimulation; right glands were not perfused or stimulated.
catecholamines. Ratios of absolute amounts of catecholamines and peptides in tissuesand inperfusates. In unstimulated tissues,
there was a mean of 4.3 times more Epi than NE (Table 6), 130 times more Epi than DA, 1,500 times more Epi than Met-Enk, 33 times more NE than DA, 350 times more NE than Met-Enk, and 12 times more DA than Met-Enk. In basal perfusates the ratio of Epi to NE was almost twice that in unstimulated tissues, the ratio of NE to DA was half its value in tissues, and the ratio of NE to MetEnk was significantly less than in tissues. The ratios of Epi to DA, Epi to Met-Enk, and DA to Met-Enk in basal perfusates were not significantly different from those in tissues. These data indicate that under basal conditions Epi, DA, and Met-Enk left the tissue in preference to NE .
0.1% -
0.0%
EFFLUX
6. Mean ? SE fractional autocoid release per minute from calculated original tissue stores during basal conditions and during nicotinic and muscarinic stimulation. Epi and DA are released preferentially to NE, except during a sufficient stimulation with DMPP, when DA is released preferentially to both Epi and NE. *Significantly different from DA; tsignificantly different from Epi fractional release at same level of stimulation (P < 0.05, analysis of variance and Student’s t test). FIG.
In perfusates during DMPP stimulation, the Epi-toNE ratio was less than that in basal perfusates and very similar to that in tissues. The Epi-to-DA ratio was less during 50 PM DMPP stimulation than in basal perfusates and in tissues; the differences between paired values did not reach significance (P = 0.09 and 0.10, respectively, by Student’s t test), but the differences from mean ratios of all basal samples (n = 24) and tissues (n = 23) were significant (P = 0.05 and 0.04, respectively, by Mann-Whitney U test). The NE-to-DA ratio in perfusates during DMPP stimulation was significantly less than in tissues and was not significantly different from basal perfusates. These data indicate that DA was preferentially released from tissues during DMPP stimulation. The ratios of Epi to Met-Enk and NE to Met-Enk were less during DMPP stimulation than in tissue, suggesting there was also a preferential release of Met-Enk from tissue during nicotinic stimulation. There was no significant difference in DA-to-Met-Enk ratios during nicotinic stimulation as compared with tissue or basal perfusate. During 1 mM pilocarpine stimulation, the ratio of Epi to NE in perfusate was less than during basal conditions but not as depressed as during DMPP stimulation. The ratios of Epi to DA and NE to DA were less in perfusates stimulated with pilocarpine than in tissues, indicating that DA was preferentially released. The ratios of Epi to Met-Enk, NE to Met-Enk, and DA to Met-Enk were not significantly different in perfusates collected during pilocarpine stimulation as compared with unstimulated tissues or basal perfusates. HPLC of peptides. HPLC separation and subsequent enzymatic cleavage of the peptides in adrenal tissues revealed a number of Met-Enk peaks (Fig. 7). A consistent peak of immunoreactivity eluted at 21-22 min, which coeluted with the free Met-Enk standard. Another consistent tissue peak eluted at 36-37 min, which was near the elution times of the 35- and 34-amino acid bovine proenkephalin fragments peptide E and peptide F. The ratio of Met-Enk measured in tissue after enzymatic
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NICOTINIC-
AND
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ADRENAL
AUTOCOID
R595
RELEASE
6. Ratios of autocoids in tissues and in perfusates during basal and stimulated conditions
TABLE
Perfusates Unstimulated Tissues
Ratios
Epi/NE Epi/DA Epi/Met-Enk NE/DA NE/Met-Enk DA/Met-Enk
Basal
3 pM DMPP (7)
(24)
4.29t0.24 132.8t11.3 1,493+229 32.5k3.0 350.2t50.0 11.6t1.5
9.71*0.77* 133.9k13.1 1,963+273 14.7t1.4* 208.5&23.1* 17.0k2.4
(23) (23) (22) (23) (22) (22)
4.74k0.92t 98.4k35.2 103.0&365*t 22.0t4.8* 61.0t61.1* 4.28t2.1
50 ,uM DMPP (5) 4.53+0.89-f 78.3t17.6 874.7&148* 17.3&2.5* 211.0t46.4* 13.5k4.0
1mM Pilocarpine
(5)
6.65k1.3t 68.0*11.8*t 5,684+3,480 11.7*2.2* 639.22288.1 128klOO
(4) (4)
Values are means t SE; no. of dogs given in parentheses. Ratios are not given for 50 PM pilocarpine stimulation; releases were small, and highly variable ratios were obtained. Ratios are also not given for NPY because of variability of releases, especially under basal conditions and during stimulation with 3 PM DMPP. * Significantly different (P c 0.05, Wilcoxon or Student’s t test) from ratio in paired unstimulated tissues. t Significantly different (P < 0.05, Wilcoxon or Student’s t test) from ratio in paired basal samples. 2
n
MET-ENK
1.8 1.6
5 ti 2 ii Fm z 6 I
1.4
l
UNSTIMULATED
x
STIMULATED
ently detectable peak (5 out of 6) occurred at 21 min, which coeluted with the free Met-Enk standard. The next most consistent peak (3 out of 6) eluted at 35-37 min, similar to peaks seen in the tissue separations. The ratio of Met-Enk measured in perfusates after enzymatic cleavage to the Met-Enk measured before cleavage ranged from 1.1 to 4.7 (n = 6). HPLC separation of tissue NPY immunoreactivity revealed two peaks, which coeluted with human NPY standard and oxidized human NPY standard (Fig. 9). Perfusate samples were not separated because of their relatively low immunoreactivity.
ADRENAL
ADRENAL
Ah
1.2
1 0.8 0.6 Oxidized
0
I 10
MET-ENK
I
I 2b
DISCUSSION
I 3;
RETENTION TIME, min
7. Met-Enk immunoreactivity in adrenal tissue homogenates of 1 dog that was stimulated with 1 mM pilocarpine. Samples were passed over a high-performance liquid chromatography (HPLC) column and then treated with trypsin and carboxypeptidase B to release encrypted Met-Enk. Standards run in parallel and their retention times (min) included oxidized Met-Enk (15), Met-Enk (21), peptide F (33), and peptide E (34-35). FIG.
800
, MET-ENK
700
x
n
BASAL PERFUSATE
x
STIMULATED
PERFUSATE
Basal secretion of adrenal autocoids. The unstimulated efflux of all autocoids from the adrenals was high during the initial hour of perfusion. The initial efflux was not shown in the figures but was usually at or above the peak of stimulated efflux. After the first hour average basal efflux had decreased to
release and peptides
S. L. CHRITTON, M. K. DOUSA, T. L. YAKSH, AND G. M. TYCE Departments of Physiology and Biophysics and of Neurosurgery, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905
S. L., M. K. DOUSA, T. L. YAKSH, AND G. M. and muscarinic-evoked release of canine adrenal catecholamines and peptides. Am. J. Physiol. 260 (Regulatory Integrative Comp. Physiol. 29): R589-R599, 1991.-The tissue content and overflow of norepinephrine (NE), epinephrine (Epi), dopamine (DA), Met-enkephalin (Met-Enk), and neuropeptide Y (NPY) from isolated, retrogradely perfused dog adrenal glands were studied. Under resting conditions, -25% of the overflow of autocoids from the glands was Ca” dependent; the cholinergic antagonists hexamethonium and atropine had no effects on basal efflux. Stimulation with the nicotinic agonist l,l-dimethyl-4-phenylpiperazinium iodide (DMPP; 3 or 50 PM) or with the muscarinic agonist pilocarpine (50 PM or 1 mM) evoked releases of autocoids. These releases were blocked or dramatically reduced by appropriate antagonists or by the removal of Ca2’ from the perfusate. Expressed as percentages of tissue stores, the rank order of overflow of autocoids was E - DA >> NE during resting conditions, DA >> E - NE during stimulation with 50 PM DMPP, and DA > Epi > NE during stimulation with 1 mM pilocarpine. These data are consistent with different mechanisms of release for the catecholamines, perhaps from different cell populations. The data support corelease of peptides and catecholamines, although clear pairing of autocoids could not be confirmed. CHRITTON,
TYCE. Nicotinic-
adrenal medulla; chromaffin cells; epinephrine; norepinephrine; dopamine; methionine-enkephalin; neuropeptide Y; pilocarpine; 1,1-dimethyl-4-phenylpiperazinium iodide
Two neuropeptides, Met-enkephalin immunoreactive material (Met-Enk) and neuropeptide Y immunoreactive material (NPY), are also released by cholinergic stimuli from isolated, perfused adrenals (1, 18, 36). There is continuing controversy regarding the localization of these peptides in chromaffin cells. Although Met-Enk has been shown to be localized mainly in bovine Epicontaining cells in histological studies (17, 19), cosecretion of Met-Enk with NE as well as with Epi has been observed with perfused bovine glands and cultured bovine chromaffin cells (1, 39). NPY has been shown to be colocalized with Epicontaining granules by biochemical .techniques (2) and with Epi-containing cells by histochemistry and in situ hybridization in bovine adrenal glands (27). The release of NPY from cat adrenals has been more closely associated with Epi release than NE release (18). However, in another report, NPY immunoreactivity in bovine adrenals was found to be restricted to NE-containing cells (19) The purpose of this study was to characterize the simultaneous efflux of the autocoids Epi, NE, DA, MetEnk, and NPY from isolated dog adrenals during basal conditions and under nicotinic or muscarinic stimulation. METHODS
secretes a variety of hormones into the blood in response to acetylcholine released by splanchnic nerves. Most abundant among these hormones are epinephrine (Epi) and norepinephrine (NE), which have been shown to be stored in two different cell populations within the adrenal (5). Release of Epi and NE from the isolated adrenal occurs in different proportions with muscarinic vs. nicotinic stimulation in both the cat (6, 26) and dog (34). This suggests that the NEand Epi-containing cells respond differently to nicotinic and muscarinic agonists. Much less studied than Epi and NE is dopamine (DA), which is also released from the adrenals on stimulation (21). It has been suggested that DA has various roles as precursor, hormone, and intra-adrenal modulator of release (9,29). DA must indeed be present in NE- and Epicontaining cells as a precursor, but it appears that there is also a separate group of small granule chromaffin cells, which may contain DA as their only catecholamine (32).
THE ADRENAL
MEDULLA
0363-6119/91
$1.50 Copyright
AdrenaL perfusion. Left and right adrenals were removed from mongrel dogs of either sex. The dogs were “pool” dogs, in that different tissues were used by a number of investigators. The dogs were anesthetized with 30 mg/kg iv pentobarbital sodium and then bled by cutting both carotid arteries. The adrenals were removed within 5 min of exsanguination and placed in ice-cold modified Krebs-Ringer (KR) solution (118.3 mM NaCl, 5.7 mM KCl, 2.5 mM CaC12, 1.2 mM KH2P04, 1.2 mM MgSOd, 25 mM NaHC03, 10 mg/lOO ml sodium metabisulfite, 1.5 mg/lOO ml EDTA, 200 mg/lOO ml dextrose), which had been bubbled with 95% oxygen-5% carbon dioxide gas. In a few experiments, 50 mg/lOO ml bovine serum albumin (BSA) was present in the KR solution. This concentration did not increase values of Met-Enk or NPY when 49 initial basal perfusate samples without BSA were compared with 34 initial basal samples with BSA. One adrenal, usually the left gland because of its more amenable anatomy, was perfused. Perfusion was started within 15 min of the adrenal harvest. The adrenal
(c> 1991 the American
Physiological
Society
R589
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R590
NICOTINIC-
AND
MUSCARINIC-EVOKED
was retrogradely perfused via a cannula secured within the adrenolumbar vein (24). Slits were made in the end of each lobe of the adrenal through the capsule and cortex to allow perfusate to exit. Perfusions were carried out in a chamber with a controlled temperature of 37°C and a humidity of X0%. The KR solution was warmed and equilibrated with 95% oxygen-5% carbon dioxide gas (final pH 7.4 t 0.1) before being pumped at l-3 ml/min through the adrenal. The pump exerted a constant pressure and was adjusted to provide flow around 1.5 ml/ min; in a few experiments, a constant-flow peristaltic pump was used. Perfusate was collected in graduated tubes on ice, and an aliquot was removed and frozen for peptide analysis; 10 ~1 of 5% sodium metabisulfite were added per milliliter of the remaining perfusate, which was reserved for catecholamine analysis. After a 65-min collection of perfusate, an initial lomin basal sample was collected. The tubing leading to the adrenal was temporarily clamped off while the stimulation solution was injected through a narrow-gauge needle inserted into tubing proximal to the adrenal. The stimulation solutions were either 3 or 50 PM l,l-dimethyl-4-phenylpiperazinium iodide (DMPP), or 50 PM or 1 mM pilocarpine, and were prepared by diluting a concentrated stock with warmed, oxygenated KR solution from the perfusion reservoir just before injection. The volume injected (-3 ml) was calculated to match the previous flow through the gland. After a 2min collection while the stimulation solution was injected, the original perfusion was resumed, and an 8min poststimulation sample was collected. This was followed by a ZOmin sample and a final IO-min basal perfusion sample. Efflux of autocoids was calculated in terms of picomoles per minute. In some experiments, stimulations were done in the presence of the nicotinic antagonist hexamethonium (100 PM), the muscarinic antagonist atropine (50 PM), or in calcium-free KR solution containing 1 mM ethylene glycol-bis(@aminoethyl ether)N,N,N’,N’-tetraacetic acid (EGTA). When using calcium-free KR, additional NaCl was added to maintain isotonicity with normal KR solution. During these experiments, additional samples were taken to evaluate the effects of the KR solution containing antagonists or calcium-free KR on basal adrenal efflux. Extraction of autocoids from adrenal tissues. The unperfused glands were kept in ice-cold KR solution until they were cleaned of surrounding tissue and frozen on dry ice. The unperfused glands were frozen within 1 h of harvest. At the end of the perfusion, the perfused glands were also cleaned, frozen on dry ice, and then both glands were stored at -20°C. The glands were homogenized in 0.1 N HCl, and an aliquot was heated in a water bath at 100°C in preparation for peptide analysis. Another aliquot was rapidly deproteinized using 0.4 M perchloric acid before catecholamine analysis. Catecholamine analysis. For catecholamine extraction, 5 ml or less of perfusate was combined with 500 mg activated alumina and 1 ml of 2 M tris(hydroxymethyl)aminomethane. HCI buffer containing 5% EDTA at pH 8.6. The mixture was adjusted to pH 8.4-8.6 with Na2COg, shaken for 10 min, and then poured into 5-mm
ADRENAL
AUTOCOID
RELEASE
diameter glass columns plugged with glass wool. The alumina was washed with 5 ml distilled water, and catecholamines were eluted with 6 ml of 0.05 N perchloric acid. Quantitation of catecholamines was by high-performance liquid chromatography (HPLC). Catecholamines were measured in diluted eluates or tissue homogenates after a reversed-phase separation using a C-18 solid phase on 5-pm diameter particles (Ultrasphere, Beckman Instruments) packed in a 4.6 mm X 25 cm column. Samples were autoinjected (Waters Intelligent Sample Processor, Millipore) into a 1 ml/min flow of mobile phase through the HPLC column. The mobile phase contained 70 mM NaH2P04, 2 mM heptane sulfonate, 0.2 mM EDTA, and 5% acetonitrile at a pH of 3.4. Amperometric detection of catecholamines was accomplished using a Bioanalytical Systems detector at a potential of 0.65 V relative to a silver-silver chloride electrode. 3,4-Dihydroxybenzylamine (DHBA) was used as an internal standard. Mean recoveries of Epi, NE, DA, and DHBA standards were 82, 83, 76, and 81% (for all, SE = 2%) from the alumina; values reported have been corrected for recovery. Peptide analysis. Peptide immunoreactivity was measured by radioimmunoassay using antibodies directed at Met-Enk (antibody no. N302) and NPY (antibody no. 221) (13, 40). The limits of detection for Met-Enk and NPY were 400 t 30 and 8 t 2 (SE) fmol/min. HPLC of peptides was performed on some samples using an LKB model 2150 with a reverse-phase C-18 column (Rainin Short One). Samples were lyophilized and reconstituted in 0.6 ml of the initial mobile phase. The mobile phase was 0.1 M sodium acetate, pH 4, with acetonitrile gradients of lo-60% for Met-Enk or 25-45% for NPY. After the sample was injected and the gradient was started, I-min fractions of 0.5 ml were collected from the column and lyophilized. Met-Enk fractions separated by HPLC were enzymatically cleaved to reveal encrypted Met-Enk immunoreactivity. Fractions were incubated at 37°C for 1 h with 25 pg trypsin followed by 1 h with 0.5 pg carboxypeptidase B and then were lyophilized and assayed for Met-Enk. NPY immunoreactivity of HPLC fractions was performed with no further processing. Calibration standards of synthetic human NPY and synthetic human NPY oxidized with 3% hydrogen peroxide were run separately. Calculations and statistics. Release was measured in the lo-min period immediately after the first basal sample; 20 min after the release period a second basal sample was measured. To calculate net evoked release of an autocoid, a calculated underlying basal efflux of the autocoid was subtracted from the total release evoked by the stimulation. To model the behavior of basal efflux, unstimulated efflux was studied. Unstimulated efflux after 60 min of perfusion declined as a single exponential curve; on a plot of logarithmic values of efflux agains t the time of perfusion, the efflux descended in a straigh t line. The slope of such a line would be ln(B2) - In(B1) tB2
-
tB1
where Bl and B2 are effluxes of the autocoid into basa .l
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NICOTINIC-
AND
MUSCARINIC-EVOKED
samples before and after stimulation, respectively, and tgl and tBZ are the midpoints in time of the basal samples. The slope of the same line can also be calculated as - ln(B1)
ln(BR) tR
-
tB1
where BR is the predicted basal efflux during evoked release and tR is the midpoint in time of the evoked release. Setting the two expressions equal and solving for BR, one finds ln(B1)+
BR = e{
[ln(B2)--ln(Bl)]
[ht tB2
-
tB1
-
tBl] 1
where net release = total release - BR. One-way analysis of variance (ANOVA) and repeatedmeasures ANOVA were performed on the fractional release data. Other statistical methods used in this study were the sign test, Wilcoxon’s signed-rank test, MannWhitney U test, paired Student’s t test, and correlations to determine Pearson’s and Spearman’s correlation coefficients. Drugs. l,l-Dimethyl-4-phenylpiperazinium iodide, pilocarpine nitrate, hexamethonium chloride, atropine sulfate, trypsin, and carboxypeptidase B were obtained from Sigma Chemical (St. Louis, MO). Peptide standards were obtained from Peninsula Labs (Belmont, CA). RESULTS
Catecholamines and peptides in perfusates under basal conditions. The unstimulated efflux of each autocoid, as
examined in five unstimulated glands, displayed two phases (data not shown). During the first 60 min of perfusion, efflux was high but dropped off rapidly at a rate of -3%/min. After 60 min of perfusion, both basal efflux and its subsequent rate of decline were low; catecholamine effluxes decreased at -0.5%/min. There were no differences in the rates of decline between catecholamines and peptides during the second phase. Basal effluxes of adrenal autocoids after 60 min of perfusion were (in pmol/min t SE; n = 23) 4,600 t 560 Epi; 520 t 81 NE; 46 t 7.7 DA; 2.7 t 0.36 Met-Enk. NPY was detected in basal samples in 13 of 23 perfusions, at a level of 8.6 t 1.7 fmol/min. When glands perfused with KR containing 2.5 mM calcium were switched to calcium-free KR, basal effluxes of catecholamines tended to decrease (Table 1). However, the decreases were not significantly different from those seen in washout control experiments with no removal of calcium. When perfusion with calcium-free KR continued for 45 min after a 2-min stimulation, then a return to KR containing 2.5 mM calcium significantly increased the effluxes of Epi, NE, and DA (Table 1). These increases in Epi, NE, and DA effluxes were significantly different from those in washout control experiments. Basal effluxes of Met-Enk and NPY did not show any clearly calcium-dependent components. Catecholamines and peptides in perfusates with nicotinic stimulation. When 3 PM DMPP was infused into
the adrenal, it significantly enhanced the effluxes of Epi, NE, DA, and Met-Enk (Fig. 1). The mean net releases
ADRENAL
AUTOCOID
RELEASE
R591
evoked by 3 PM DMPP of Epi, NE, DA, Met-Enk, and NPY were 33, 57, 63, 67, and 20%, respectively, above the predicted baselines. At a concentration of 50 PM, DMPP evoked a brisk release of autocoids from the adrenal (Fig. 2). Mean net releases of Epi, NE, DA, and Met-Enk were 230, 520, 500, and 580%) respectively, above predicted baselines. The highest level of release measured for each autocoid occurred during the 2 min of actual infusion, although during the 8 min after infusion autocoids remained substantially above baseline. NPY was not consistently detected except during the lo-min release period in these experiments. Releases with 50 PM DMPP were blocked by the addition of 100 PM hexamethonium to the perfusate (Fig. 3). Introduction of hexamethonium did not affect the basal overflows of autocoids. Releases of Epi, NE, and DA with 50 PM DMPP were dependent on the presence of calcium in the medium (Table 2). Release of Met-Enk with no calcium was reduced to 4% of the mean release with calcium, which was NE (Fig. 6). Thus, during basal conditions, Epi and DA preferentially overflowed from stores in relation to NE. Met-Enk and NPY overflowed from stores at levels between NE and the other catecholamines.
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NICOTINIC-
AND
MUSCARINIC-EVOKED
ADRENAL
100,Wo
AUTOCOID
R593
RELEASE
lW,Wo
r_L,,
10,Wo
T
EPI 1
1
I
10,Wo
NE
1,~
1
EPI NE
‘,ooo
* E E ‘3i I! i DC 3
loo
1,L
1
I
I0
MET-ENK 1
1
1
E E ‘3i 2 i
*
1
1
I0 1
34 3 k
~\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\~~~
k
100
MET-ENK 1
1
W
HEXAMETHONIUM 0.1
0.1
1 0.01
I
0.01
0.001
I
60
IH
I
6ola DMPP MINUTES
I
I
100
I
0.001
I
I
60
120
N
I
I
Rho PILOCARPINE
NPY 1
I
I
loo
I
I
120
OF PERFUSION
FIG. 3. Mean k SE rate of efflux of autocoids in perfusates after introduction of 100 PM hexamethonium and with infusion of 50 PM DMPP for 2 min (n = 2). DMPP-evoked release was antagonized by hexamethonium.
FIG. 4. Mean t SE rate of efflux of autocoids in perfusates with infusion of pilocarpine (1 mM) for 2 min (n = 5). Peak release occurred during 8 min after infusion. *Levels of autocoid were significantly increased during IO-min release period (P < 0.05, Student’s t test after logarithmic transformation).
2. Calcium dependency of DMPPand pilocarpine-evoked releases
TABLE
Basal
lo-Min Release
Net Release
50 PM DMPP stimulation (n = 2, except for NPY where n = 1) No calcium + 1 mM EGTA Epi, nmol/min NE, pmol/min DA, pmol/min Met-Enk, pmol/min NPY, fmol/min
2.63 470 31 1.85 5.9
2.36 430 26.48 2.11 6.2
-0.27 -30 -5 0.26 0.3
#E
K
EPI t
t
NE -1
loo
DA
E
P
f
1
I’
v
MET-ENK I0
m
5
1 mM pilocarpine No calcium Epi, nmol/min NE, pmol/min DA, pmol/min Met-Enk, pmol/min NPY, fmol/min
stimulation (n = 1) + 1 mM EGTA 3.1 430 20 1.16 6.8
3.1 450 24 1.15 5.3
1
i
kl
-0.02 20 4 -0.01 -1.5
Perfusion was carried out for -70 min with KR containing 2.5 mM calcium, then for 35 min in KR containing no calcium and 1 mM EGTA. Glands were then stimulated for 2 min with 50 PM DMPP or 1 mM pilocarpine. Perfusion with no calcium and 1 mM EGTA continued until 45 min after stimulation. Basal efflux in calcium-free medium was calculated from logarithmic slope between samples taken 5 min before and 40 min after stimulation.
Stimulation with DMPP resulted in a different pattern: DA > Epi - NE (Fig. 6). Epi did not increase as much as the other autocoids, so that DA was preferentially released from stores in relation to Epi and NE. The peptides increased, but their variability did not allow them to be differentiated from the catecholamines. During stimulation with 1 mM pilocarpine the preferential fractional overflow of DA over NE seen during basal efflux remained (Fig. 6). The fractional efflux of
0.1
0.001
1
60
I
I
60 PILOCARPINE MINUTES
I
1
loo
I
I
120
OF PERFUSION
FIG. 5. Mean t SE rate of efflux of autocoids in perfusates after introduction of 50 PM atropine and with infusion of 1 mM pilocarpine for 2 min (n = 2). Pilocarpine-evoked release was antagonized by atropine.
Epi was lower than that of DA but did not fall to the level of NE as seen with DMPP stimulation, even though releases of catecholamines with 1 mM pilocarpine were larger than those evoked by 50 PM DMPP. The peptides, Met-Enk and NPY, increased along with the catecholamines, but their variability did not allow them to be differentiated from the fractional releases of individual
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R594
NICOTINIC-
AND
MUSCARINIC-EVOKED
ADRENAL 0.5%
3. Tissue concentrations of catecholamines, Met-Enk, and NPY with nicotinic stimulation TABLE
Unperfused Unstimulated EPi
wwg
NE, wWg DA, nmol/g Met-Enk, nmol/g NPY, pmol/g
and
Stimulated With 50 ,uM DMPP
1.99kO.50 55.35t10.84 3.31kO.68
12.5t4.9
Met-Enk,
and NPY with muscarinic stimulation Unperfused Unstimulated
EPi,
wok
NE, wok DA, nmol/g Met-Enk, nmol/g NPY, pmol/g
and With
5.50-r-0.63 1.21t0.12 51.98t4.83
Stimulated Pilocarpine
4.09kO.43 0.89kO.06" 74.91k6.50
6.32t1.15
4.91kO.88
22.4t4.4
16.0&4.2*
Values are means t SE; n = 6 dogs. One of a pair right glands, 1 left gland) was not perfused or stimulated. was perfused for 125-260 min and stimulated l-4 times pine. In correlation studies of these glands, it was found number of stimulations or a longer time of perfusion was with greater depletion of adrenal autocoids. * Concentration cantly different in stimulated gland as compared with fused gland (P < 0.05, Wilcoxon and Student’s t test).
of adrenals (5 Other gland with pilocarthat a larger not associated is signifipaired unper-
5. Tissue concentrations of catecholamines, Met-Enk, and NPY with perfusion but no stimulation TABLE
Unperfused Unstimulated W,
wol/g
NE, wok DA, nmol/g Met-Enk, nmol/g NPY, pmol/g
8 8
Right gland of a pair of adrenals gland was perfused for 195-205 PM DMPP. * Concentration is gland as compared with paired t test).
4. Tissue concentrations of catecholamines,
TABLE
and
7.24kO.75 1.55kO.11 61.92t5.97 5.78-e0.28 13.2k2.3
-
RELEASE
gg EPINEPHRINE Ed NOREPINEPHRINE DOPAMINE MET-ENKEPHALIN cl NEUROPEPTIDE
Y
8.9Ok 1.85 1.89k0.36 83.5Ok10.67" 3.84k1.34 16.4k1.7
10.10cL95
Values are means t SE; n = 5 dogs. was not perfused or stimulated. Left lnin and stimulated 2 times with 50 significantly different in stimulated unperfused gland (P < 0.001, Student’s
AUTOCOID
Perfused and Unstimulated
6.90t0.82 1.62k0.12 81.78t8.26 6.16kO.80 26.Ok6.4
Values are means & SE; n = 5 animals. Autocoid tissue levels were measured in adrenal glands after an abbreviated perfusion. Left glands of 5 pairs of adrenals were perfused for 75 min with no stimulation; right glands were not perfused or stimulated.
catecholamines. Ratios of absolute amounts of catecholamines and peptides in tissuesand inperfusates. In unstimulated tissues,
there was a mean of 4.3 times more Epi than NE (Table 6), 130 times more Epi than DA, 1,500 times more Epi than Met-Enk, 33 times more NE than DA, 350 times more NE than Met-Enk, and 12 times more DA than Met-Enk. In basal perfusates the ratio of Epi to NE was almost twice that in unstimulated tissues, the ratio of NE to DA was half its value in tissues, and the ratio of NE to MetEnk was significantly less than in tissues. The ratios of Epi to DA, Epi to Met-Enk, and DA to Met-Enk in basal perfusates were not significantly different from those in tissues. These data indicate that under basal conditions Epi, DA, and Met-Enk left the tissue in preference to NE .
0.1% -
0.0%
EFFLUX
6. Mean ? SE fractional autocoid release per minute from calculated original tissue stores during basal conditions and during nicotinic and muscarinic stimulation. Epi and DA are released preferentially to NE, except during a sufficient stimulation with DMPP, when DA is released preferentially to both Epi and NE. *Significantly different from DA; tsignificantly different from Epi fractional release at same level of stimulation (P < 0.05, analysis of variance and Student’s t test). FIG.
In perfusates during DMPP stimulation, the Epi-toNE ratio was less than that in basal perfusates and very similar to that in tissues. The Epi-to-DA ratio was less during 50 PM DMPP stimulation than in basal perfusates and in tissues; the differences between paired values did not reach significance (P = 0.09 and 0.10, respectively, by Student’s t test), but the differences from mean ratios of all basal samples (n = 24) and tissues (n = 23) were significant (P = 0.05 and 0.04, respectively, by Mann-Whitney U test). The NE-to-DA ratio in perfusates during DMPP stimulation was significantly less than in tissues and was not significantly different from basal perfusates. These data indicate that DA was preferentially released from tissues during DMPP stimulation. The ratios of Epi to Met-Enk and NE to Met-Enk were less during DMPP stimulation than in tissue, suggesting there was also a preferential release of Met-Enk from tissue during nicotinic stimulation. There was no significant difference in DA-to-Met-Enk ratios during nicotinic stimulation as compared with tissue or basal perfusate. During 1 mM pilocarpine stimulation, the ratio of Epi to NE in perfusate was less than during basal conditions but not as depressed as during DMPP stimulation. The ratios of Epi to DA and NE to DA were less in perfusates stimulated with pilocarpine than in tissues, indicating that DA was preferentially released. The ratios of Epi to Met-Enk, NE to Met-Enk, and DA to Met-Enk were not significantly different in perfusates collected during pilocarpine stimulation as compared with unstimulated tissues or basal perfusates. HPLC of peptides. HPLC separation and subsequent enzymatic cleavage of the peptides in adrenal tissues revealed a number of Met-Enk peaks (Fig. 7). A consistent peak of immunoreactivity eluted at 21-22 min, which coeluted with the free Met-Enk standard. Another consistent tissue peak eluted at 36-37 min, which was near the elution times of the 35- and 34-amino acid bovine proenkephalin fragments peptide E and peptide F. The ratio of Met-Enk measured in tissue after enzymatic
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NICOTINIC-
AND
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ADRENAL
AUTOCOID
R595
RELEASE
6. Ratios of autocoids in tissues and in perfusates during basal and stimulated conditions
TABLE
Perfusates Unstimulated Tissues
Ratios
Epi/NE Epi/DA Epi/Met-Enk NE/DA NE/Met-Enk DA/Met-Enk
Basal
3 pM DMPP (7)
(24)
4.29t0.24 132.8t11.3 1,493+229 32.5k3.0 350.2t50.0 11.6t1.5
9.71*0.77* 133.9k13.1 1,963+273 14.7t1.4* 208.5&23.1* 17.0k2.4
(23) (23) (22) (23) (22) (22)
4.74k0.92t 98.4k35.2 103.0&365*t 22.0t4.8* 61.0t61.1* 4.28t2.1
50 ,uM DMPP (5) 4.53+0.89-f 78.3t17.6 874.7&148* 17.3&2.5* 211.0t46.4* 13.5k4.0
1mM Pilocarpine
(5)
6.65k1.3t 68.0*11.8*t 5,684+3,480 11.7*2.2* 639.22288.1 128klOO
(4) (4)
Values are means t SE; no. of dogs given in parentheses. Ratios are not given for 50 PM pilocarpine stimulation; releases were small, and highly variable ratios were obtained. Ratios are also not given for NPY because of variability of releases, especially under basal conditions and during stimulation with 3 PM DMPP. * Significantly different (P c 0.05, Wilcoxon or Student’s t test) from ratio in paired unstimulated tissues. t Significantly different (P < 0.05, Wilcoxon or Student’s t test) from ratio in paired basal samples. 2
n
MET-ENK
1.8 1.6
5 ti 2 ii Fm z 6 I
1.4
l
UNSTIMULATED
x
STIMULATED
ently detectable peak (5 out of 6) occurred at 21 min, which coeluted with the free Met-Enk standard. The next most consistent peak (3 out of 6) eluted at 35-37 min, similar to peaks seen in the tissue separations. The ratio of Met-Enk measured in perfusates after enzymatic cleavage to the Met-Enk measured before cleavage ranged from 1.1 to 4.7 (n = 6). HPLC separation of tissue NPY immunoreactivity revealed two peaks, which coeluted with human NPY standard and oxidized human NPY standard (Fig. 9). Perfusate samples were not separated because of their relatively low immunoreactivity.
ADRENAL
ADRENAL
Ah
1.2
1 0.8 0.6 Oxidized
0
I 10
MET-ENK
I
I 2b
DISCUSSION
I 3;
RETENTION TIME, min
7. Met-Enk immunoreactivity in adrenal tissue homogenates of 1 dog that was stimulated with 1 mM pilocarpine. Samples were passed over a high-performance liquid chromatography (HPLC) column and then treated with trypsin and carboxypeptidase B to release encrypted Met-Enk. Standards run in parallel and their retention times (min) included oxidized Met-Enk (15), Met-Enk (21), peptide F (33), and peptide E (34-35). FIG.
800
, MET-ENK
700
x
n
BASAL PERFUSATE
x
STIMULATED
PERFUSATE
Basal secretion of adrenal autocoids. The unstimulated efflux of all autocoids from the adrenals was high during the initial hour of perfusion. The initial efflux was not shown in the figures but was usually at or above the peak of stimulated efflux. After the first hour average basal efflux had decreased to
