- Email: [email protected]
Non-adrenergic sympathetic vasoconstriction in the eye and some other facial tissues in the rabbit
European Journal of Pharmacology, 175 (1990) 175-186
175
Elsevier EJP 51124
Non-adrenergic sympathetic vasoconstriction in the eye and some other facial tissues in the rabbit E l i s a b e t G r a n s t a m a n d Siv F.E. N i l s s o n Department of Physiology and Medical Biophysics, University of Uppsala, Uppsala, Sweden Received 26 June 1989, revised MS received 12 September 1989, accepted 24 October 1989
The effects of unilateral sympathetic nerve stimulation (SNS) on regional blood flow in the rabbit were studied with radioactive microspheres. SNS at 10 or 4 Hz caused an approximately 60% reduction in choroidal blood flow, which was partly resistant to a-adrenoceptor blockade with phenoxybenzamine. The vasoconstriction evoked by SNS at 2 Hz was completely abolished by a-adrenoceptor blockade. A similar response was seen in the iris, ciliary body, masseter muscle and lacrimal gland. In the harderian gland, however, SNS (2 Hz) after a-adrenoceptor blockade caused a significant reduction in blood flow. In the salivary glands, combined /3- and a-adrenoceptor blockade with propranolol and phenoxybenzamine revealed a slight non-adrenergic vasoconstriction during SNS at 10 Hz; however, the blood flow was significantly increased during SNS at 4 and 2 Hz following a-adrenoceptor blockade. These results indicate that there is a frequency-dependent, non-adrenergic component in the sympathetic vasoconstriction of the eye and several facial tissues. In the salivary glands, /3-adrenoceptor-mediated vasodilatation tends to mask a non-adrenergic vasoconstriction. a-Adrenoceptor blockade; fl-Adrenoceptor blockade; Eye; Neuropeptide Y; Sympathetic nerve stimulation; Vasoconstriction
1. Introduction The intraocular tissues are supplied with nutrients from two different vascular beds, the retinal and the uveal blood vessels. The retinal blood vessels are not innervated and the retinal blood flow is autoregulated in a similar way as the cerebral blood flow. In the rabbit, the uveal blood vessels, which comprise the vessels of the choroid, the iris and the ciliary body, show hardly any autoregulation. However, these vessels receive a rich innervation from the autonomic nervous system (see Bill, 1984). The facial nerve and the oculomotor nerve both contribute to the parasympathetic innervation of
Correspondence to: E. Granstam, Department of Physiology and Medical Biophysics, Box 572, S-751 23 Uppsala, Sweden.
the uveal blood vessels. The sympathetic innervation is supplied from the superior cervical ganglion. Stimulation of the oculomotor nerve is known to cause vasoconstriction, which is partly cholinergic, in the iris and vasoconstriction/ vasodilatation in other parts of the uvea, depending on the species studied (see Bill, 1984). Stimulation of the facial nerve causes vasodilatation of uveal vessels. This dilatation seems to be mediated mainly by vasoactive intestinal polypeptide (VIP) and possibly by peptide histidine isoleucine (PHI) (see Nilsson, 1986). Although it is well-documented that sympathetic stimulation causes vasoconstriction in the uvea (Bill, 1962; Alm and Bill, 1973; Alto, 1977), less is known about the extent to which the vasoconstriction is affected by a-adrenoceptor blockade and the possible involvement of neuropeptides.
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
176 The aim of the present study was to evaluate the extent to which the sympathetically induced vasomotor response in the eye and some other facial tissues is resistant to c~- and fl-adrenoceptor blockade and to investigate the effect of different stimulation frequencies. Preliminary reports of part of this investigation have been given elsewhere (Nilsson, 1988; Granstam and Nilsson, 1989). 2. Materials and methods
All experiments were performed on New Zealand White rabbits of either sex, weighing 1.9-3.5 kg. Regional blood flows were determined with the radioactively labelled microsphere method.
2.1. Anaesthesia and general operative procedures All animals were anaesthetized with urethane ( - 1.75 m g / k g bw) infused into a marginal ear vein. Small additional doses were given if required. The animals were tracheostomized and artificially ventilated. Both femoral veins were cannulated with polyethylene tubing for the infusion of drugs. The femoral arteries were cannulated bilaterally for continous blood pressure recording and for the collection of blood samples. A polyethylene tube, with the tip bent at a 120 degree angle, was placed into the left heart ventricle via a brachial artery for the injection of microspheres. Heparin (Heparin ®, KabiVitrum, Stockholm, Sweden), 1000 I E / k g bw, was given i.v. before the experiments. Muscle relaxation was induced by the i.v. administration of tubocurarine (Tubocuran "~, Nordisk Droge, Copenhagen, Denmark) (0.5-1.0 m g / k g bw). Arterial blood samples were collected before and during the experiments to determine the acid-base balance. The blood sampies were analysed in an ABL 300 acid-base analyser (Radiometer, Copenhagen, Denmark). Disturbances of the balance were corrected by administering sodium bicarbonate a n d / o r changing the ventilation rate.
2.2. Drugs c~-Adrenoceptor blockade was induced by the administration of phenoxybenzamine (Dibe-
nyline "~, S K & F , Welwyn Garden City, England) 50 m g / k g bw, which was slowly infused i.v. The arterial blood pressure fell during drug administration but was partly restored by the i.v. infusion of 30-40 ml of a mixture (1:1) of Macrodex (Pharmacia, Uppsala, Sweden) and saline or Rehydrex (Pharmacia, Uppsala, Sweden). Blood flow was determined 15-30 rain after the administration of phenoxybenzamine. Phenoxybenzamine is a non-specific blocking agent at the dose used, blocking not only cq- and c~2-adrenoceptors but also the effects of histamine, serotonin and acetylcholine. This high dose of phenoxybenzamine was chosen because it was used in a previous study on the effects of sympathetic nerve stimulation on uveal blood flow (Bill, 1962). However, the interference of phenoxybenzamine with receptors other than a-adrenoceptors does not affect the interpretation of the present results since the aim of the study was to determine the possible existence of a peptidergic component in the sympathetic vasoconstriction. fl-Adrenoceptor blockade was achieved by the i.v. administration of propranolol (Inderal ®, ICIPharma, Macclesfield, England) (2 m g / k g bw). At least 15 min elapsed before the blood flow was determined.
2.3. Stimulation of the cervical sympathetic nerve The cervical sympathetic nerves were sectioned bilaterally and stimulated unilaterally on the left or right side. In the first two series of experiments, a bipolar platinum electrode attached to a Digitimer Stimulator DS9A (Digitimer Ltd., Welwyn Garden City, England) was used for stimulation. The electrode tips were insulated with a small plastic holder, which also made it possible to anchor the electrode to the adjacent tissues. After the electrode had been attached to the nerve, the rabbit was placed in a prone position. In the third and fourth series of experiments the rabbits were placed in a supine position and stimulated with a bipolar silver electrode connected to the Digitimer Stimulator. Care was taken to isolate the electrode from adjacent structures. In these experiments the nerve was kept moist with paraffin. The stimulation intensity (5-8 V) was adjusted to give an immediate and marked dilatation of the pupil.
177 The pulse lasted 1-2 ms and the stimulation period was 3 or 4 min. Blood flow was measured 2 rain after the start of nerve stimulation.
2.4. Determination of regional blood flows with radioactive microspheres Regional blood flows were determined with 15 /~m radioactive microspheres (NEN, Boston, MA, USA), according to the reference flow method (see Alm and Bill, 1972; Hillerdal et al., 1987). Spheres labeled with three different radionuclides, 141Ce, 113Sn and l°3Ru, were used. This made it possible to make three blood flow determinations in each animal. Between 1 0 6 and 2 • 1 0 6 microspheres were injected each time. The spheres were normally injected over 10-15 s and blood sampling was started at the same time and continued to 1 rain. If the mean arterial blood pressure was very low, the spheres were injected over 20-30 s and the sampling time was extended to 2 min. The animals were killed after the experiments by an intracardiac or i.v. injection of a KC1 solution. Both eyes were enucleated and the retina, choroid, iris and the ciliary body were dissected. Samples were also taken from other tissues innervated by the cervical sympathetic nerve and from some peripheral tissues. The blood samples and the tissue samples were then weighed and analysed in a three-channel gamma-spectrometer.
2.5. Experimental protocols (A) In the first series of experiments, the blood flow was determined during sympathetic nerve stimulation at 10 Hz, after a-adrenoceptor blockade but without stimulation and after a-adrenoceptor blockade during stimulation at 10 Hz (n = 6). (B) In the second series of experiments, the blood flow was determined during sympathetic nerve stimulation at 10 Hz; without blockade, after /3-adrenoceptor blockade and then after /3and a-adrenoceptor blockade (n = 8). (C) In the third series of experiments, the blood flow was determined during sympathetic nerve stimulation at 4 Hz before a-adrenoceptor blockade and after a-adrenoceptor blockade during sympathetic nerve stimulation at 4 and 10 Hz (n = 7).
TABLE 1 Blood pressure (ram Hg) during blood flow measurements in the four different experimental series (means-4-S.E.). 1st blood flow 2nd blood flow 3rd blood flow measurement measurement measurement SeriesA Series B Series C Series D
65_+8 73_+5 74 ___7 69 4-3
47+2 61+3 43 __+3 39 + 2
46+2 34+3 43 + 3 37 _+2
(D) In the fourth series of experiments, the local blood flows were determined during sympathetic nerve stimulation at 2 Hz and again after a-adrenoceptor blockade during sympathetic nerve stimulation at 2 and 4 Hz (n = 10).
2.6. Statistical analysis It is difficult to completely separate the vitreous body from the intraocular tissues, which results in an over-estimation of the weight of these tissues. To avoid this problem, the blood flow in the choroid, iris and ciliary body was calculated as the total blood flow per tissue and expressed as m g / m i n . For the other tissues, the blood flow was related to tissue weight ( g / m i n × g). In the rabbit, changes in arterial blood pressure greatly affect uveal blood flow, since this blood flow is almost without autoregulation. In the present experiments, the mean arterial blood pressure fell after a-adrenoceptor blockade and after combined/3- and a-adrenoceptor blockade (table 1) in spite of rehydration. The effects of sympathetic nerve stimulation were therefore evaluated by comparing the stimulated side and the control side. Statistical analysis was performed with the two-tailed Student's t-test for paired data. P values less than 0.05 were considered significant. All values are given as means + S.E.
3. Results
3.1. Effect of sympathetic nerve stimulation before and after a- and/3-adrenoceptor blockade Sympathetic nerve stimulation at 10 Hz markedly decreased the blood flow in the eye and
178 TABLE 2
TABLE 3
Absolute blood flow rates in ocular tissues during stimulation at 10 Hz, control and stimulated sides, respectively. Blood flows are given as m g / m i n , means + S.E.
Absolute blood flow rates in facial tissues during stimulation at 10 Hz, control and stimulated sides, respectively. Blood flows are given as g / m i n × g, means +- S.E.
Tissue
Stimulated side
Control side
Difference
Tissue
Stimulated side
Control side
Difference
Choroid Iris Ciliary body
340+_85 14+_ 4 27+ 6
862+_144 100+- 20 107+_ 20
-522+-85 a -86+-19 a -80+_15 a
Parotid gland Submandibular gland Harderian gland Lacrimal gland Masseter muscle
0.01 +-0.00
0.41 +-0.10
-0.40+_0.10 c
0.02+_0.01 0.07_+0.02 0.06+_0.05 0.04+_0.02
0.84+_0.17 0.65+0.18 0.36+_0.07 0.56+__0.01
-0.82_+0.17 -0.58+_0.17 -0.30+_0.05 -0.52_+0.08
P ~<0.01.
a p ~< 0.05, b p ~<0.01, c p ~< 0.001.
several other facial tissues. The absolute rates of blood flow for control and stimulated side in various tissues during stimulation at 10 Hz are given in tables 2 and 3. Sympathetic nerve stimulation at 10 Hz decreased the blood flow in the choroid by 62 + 5% (P < 0.001; n = 6) compared to the non-stimulated control side. After a-adrenoceptor blockade, sympathetic nerve stimulation caused a 43 + 7% (P < 0.01; n = 6) reduction of choroidal blood flow (fig. 1). The other parts of the uvea, the iris and the ciliary body, responded in a similar way. The corresponding values for the iris and the ciliary body were 86 +_ 4% (P < 0.001; n = 6) and 76 +_ 4% (P < 0.001; n = 6) before the blockade and 60 +_ 10% (P < 0.01; n = 6) and 45 +_ 9% (P < 0.01; n = Choroid
1oo
Masseter
h a ~ b
6), respectively, after the blockade. In these tissues, the control microsphere injection following a-adrenoceptor blockade showed that there was an equal blood flow on both the control and experimental sides. Sympathetic nerve stimulation after /3-adrenoceptor blockade caused a somewhat greater decrease in choroidal blood flow on the stimulated side compared to the control side than during stimulation at 10 Hz without any blockade (69 _+ 7 versus 60 + 8, P < 0.05; n = 8) (fig. 2). However, the effect of sympathetic nerve stimulation on choroidal blood flow after combined /3- and c~m
Lacrimal
gl
Parotid
gl
80 ~= 60 O
~-
40
o 20 _o .Q 0
-= -2o
& -4oI e-
-8o!
'*
m
-x
-100 -120
[] [] []
lO Hz After alpha-adrenoceptor blockade 10 Hz after alpha-adrenoceptor blockade
Fig. 1. Effects of sympathetic nerve stimulation at 10 Hz on blood flow in the choroid, masseter muscle, lacrimal and harderian glands. Local blood flow was determined during stimulation at 10 Hz, without stimulation after a-adrenoceptor blockade and during stimulation at 10 Hz after the blockade. Blood flow values are given as percentage change from the non-stimulated control side. Mean values and S.E. are given (n = 6). * P ~<0.05; * * P ~< 0.01 and * * * P ~< 0.001.
179 Choroid
1O0
Masseter i
m
Lacrimal
gl
i
Parotid
gl
J
80 60 --
"el
o
o
e.,
40 20 0
E
-2o
¢~
-40
m
-60 -80
o
ell 'E,
-I O0 -120
[]
10Hz
[] []
10 Hz after beta-adrenoceptor blockade 10 Hz after alpha- and beta-adrenoceptor blockade
Fig. 2. Effects of sympathetic nerve stimulation at 10 Hz; without blockade, during fl-adrenoceptor blockade and during combined fl- and a-adrenoceptor blockade. Blood flow values are given as percentage change from the non-stimulated control side. Mean values and S.E. are given ( n = 8 except for the masseter muscle and lacrimal gland, where n = 7 ) . * P ~ O . 0 5 ; ** P~<0.01; • ** P ~< 0.001.
adrenoceptor blockade was not different from that after a-adrenoceptor blockade alone (compare figs. I and 2). Blood flow in the masseter muscle and in the lacrimal and harderian glands decreased by 95 _+ 2, 88_+8 and 8 8 + 2 % ( P < 0 . 0 0 1 ; n = 6 ) , respectively, during sympathetic nerve stimulation at 10 Hz (fig. 1). There was a significant decrease in blood flow during sympathetic nerve stimulation at 10 Hz after a-adrenoceptor blockade in the masseter muscle (60 _+ 9%, P < 0.001; n = 6) (fig. 1) and in the harderian gland (57 + 3%, P < 0.001; n = 6) but the change in the lacrimal gland was not statistically significant (fig. 1). The control injection of microspheres following a-adrenoceptor blockade showed a comparable blood flow on control and experimental sides in the glandular tissues (fig. 1). In the masseter msucle, however, the blood flow was significantly reduced on the experimental side compared to the control side even though the muscle had not been stimulated (15 _+ 2%, P < 0.001; n = 6) (fig. 1). The responses of the masseter muscle (fig. 2) and harderian gland to sympathetic nerve stimulation after combined/3- and a-adrenoceptor blockade were not different from those following aadrenoceptor blockade alone. In this series of experiments the combined blockade revealed a
32 _+ 9% (P < 0.05; n -- 7) reduction in blood flow in the lacrimal gland during sympathetic nerve stimulation at 10 Hz (fig. 2). Sympathetic nerve stimulation at 10 Hz markedly decreased the blood flow to the salivary glands: a 96 + 2% (P < 0.001; n = 6) decrease in the parotid gland (fig. 1) and a 97 + 1% (P < 0.001; n = 6) decrease in the submandibular gland. This effect was completely abolished in the submandibular gland by a-adrenoceptor blockade. The blood flow in the parotid gland increased but not significantly during sympathetic nerve stimulation at 10 Hz after a-adrenoceptor blockade (41 _+ 29%; n = 6) (fig. 1). After combined /3- and a-adrenoceptor blockade, the blood flow in the parotid gland decreased by 32 + 7% (P < 0.01; n = 8) (fig. 2). A slight but not statistically significant decrease in blood flow was seen in the submandibular gland after combined /3- and c~-adrenoceptor blockade.
3.2. Effects of sympathetic nerve stimulation at different frequencies before and after a-adrenoceptor blockade The effects of sympathetic nerve stimulation at 4 Hz on uveal blood flow were similar to those at 10 Hz: choroidal blood flow was reduced by 58 + 8% ( P < 0 . 0 0 ] ; n = 7 ) (fig. 3). In the iris and
180
Choroid
500
Masseter
m
Lacrimal
gl i
Parotid
gl
400 0
3OO
o o .el "
200 1O0 o
'~
-100 -200
[] [] []
4 Hz 4 Hz after alpha-adrenoceptor blockade 10 Hz after alpha-adrenoceptor blockade
Fig. 3. Effects of sympathetic nerve stimulation at 4 Hz before a-adrenoceptor blockade and at 4 and 10 Hz after et-adrenoceptor blockade. Blood flow values are given as percentage change from the non-stimulated control side. Mean values and S.E. are given ( n = 7 ) . * P~<0.05;** ~<0.01; * * * P~<0.001.
ciliary body, the blood flow was reduced by 82 +_ 4% ( P < 0 . 0 0 1 ; n = 7 ) and 81_+5% ( P < 0 . 0 0 1 ; n = 7) respectively. Sympathetic nerve stimulation at 2 Hz caused a smaller reduction in choroidal blood flow (41 ± 8%, P <0.001; n = 10) (fig. 4) and a significant reduction in irideal and ciliary blood flows. After phenoxybenzamine administration, sympathetic nerve stimulation at 4 Hz de-
500 ~g 0
400
,
Choroid
|
Maaseter
creased the local blood flow in the iris by 24 ± 8% (P < 0.05; n = 17) and in the ciliary body by 30 _+ 8% (P < 0.01; n = 17) while the choroidal blood flow did not decrease significantly (fig. 5). Sympathetic nerve stimulation at 2 Hz after a-adrenoceptor blockade did not cause a statistically significant change in blood flow in any part of the uvea (fig. 5).
m
a
Lacrimal
gl
i
Parotid
gl
r
300 0 _o
7-
2OO
C OI
1O0
C
J=
0
U ,k~*
d,
-1 O0 -200
[] [] []
2 Hz 2 Hz after alpha-adrenoceptor blockade 4 Hz after alpha-adrenoceptor blockade
Fig. 4. Effects of sympathetic nerve stimulation at 2 Hz before and after a-adrenoceptor blockade and at 4 Hz after the blockade. Blood flow values are given as percentage change from the non-stimulated control side. Mean values and S.E. are given (n = 10). * P < 0 . 0 5 ; * * P~<0.01;*** P~<0,001.
181
20 (n=lO) ~
Choroid
0
o
"o
8
-20
,o
-40
(n=lO)
•
(n=13)
0
-60
m ,,C
(n=14) (n=7)
-80 - 1 O0
I
I
I
2
4
10
2O Iris 0 o
_g
-20
¢
-40
Q 0 'dll ,¢ 0
-60
(n=lO)
,o
(n=l O) (n=l 3)
-8o
(n=14) (n=7)
,,,
I
I
I
2
4
10
- 1 O0
2O
Ciliary , 0 0 x~
8
(n=lO) -20 •
-~
-40 • ¢a l
body
, (n=lO)
-60
(n=13)
o
(n=14)
-8o
I
~
'
e**
(n=7) - 1 O0
I
t
I
2
4
10 Frequency (Hz)
Fig. 5. Effects of sympathetic nerve stimulation at different frequencies on blood flow in the choroid, iris and ciliary body with and without a-adrenoceptor blockade. Blood flow values are given as percentage change from the non-stimulated control side. Mean values and S.E. are given. The data presented are the summarized results of experimental series A-D. Squares represent changes in blood flow before a-adrenoceptor blockade. Diamonds represent changes in blood flow after a-adrenoceptor blockade. * P ~< 0.05; • * P ~ < 0 . 0 1 ; * * * P~<0.001.
182
20,
O:
Masseter muscle
(n=lO) ,
-2Ol
-~
-40:
-6o! -80
,,,
j.
(n=l O) ~,,,.~
-"'--o
-100 -120
**,
(n=l 3)
*** •I"1 (n=13)
(n=7)
I
I
I
2
4
10
20 0
Lacrimal
gland
(n=lO)
-20 .~ C
-40
***
®
-60
(n=lO) ~ * * *
o a ,1= 0
(n=17)
.~
-80
(n=13)
(n=13)
-loa
(n=7)
-120
I
I
I
2
4
10
2O Harderlan gland
0 -2o .~
(n=l O)
***
-40
!
(n=17) (n=l O) ~
.~
,
-80
(n=13)
0
(n=13)
(n=7)
-100 -120
"~
I
I
2
4
I
Frequency (Hz)
10
Fig. 6. Effects of sympathetic nerve stimulation at different frequencies on blood flow in the masseter muscle, lacrimal and harderian glands with and without a-adrenoceptor blockade. Blood flow values are given as percentage change from the non-stimulated control side. Mean values and S.E. are given. The data presented are the summarized results of experimental series A-D. Squares represent changes in blood flow before a-adrenoceptor blockade. D i a m o n d s represent changes in blood flow after a-adrenoceptor blockade. * P ~< 0.05; * * P ~< 0.01; * * * P ~< 0.001.
183
In the masseter muscle, sympathetic nerve stimulation at 4 Hz caused a 97 + 1% (P < 0.001; n = 7) (fig. 3) decrease in blood flow compared to the control side while sympathetic nerve stimulation at 2 Hz decreased the blood flow by 82 _ 3% (P < 0.001; n = 10) (fig. 4). After phenoxybenzamine treatment, sympathetic nerve stimulation at 4 Hz reduced the blood flow by 29 _+ 8% (P < 0.01; n = 17) (fig. 6) whereas no reduction was observed at 2 Hz. A similar response was seen in the lacrimal gland (fig. 6). In the harderian gland, however, stimulation at 2 Hz after a-adrenoceptor blockade reduced the blood flow by 30 + 5% (P < 0.001; n = 10) (fig. 6). 480 420 360 0
300
~
240
"R
180
!
120
t-
60
Q OI
(n=l O)
Sympathetic nerve stimulation at 4 and 2 Hz markedly reduced the blood flow in the salivary glands (figs. 3 and 4). Sympathetic nerve stimulation at 4 Hz after c~-adrenoceptor blockade increased the blood flow on the stimulated side compared to the control side. In the parotid gland, the increase was 258 _+ 60% (P < 0.001; n = 17) (fig. 7) and in the submandibular gland 56 _+ 8% (P < 0.001; n = 17) (fig. 7). Stimulation at 2 Hz following a-adrenoceptor blockade also resulted in a marked increase in blood flow: 337 _+ 81% (P < 0.01; n = 10) (fig. 7) in the parotid gland and 63_+ 10% (P <0.001; n = 9) (fig. 7) in the submandibular gland.
~
tid g l a n d
(n=l 7) (n=13)
¢11
•== o
0 -60
(n=l O) I ~ ' - - - . - - . _ ~ * _ * * (n=7) t
-120
*,"I* * i
2
i
Y
4
(n=14)
10
480 420 S u b m a n d i b u l a r gland
360 o r-
0 _o .D t-
300 24O 180 120
*lr*
=
60
(n=g) =t
o
0
Q
-60
(n=17) ***
(n:9)
~
1
(n=l 3) *t*
(n:7)
(n:14)
-120 2
4
10 F r e q u e n c y (Hz)
Fig. 7. Effects of sympathetic nerve stimulation at different frequencies on blood flow in the parotid and submandibular glands with and without a-adrenoceptor blockade. Blood flow values are given as percentage change from the non-stimulated control side. Mean values and S.E. are given. The data presented are the summarized results of experimental series A-D. Squares represent changes in blood flow before a-adrenoceptor blockade. Diamonds represent changes in blood flow after a-adrenoceptor blockade. * P ~< 0.05; • * P~<0.01; * * * P~< 0.001.
184
The completeness of the a-adrenoceptor blockade was tested in two different ways. An i.v. bolus injection of noradrenaline, given at the end of some of the experiments with a-adrenoceptor blockade, decreased the arterial blood pressure, indicating that there was a complete a-adrenoceptor blockade. Sympathetic nerve stimulation after a-adrenoceptor blockade did not affect the size of the pupils, also indicating an effective blockade. However, a slight increase in pupillary diameter was observed when the sympathetic nerve was stimulated after a combined a- and /3-adrenoceptor blockade.
4. Discussion
Several different explanations have been suggested for the inability of adrenoceptor blocking agents to completely abolish the effects of sympathetic nerve stimulation. It has been suggested that noradrenaline might be released from nerve endings in such large amounts that the antagonists fail to achieve a competetive blockade of the adrenoceptors (Bevan, 1969). Another explanation is that anti-adrenergic drugs block presynaptic aa-adrenoceptors , which results in an enhanced stimulation-evoked release of noradrenaline (Schoups et al., 1988). This could be sufficient to compensate for the blockade of a-adrenoceptors. It has also been implied that noradrenaline from vascular nerves might act through receptors other than a-adrenoceptors (Hirst and Neild, 1980). During the last few years it has become evident that the perivascular sympathetic nerve fibers in most tissues contain neuropeptide Y (NPY) in addition to noradrenaline (for references see Wahlestedt, 1987; Pernow, 1988). NPY, originally isolated from the porcine brain, consists of 36 amino acids (Tatemoto, 1982) and is a potent hypertensive agent, causing marked vasoconstriction in several tissues (Lundberg and Tatemoto, 1982; Lundberg et al., 1985; Rudehill et al., 1987). NPY is released from sympathetic nerves upon sympathetic nerve stimulation (Lundberg et al., 1986) and has been shown to cause adrenoceptor blockade-resistant vasoconstriction (Lundberg and
Tatemoto, 1982). This makes it a likely mediator of the a-adrenoceptor blockade-resistant part of sympathetic vasoconstriction. In the rabbit eye, Bill (1962) observed that sympathetic nerve stimulation after a-adrenoceptor blockade caused a slight increase in the uveal vascular resistance. Our results are consistent with this observation and show that there is a rather marked uveal vasoconstriction even after a-adrenoceptor blockade when the sympathetic nerve is stimulated at high frequencies. It seems unlikely that this non-adrenergic vasoconstriction should be due to incomplete blockade of a-adrenoceptors; noradrenaline given after the a-adrenoceptor blockade decreased the arterial blood pressure, and the pupillary response to sympathetic nerve stimulation was completely abolished by the aadrenoceptor blockade. In the eye, NPY-like immunoreactive (IR) nerve fibres have been shown to innervate the uveal blood vessels, the ciliary processes and the iris dilator muscle (Bruun et al., 1984; Stone et al., 1986). The distribution of NPY-like IR nerves closely parallels that of sympathetic, noradrenergic nerves (Gibbins and Morris, 1987). Chronic sympathetic denervation has been shown to markedly reduce the content of NPY in the iris/ciliary body (Allen et al., 1983) and to reduce the density of NPY-IR fibres in the iris (Zhang et al., 1984), thereby giving further support to the hypothesis that NPY is co-localized with noradrenaline in ocular sympathetic nerves. The present results suggest that NPY could contribute to the uveal vasoconstriction caused by sympathetic nerve stimulation, especially at high frequencies. This notion is further supported by the marked effect of NPY on uveal blood flow in the rabbit (Nilsson, 1987; 1988). This effect of NPY was resistant to a-adrenoceptor blockade with phenoxybenzamine in doses equivalent to those used in our experiments (Nilsson, unpublished results). NPY does not seem to contribute significantly to the pupillary dilatation observed during sympathetic nerve stimulation as the dilatation was abolished completely by a-adrenoceptor blockade. This is in agreement with the results of earlier experiments with the rabbit dilator muscle in vitro; NPY had no effect of its own but
185 potentiated the effect of phenylephrine (Piccone et al., 1988). In the pig spleen, the release of noradrenaline and NPY is frequency-dependent (Lundberg et al., 1986). Noradrenaline is mainly released at low stimulation frequencies whereas at higher frequencies both noradrenaline and N P Y are released. In the present experiments, high-frequency stimulation at 10 Hz caused a decrease in uveal blood flow which was resistant to a-adrenoceptor blokade whereas the decrease was partly resistant to (4 Hz) or completely abolished by (2 Hz) the a-adrenoceptor blockade when lower stimulation frequencies were used. This suggests that the release of noradrenaline and N P Y is also frequencydependent in the rabbit uvea. It has been suggested that N P Y release is influenced by noradrenaline acting via a negative feedback mechanism involving prejunctional a 2adrenoceptors. Sympathetic nerve stimulation during blockade of the a2-adrenoceptors has been shown to enhance the output of NPY-like immunoreactivity (Dahl6f et al., 1986; Lundberg et al., 1986). A non-competetive a 1- and a2-adrenoceptor blockade was achieved with the adrenoceptor-blocking agent used in this study, phenoxybenzamine. This could result in an enhanced release of N P Y during sympathetic nerve stimulation, leading to an over-estimation of the contribution of the non-adrenergic transmitter to sympathetic vasoconstriction. The blood flow changes in the masseter muscle and lacrimal gland during sympathetic nerve stimulation at different frequencies were very similar to those measured in the uvea. However, in the harderian gland, the adrenoceptor blockaderesistant vasoconstriction was significant even at the lowest stimulation frequency. This indicates that the extent of non-adrenergic sympathetic vasoconstriction during low-frequency stimulation varies between tissues. In the masseter muscle, there was a significant difference between the blood flow on the experimental side and the control side even without stimulation in the first series of experiments. The reason for this is obscure. One possible explanation is that there was insufficient time between the first and the second blood flow determinations to
allow a complete wash-out of transmitter from the extra vascular space. As the masseter muscle has continuous capillaries while the uvea and the salivary glands have fenestrated capillaries, the wash-out of transmitter is likely to be slower in the masseter muscle, especially if the transmitter involved has a high molecular weight as N P Y does. fl-Adrenoceptor-mediated dilatation has been shown to mask a non-adrenergic sympathetic vasoconstriction in the cat submandibular gland (Lundberg and Tatemoto, 1982). Similar results were also obtained in the present experiments. In the salivary glands, a combined fl- and a-adrenoceptor blockade was necessary to unmask a slight non-adrenergic component in the sympathetically induced vasomotor response during stimulation at 10 Hz. Vasodilatation was seen in the salivary glands during low frequency stimulation (2 and 4 Hz) after a-adrenoceptor blockade. In the uvea, however, a clear non-adrenergic vasoconstriction was observed after only a-adrenoceptor blockade. This gives further support to the suggestion that the uveal blood vessels lack /3adrenoceptors; noradrenaline and adrenaline cause uveal vasoconstriction (Aim, 1980; Morgan et al., 1981; 1983), while isoproterenol is without effect on uveal blood flow (Bill, 1962; Morgan et al., 1981). Also c~-adrenoceptor blockade alone was sufficient to reveal a non-adrenergic vasoconstriction in the masseter muscle and the harderian gland. In conclusion, our results show that sympathetic nerve stimulation at higher frequencies decreases the blood flow in the eye and most facial tissues. This decrease is mainly resistant to a-adrenoceptor blockade. This non-adrenergic component of sympathetic vasoconstriction is less pronounced when stimulation is performed at lower frequencies. The most likely mediator of this adrenoceptor blockade-resistant part of sympathetic vasoconstriction seems to be NPY.
Acknowledgements We wish to thank Professor Anders Bill for valuable discussions and Ms. Annsofi Hoist for expert technical assistance. This study was finacially supported by Grant 5 RO1 EY 00475
186 from the National Eye Institute, U.S. Public Health Service and by Grant 85-14X-00147 from the Swedish Medical Research Council.
References Allen, J.M., G.P. McGregor, T.E. Adrian, S.R. Bloom, S.Q. Zhang, K.W. Ennis and W.G. Unger, 1983, Reduction of neuropeptide Y (NPY) in the rabbit iris-ciliary body after chronic sympathectomy, Exp. Eye Res. 37, 213. Alm, A., 1977, The effect of sympathetic stimulation on blood flow through the uvea, retina and optic nerve in monkeys (Macaca irus), Exp. Eye Res. 25, 19. Alm, A., 1980, The effect of topical 1-epinephrine on regional ocular blood flow in monkeys, Invest. Opthalmol. Vis, Sci. 19, 487. Alm, A. and A. Bill, 1972, The oxygen supply to the retina. II. Effects of high intraocular pressure and of increased arterial carbon dioxide tension on uveal and retinal blood flow in cats. A study with radioactively labelled microspheres including flow determinations in brain and some other tissues, Acta Physiol. Scand. 84, 306. Aim, A. and A. Bill, 1973, The effect of stimulation of the cervical sympathetic chain on retinal oxygen tension and on uveal, retinal and cerebral blood flow in cats, Acta Physiol. Scand. 88, 84. Bevan, J.A., 1969, Some structural considerations of the reactivity of vascular smooth muscle, Microvascular Res. 1, 329. Bill, A., 1962, Autonomic nervous control of uveal blood flow, Acta Physiol. Scand. 56, 70. Bill, A., 1984, Circulation in the eye, in: Handbook of Physiology - The Cardiovascular System IV, eds. E.M. Renkin, C.C. Michel and S.R. Geiger (American Physiological Society, Bethesda, MD, U.S.A.) p. 1001. Bruun, A., B. Ehinger, F. Sundler, K. Tornqvist and R. Uddman, 1984, Neuropeptide Y immunoreactive neurons in the guinea-pig uvea and retina, Invest. Ophthalmol. Vis. Sci. 25, 1113. Dahl0f, C., P. Dahl/3f and J.M. Lundberg, 1986, Alpha 2Adrenoceptor-mediated inhibition of nerve stimulationevoked release of neuropeptide Y (NPY)-like immunoreactivity in the pithed guinea-pig, European J. Pharmacol. 131, 279, Gibbins, 1.L. and J.L. Morris, 1987, Co-existence of neuropeptides in sympathetic, cranial autonomic and sensory neurons innervating the iris of the guinea-pig, J. Autonom. Nerv. Syst. 21, 67. Granstam, E. and S.F.E. Nilsson, 1989, Non-adrenergic vasoconstriction in facial tissues, Proc. of the International Union of Physiological Sciences XVII, p. 66. Hillerdal, M., G.O. Sperber and A. Bill, 1987, The microsphere method for measuring low blood flows: theory and computer simulations applied to findings in the rat cochlea, Acta Physiol. Scand. 130, 229. Hirst, G.D.S. and T.O. Neild, 1980, Evidence for two populations of excitatory receptors for noradrenaline on arteriolar smooth muscle, Nature 283, 767.
Lundberg, J.M., A. AnggS, rd, J. Pernow and T. Hi3kfelt, 1985, Neuropeptide Y-, substance P- and VIP-immunoreactive nerves in cat spleen in relation to autonomic vascular and volume control, Cell Tissue Res. 239, 9. Lundberg, J.M., A. Rudehill, A. Sollevi, E. TheodorssonNorheim and B. Hamberger, 1986, Frequency- and reserpine-dependent chemical coding of sympathetic transmission: Differential release of noradrenaline and neuropeptide Y from pig spleen, Neurosci. Lett. 63, 96. Lundberg, J.M. and K. Tatemoto, 1982, Pancratic polypeptide family (APP, BPP, NPY, and PYY) in relation to sympathetic vasoconstriction resistant to alpha-adrenoceptor blockade, Acta Physiol. Scand. 116, 393. Morgan, T.R., K. Green and K. Bowman, 1981, Effects of adrenergic agonists upon regional ocular blood flow in normal and ganglionectomized rabbits, Exp. Eye Res. 32, 691. Morgan, T.R., D.J. Mirate, K. Bowman and K. Green, 1983, Topical epinephrine and regional ocular blood flow in aphakic eyes of rabbits, Arch. Ophthalmol. 101, 112. Nilsson, S.F.E., 1986, Studies on ocular blood flow and aqueous humor dynamics: Effects of VIP and PHI related to the effects of facial nerve stimulation, Acta Univ. Ups. 43, 1. Nitsson, S.F.E., 1987, Effects of NPY on ocular blood flow and local blood flow in some other tissues in the rabbit, J. Physiol. 390, 119P. Nilsson, S.F.E., 1988, NPY as a possible mediator of sympathetic vasoconstriction in the rabbit urea, Invest. Ophthalmol. Vis. Sci. 29 (Suppl.) 323. Pernow, J., 1988, Co-release and functional interactions of neuropeptide Y and noradrenaline in peripheral sympathetic vascular control, Acta Physiol. Scand. 133 (Suppl. 568), 1-56. Piccone, M., J. Littzi, T. Krupin, R.A. Stone, M. Davis and M.B. Wax, 1988, Effects of neuropeptide Y on the isolated rabbit iris dilator muscle, Invest. Ophthalmol. Vis. Sci. 29, 330. Rudehill, A., M. Olcrn, A. Sollevi, B. Hamberger and J.M. Lundberg, 1987, Release of neuropeptide Y upon haemorrhagic hypovolaemia in relation to vasoconstrictor effects in the pig, Acta Physiol. Scand. 131,517. Schoups, A.A., V.K. Saxena, K. Tombeur and W.P. De Potter, 1988, Facilitation of the release of noradrenaline and neuropeptide Y by the alpha2-adrenoceptor blocking agents idazoxan and hydergine in the dog spleen, Life Sci. 42, 517. Stone, R.A., A.M. Laties and P.C. Emson, 1986, Neuropeptide Y and the ocular innervation of rat, guinea pig, cat and monkey, Neuroscience 17, 1207. Tatemoto, K., 1982, Neuropeptide Y: Complete amino acid sequence of the brain peptide, Proc. Natl. Acad. Sci. U.S.A. 79, 5485. Wahlestedt, C., 1987, Neuropeptide Y (NPY): Actions and interactions in neurotransmission (Doctoral thesis University of Lund). Zhang, S.Q., G. Terenghi, W.G. Unger, K.W. Ennis and J. Polak, 1984, Changes in substance P- and neuropeptide Y-immunoreactive fibres in rat and guinea-pig irides following unilateral sympathectomy, Exp. Eye Res. 39, 365.
175
Elsevier EJP 51124
Non-adrenergic sympathetic vasoconstriction in the eye and some other facial tissues in the rabbit E l i s a b e t G r a n s t a m a n d Siv F.E. N i l s s o n Department of Physiology and Medical Biophysics, University of Uppsala, Uppsala, Sweden Received 26 June 1989, revised MS received 12 September 1989, accepted 24 October 1989
The effects of unilateral sympathetic nerve stimulation (SNS) on regional blood flow in the rabbit were studied with radioactive microspheres. SNS at 10 or 4 Hz caused an approximately 60% reduction in choroidal blood flow, which was partly resistant to a-adrenoceptor blockade with phenoxybenzamine. The vasoconstriction evoked by SNS at 2 Hz was completely abolished by a-adrenoceptor blockade. A similar response was seen in the iris, ciliary body, masseter muscle and lacrimal gland. In the harderian gland, however, SNS (2 Hz) after a-adrenoceptor blockade caused a significant reduction in blood flow. In the salivary glands, combined /3- and a-adrenoceptor blockade with propranolol and phenoxybenzamine revealed a slight non-adrenergic vasoconstriction during SNS at 10 Hz; however, the blood flow was significantly increased during SNS at 4 and 2 Hz following a-adrenoceptor blockade. These results indicate that there is a frequency-dependent, non-adrenergic component in the sympathetic vasoconstriction of the eye and several facial tissues. In the salivary glands, /3-adrenoceptor-mediated vasodilatation tends to mask a non-adrenergic vasoconstriction. a-Adrenoceptor blockade; fl-Adrenoceptor blockade; Eye; Neuropeptide Y; Sympathetic nerve stimulation; Vasoconstriction
1. Introduction The intraocular tissues are supplied with nutrients from two different vascular beds, the retinal and the uveal blood vessels. The retinal blood vessels are not innervated and the retinal blood flow is autoregulated in a similar way as the cerebral blood flow. In the rabbit, the uveal blood vessels, which comprise the vessels of the choroid, the iris and the ciliary body, show hardly any autoregulation. However, these vessels receive a rich innervation from the autonomic nervous system (see Bill, 1984). The facial nerve and the oculomotor nerve both contribute to the parasympathetic innervation of
Correspondence to: E. Granstam, Department of Physiology and Medical Biophysics, Box 572, S-751 23 Uppsala, Sweden.
the uveal blood vessels. The sympathetic innervation is supplied from the superior cervical ganglion. Stimulation of the oculomotor nerve is known to cause vasoconstriction, which is partly cholinergic, in the iris and vasoconstriction/ vasodilatation in other parts of the uvea, depending on the species studied (see Bill, 1984). Stimulation of the facial nerve causes vasodilatation of uveal vessels. This dilatation seems to be mediated mainly by vasoactive intestinal polypeptide (VIP) and possibly by peptide histidine isoleucine (PHI) (see Nilsson, 1986). Although it is well-documented that sympathetic stimulation causes vasoconstriction in the uvea (Bill, 1962; Alm and Bill, 1973; Alto, 1977), less is known about the extent to which the vasoconstriction is affected by a-adrenoceptor blockade and the possible involvement of neuropeptides.
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
176 The aim of the present study was to evaluate the extent to which the sympathetically induced vasomotor response in the eye and some other facial tissues is resistant to c~- and fl-adrenoceptor blockade and to investigate the effect of different stimulation frequencies. Preliminary reports of part of this investigation have been given elsewhere (Nilsson, 1988; Granstam and Nilsson, 1989). 2. Materials and methods
All experiments were performed on New Zealand White rabbits of either sex, weighing 1.9-3.5 kg. Regional blood flows were determined with the radioactively labelled microsphere method.
2.1. Anaesthesia and general operative procedures All animals were anaesthetized with urethane ( - 1.75 m g / k g bw) infused into a marginal ear vein. Small additional doses were given if required. The animals were tracheostomized and artificially ventilated. Both femoral veins were cannulated with polyethylene tubing for the infusion of drugs. The femoral arteries were cannulated bilaterally for continous blood pressure recording and for the collection of blood samples. A polyethylene tube, with the tip bent at a 120 degree angle, was placed into the left heart ventricle via a brachial artery for the injection of microspheres. Heparin (Heparin ®, KabiVitrum, Stockholm, Sweden), 1000 I E / k g bw, was given i.v. before the experiments. Muscle relaxation was induced by the i.v. administration of tubocurarine (Tubocuran "~, Nordisk Droge, Copenhagen, Denmark) (0.5-1.0 m g / k g bw). Arterial blood samples were collected before and during the experiments to determine the acid-base balance. The blood sampies were analysed in an ABL 300 acid-base analyser (Radiometer, Copenhagen, Denmark). Disturbances of the balance were corrected by administering sodium bicarbonate a n d / o r changing the ventilation rate.
2.2. Drugs c~-Adrenoceptor blockade was induced by the administration of phenoxybenzamine (Dibe-
nyline "~, S K & F , Welwyn Garden City, England) 50 m g / k g bw, which was slowly infused i.v. The arterial blood pressure fell during drug administration but was partly restored by the i.v. infusion of 30-40 ml of a mixture (1:1) of Macrodex (Pharmacia, Uppsala, Sweden) and saline or Rehydrex (Pharmacia, Uppsala, Sweden). Blood flow was determined 15-30 rain after the administration of phenoxybenzamine. Phenoxybenzamine is a non-specific blocking agent at the dose used, blocking not only cq- and c~2-adrenoceptors but also the effects of histamine, serotonin and acetylcholine. This high dose of phenoxybenzamine was chosen because it was used in a previous study on the effects of sympathetic nerve stimulation on uveal blood flow (Bill, 1962). However, the interference of phenoxybenzamine with receptors other than a-adrenoceptors does not affect the interpretation of the present results since the aim of the study was to determine the possible existence of a peptidergic component in the sympathetic vasoconstriction. fl-Adrenoceptor blockade was achieved by the i.v. administration of propranolol (Inderal ®, ICIPharma, Macclesfield, England) (2 m g / k g bw). At least 15 min elapsed before the blood flow was determined.
2.3. Stimulation of the cervical sympathetic nerve The cervical sympathetic nerves were sectioned bilaterally and stimulated unilaterally on the left or right side. In the first two series of experiments, a bipolar platinum electrode attached to a Digitimer Stimulator DS9A (Digitimer Ltd., Welwyn Garden City, England) was used for stimulation. The electrode tips were insulated with a small plastic holder, which also made it possible to anchor the electrode to the adjacent tissues. After the electrode had been attached to the nerve, the rabbit was placed in a prone position. In the third and fourth series of experiments the rabbits were placed in a supine position and stimulated with a bipolar silver electrode connected to the Digitimer Stimulator. Care was taken to isolate the electrode from adjacent structures. In these experiments the nerve was kept moist with paraffin. The stimulation intensity (5-8 V) was adjusted to give an immediate and marked dilatation of the pupil.
177 The pulse lasted 1-2 ms and the stimulation period was 3 or 4 min. Blood flow was measured 2 rain after the start of nerve stimulation.
2.4. Determination of regional blood flows with radioactive microspheres Regional blood flows were determined with 15 /~m radioactive microspheres (NEN, Boston, MA, USA), according to the reference flow method (see Alm and Bill, 1972; Hillerdal et al., 1987). Spheres labeled with three different radionuclides, 141Ce, 113Sn and l°3Ru, were used. This made it possible to make three blood flow determinations in each animal. Between 1 0 6 and 2 • 1 0 6 microspheres were injected each time. The spheres were normally injected over 10-15 s and blood sampling was started at the same time and continued to 1 rain. If the mean arterial blood pressure was very low, the spheres were injected over 20-30 s and the sampling time was extended to 2 min. The animals were killed after the experiments by an intracardiac or i.v. injection of a KC1 solution. Both eyes were enucleated and the retina, choroid, iris and the ciliary body were dissected. Samples were also taken from other tissues innervated by the cervical sympathetic nerve and from some peripheral tissues. The blood samples and the tissue samples were then weighed and analysed in a three-channel gamma-spectrometer.
2.5. Experimental protocols (A) In the first series of experiments, the blood flow was determined during sympathetic nerve stimulation at 10 Hz, after a-adrenoceptor blockade but without stimulation and after a-adrenoceptor blockade during stimulation at 10 Hz (n = 6). (B) In the second series of experiments, the blood flow was determined during sympathetic nerve stimulation at 10 Hz; without blockade, after /3-adrenoceptor blockade and then after /3and a-adrenoceptor blockade (n = 8). (C) In the third series of experiments, the blood flow was determined during sympathetic nerve stimulation at 4 Hz before a-adrenoceptor blockade and after a-adrenoceptor blockade during sympathetic nerve stimulation at 4 and 10 Hz (n = 7).
TABLE 1 Blood pressure (ram Hg) during blood flow measurements in the four different experimental series (means-4-S.E.). 1st blood flow 2nd blood flow 3rd blood flow measurement measurement measurement SeriesA Series B Series C Series D
65_+8 73_+5 74 ___7 69 4-3
47+2 61+3 43 __+3 39 + 2
46+2 34+3 43 + 3 37 _+2
(D) In the fourth series of experiments, the local blood flows were determined during sympathetic nerve stimulation at 2 Hz and again after a-adrenoceptor blockade during sympathetic nerve stimulation at 2 and 4 Hz (n = 10).
2.6. Statistical analysis It is difficult to completely separate the vitreous body from the intraocular tissues, which results in an over-estimation of the weight of these tissues. To avoid this problem, the blood flow in the choroid, iris and ciliary body was calculated as the total blood flow per tissue and expressed as m g / m i n . For the other tissues, the blood flow was related to tissue weight ( g / m i n × g). In the rabbit, changes in arterial blood pressure greatly affect uveal blood flow, since this blood flow is almost without autoregulation. In the present experiments, the mean arterial blood pressure fell after a-adrenoceptor blockade and after combined/3- and a-adrenoceptor blockade (table 1) in spite of rehydration. The effects of sympathetic nerve stimulation were therefore evaluated by comparing the stimulated side and the control side. Statistical analysis was performed with the two-tailed Student's t-test for paired data. P values less than 0.05 were considered significant. All values are given as means + S.E.
3. Results
3.1. Effect of sympathetic nerve stimulation before and after a- and/3-adrenoceptor blockade Sympathetic nerve stimulation at 10 Hz markedly decreased the blood flow in the eye and
178 TABLE 2
TABLE 3
Absolute blood flow rates in ocular tissues during stimulation at 10 Hz, control and stimulated sides, respectively. Blood flows are given as m g / m i n , means + S.E.
Absolute blood flow rates in facial tissues during stimulation at 10 Hz, control and stimulated sides, respectively. Blood flows are given as g / m i n × g, means +- S.E.
Tissue
Stimulated side
Control side
Difference
Tissue
Stimulated side
Control side
Difference
Choroid Iris Ciliary body
340+_85 14+_ 4 27+ 6
862+_144 100+- 20 107+_ 20
-522+-85 a -86+-19 a -80+_15 a
Parotid gland Submandibular gland Harderian gland Lacrimal gland Masseter muscle
0.01 +-0.00
0.41 +-0.10
-0.40+_0.10 c
0.02+_0.01 0.07_+0.02 0.06+_0.05 0.04+_0.02
0.84+_0.17 0.65+0.18 0.36+_0.07 0.56+__0.01
-0.82_+0.17 -0.58+_0.17 -0.30+_0.05 -0.52_+0.08
P ~<0.01.
a p ~< 0.05, b p ~<0.01, c p ~< 0.001.
several other facial tissues. The absolute rates of blood flow for control and stimulated side in various tissues during stimulation at 10 Hz are given in tables 2 and 3. Sympathetic nerve stimulation at 10 Hz decreased the blood flow in the choroid by 62 + 5% (P < 0.001; n = 6) compared to the non-stimulated control side. After a-adrenoceptor blockade, sympathetic nerve stimulation caused a 43 + 7% (P < 0.01; n = 6) reduction of choroidal blood flow (fig. 1). The other parts of the uvea, the iris and the ciliary body, responded in a similar way. The corresponding values for the iris and the ciliary body were 86 +_ 4% (P < 0.001; n = 6) and 76 +_ 4% (P < 0.001; n = 6) before the blockade and 60 +_ 10% (P < 0.01; n = 6) and 45 +_ 9% (P < 0.01; n = Choroid
1oo
Masseter
h a ~ b
6), respectively, after the blockade. In these tissues, the control microsphere injection following a-adrenoceptor blockade showed that there was an equal blood flow on both the control and experimental sides. Sympathetic nerve stimulation after /3-adrenoceptor blockade caused a somewhat greater decrease in choroidal blood flow on the stimulated side compared to the control side than during stimulation at 10 Hz without any blockade (69 _+ 7 versus 60 + 8, P < 0.05; n = 8) (fig. 2). However, the effect of sympathetic nerve stimulation on choroidal blood flow after combined /3- and c~m
Lacrimal
gl
Parotid
gl
80 ~= 60 O
~-
40
o 20 _o .Q 0
-= -2o
& -4oI e-
-8o!
'*
m
-x
-100 -120
[] [] []
lO Hz After alpha-adrenoceptor blockade 10 Hz after alpha-adrenoceptor blockade
Fig. 1. Effects of sympathetic nerve stimulation at 10 Hz on blood flow in the choroid, masseter muscle, lacrimal and harderian glands. Local blood flow was determined during stimulation at 10 Hz, without stimulation after a-adrenoceptor blockade and during stimulation at 10 Hz after the blockade. Blood flow values are given as percentage change from the non-stimulated control side. Mean values and S.E. are given (n = 6). * P ~<0.05; * * P ~< 0.01 and * * * P ~< 0.001.
179 Choroid
1O0
Masseter i
m
Lacrimal
gl
i
Parotid
gl
J
80 60 --
"el
o
o
e.,
40 20 0
E
-2o
¢~
-40
m
-60 -80
o
ell 'E,
-I O0 -120
[]
10Hz
[] []
10 Hz after beta-adrenoceptor blockade 10 Hz after alpha- and beta-adrenoceptor blockade
Fig. 2. Effects of sympathetic nerve stimulation at 10 Hz; without blockade, during fl-adrenoceptor blockade and during combined fl- and a-adrenoceptor blockade. Blood flow values are given as percentage change from the non-stimulated control side. Mean values and S.E. are given ( n = 8 except for the masseter muscle and lacrimal gland, where n = 7 ) . * P ~ O . 0 5 ; ** P~<0.01; • ** P ~< 0.001.
adrenoceptor blockade was not different from that after a-adrenoceptor blockade alone (compare figs. I and 2). Blood flow in the masseter muscle and in the lacrimal and harderian glands decreased by 95 _+ 2, 88_+8 and 8 8 + 2 % ( P < 0 . 0 0 1 ; n = 6 ) , respectively, during sympathetic nerve stimulation at 10 Hz (fig. 1). There was a significant decrease in blood flow during sympathetic nerve stimulation at 10 Hz after a-adrenoceptor blockade in the masseter muscle (60 _+ 9%, P < 0.001; n = 6) (fig. 1) and in the harderian gland (57 + 3%, P < 0.001; n = 6) but the change in the lacrimal gland was not statistically significant (fig. 1). The control injection of microspheres following a-adrenoceptor blockade showed a comparable blood flow on control and experimental sides in the glandular tissues (fig. 1). In the masseter msucle, however, the blood flow was significantly reduced on the experimental side compared to the control side even though the muscle had not been stimulated (15 _+ 2%, P < 0.001; n = 6) (fig. 1). The responses of the masseter muscle (fig. 2) and harderian gland to sympathetic nerve stimulation after combined/3- and a-adrenoceptor blockade were not different from those following aadrenoceptor blockade alone. In this series of experiments the combined blockade revealed a
32 _+ 9% (P < 0.05; n -- 7) reduction in blood flow in the lacrimal gland during sympathetic nerve stimulation at 10 Hz (fig. 2). Sympathetic nerve stimulation at 10 Hz markedly decreased the blood flow to the salivary glands: a 96 + 2% (P < 0.001; n = 6) decrease in the parotid gland (fig. 1) and a 97 + 1% (P < 0.001; n = 6) decrease in the submandibular gland. This effect was completely abolished in the submandibular gland by a-adrenoceptor blockade. The blood flow in the parotid gland increased but not significantly during sympathetic nerve stimulation at 10 Hz after a-adrenoceptor blockade (41 _+ 29%; n = 6) (fig. 1). After combined /3- and a-adrenoceptor blockade, the blood flow in the parotid gland decreased by 32 + 7% (P < 0.01; n = 8) (fig. 2). A slight but not statistically significant decrease in blood flow was seen in the submandibular gland after combined /3- and c~-adrenoceptor blockade.
3.2. Effects of sympathetic nerve stimulation at different frequencies before and after a-adrenoceptor blockade The effects of sympathetic nerve stimulation at 4 Hz on uveal blood flow were similar to those at 10 Hz: choroidal blood flow was reduced by 58 + 8% ( P < 0 . 0 0 ] ; n = 7 ) (fig. 3). In the iris and
180
Choroid
500
Masseter
m
Lacrimal
gl i
Parotid
gl
400 0
3OO
o o .el "
200 1O0 o
'~
-100 -200
[] [] []
4 Hz 4 Hz after alpha-adrenoceptor blockade 10 Hz after alpha-adrenoceptor blockade
Fig. 3. Effects of sympathetic nerve stimulation at 4 Hz before a-adrenoceptor blockade and at 4 and 10 Hz after et-adrenoceptor blockade. Blood flow values are given as percentage change from the non-stimulated control side. Mean values and S.E. are given ( n = 7 ) . * P~<0.05;** ~<0.01; * * * P~<0.001.
ciliary body, the blood flow was reduced by 82 +_ 4% ( P < 0 . 0 0 1 ; n = 7 ) and 81_+5% ( P < 0 . 0 0 1 ; n = 7) respectively. Sympathetic nerve stimulation at 2 Hz caused a smaller reduction in choroidal blood flow (41 ± 8%, P <0.001; n = 10) (fig. 4) and a significant reduction in irideal and ciliary blood flows. After phenoxybenzamine administration, sympathetic nerve stimulation at 4 Hz de-
500 ~g 0
400
,
Choroid
|
Maaseter
creased the local blood flow in the iris by 24 ± 8% (P < 0.05; n = 17) and in the ciliary body by 30 _+ 8% (P < 0.01; n = 17) while the choroidal blood flow did not decrease significantly (fig. 5). Sympathetic nerve stimulation at 2 Hz after a-adrenoceptor blockade did not cause a statistically significant change in blood flow in any part of the uvea (fig. 5).
m
a
Lacrimal
gl
i
Parotid
gl
r
300 0 _o
7-
2OO
C OI
1O0
C
J=
0
U ,k~*
d,
-1 O0 -200
[] [] []
2 Hz 2 Hz after alpha-adrenoceptor blockade 4 Hz after alpha-adrenoceptor blockade
Fig. 4. Effects of sympathetic nerve stimulation at 2 Hz before and after a-adrenoceptor blockade and at 4 Hz after the blockade. Blood flow values are given as percentage change from the non-stimulated control side. Mean values and S.E. are given (n = 10). * P < 0 . 0 5 ; * * P~<0.01;*** P~<0,001.
181
20 (n=lO) ~
Choroid
0
o
"o
8
-20
,o
-40
(n=lO)
•
(n=13)
0
-60
m ,,C
(n=14) (n=7)
-80 - 1 O0
I
I
I
2
4
10
2O Iris 0 o
_g
-20
¢
-40
Q 0 'dll ,¢ 0
-60
(n=lO)
,o
(n=l O) (n=l 3)
-8o
(n=14) (n=7)
,,,
I
I
I
2
4
10
- 1 O0
2O
Ciliary , 0 0 x~
8
(n=lO) -20 •
-~
-40 • ¢a l
body
, (n=lO)
-60
(n=13)
o
(n=14)
-8o
I
~
'
e**
(n=7) - 1 O0
I
t
I
2
4
10 Frequency (Hz)
Fig. 5. Effects of sympathetic nerve stimulation at different frequencies on blood flow in the choroid, iris and ciliary body with and without a-adrenoceptor blockade. Blood flow values are given as percentage change from the non-stimulated control side. Mean values and S.E. are given. The data presented are the summarized results of experimental series A-D. Squares represent changes in blood flow before a-adrenoceptor blockade. Diamonds represent changes in blood flow after a-adrenoceptor blockade. * P ~< 0.05; • * P ~ < 0 . 0 1 ; * * * P~<0.001.
182
20,
O:
Masseter muscle
(n=lO) ,
-2Ol
-~
-40:
-6o! -80
,,,
j.
(n=l O) ~,,,.~
-"'--o
-100 -120
**,
(n=l 3)
*** •I"1 (n=13)
(n=7)
I
I
I
2
4
10
20 0
Lacrimal
gland
(n=lO)
-20 .~ C
-40
***
®
-60
(n=lO) ~ * * *
o a ,1= 0
(n=17)
.~
-80
(n=13)
(n=13)
-loa
(n=7)
-120
I
I
I
2
4
10
2O Harderlan gland
0 -2o .~
(n=l O)
***
-40
!
(n=17) (n=l O) ~
.~
,
-80
(n=13)
0
(n=13)
(n=7)
-100 -120
"~
I
I
2
4
I
Frequency (Hz)
10
Fig. 6. Effects of sympathetic nerve stimulation at different frequencies on blood flow in the masseter muscle, lacrimal and harderian glands with and without a-adrenoceptor blockade. Blood flow values are given as percentage change from the non-stimulated control side. Mean values and S.E. are given. The data presented are the summarized results of experimental series A-D. Squares represent changes in blood flow before a-adrenoceptor blockade. D i a m o n d s represent changes in blood flow after a-adrenoceptor blockade. * P ~< 0.05; * * P ~< 0.01; * * * P ~< 0.001.
183
In the masseter muscle, sympathetic nerve stimulation at 4 Hz caused a 97 + 1% (P < 0.001; n = 7) (fig. 3) decrease in blood flow compared to the control side while sympathetic nerve stimulation at 2 Hz decreased the blood flow by 82 _ 3% (P < 0.001; n = 10) (fig. 4). After phenoxybenzamine treatment, sympathetic nerve stimulation at 4 Hz reduced the blood flow by 29 _+ 8% (P < 0.01; n = 17) (fig. 6) whereas no reduction was observed at 2 Hz. A similar response was seen in the lacrimal gland (fig. 6). In the harderian gland, however, stimulation at 2 Hz after a-adrenoceptor blockade reduced the blood flow by 30 + 5% (P < 0.001; n = 10) (fig. 6). 480 420 360 0
300
~
240
"R
180
!
120
t-
60
Q OI
(n=l O)
Sympathetic nerve stimulation at 4 and 2 Hz markedly reduced the blood flow in the salivary glands (figs. 3 and 4). Sympathetic nerve stimulation at 4 Hz after c~-adrenoceptor blockade increased the blood flow on the stimulated side compared to the control side. In the parotid gland, the increase was 258 _+ 60% (P < 0.001; n = 17) (fig. 7) and in the submandibular gland 56 _+ 8% (P < 0.001; n = 17) (fig. 7). Stimulation at 2 Hz following a-adrenoceptor blockade also resulted in a marked increase in blood flow: 337 _+ 81% (P < 0.01; n = 10) (fig. 7) in the parotid gland and 63_+ 10% (P <0.001; n = 9) (fig. 7) in the submandibular gland.
~
tid g l a n d
(n=l 7) (n=13)
¢11
•== o
0 -60
(n=l O) I ~ ' - - - . - - . _ ~ * _ * * (n=7) t
-120
*,"I* * i
2
i
Y
4
(n=14)
10
480 420 S u b m a n d i b u l a r gland
360 o r-
0 _o .D t-
300 24O 180 120
*lr*
=
60
(n=g) =t
o
0
Q
-60
(n=17) ***
(n:9)
~
1
(n=l 3) *t*
(n:7)
(n:14)
-120 2
4
10 F r e q u e n c y (Hz)
Fig. 7. Effects of sympathetic nerve stimulation at different frequencies on blood flow in the parotid and submandibular glands with and without a-adrenoceptor blockade. Blood flow values are given as percentage change from the non-stimulated control side. Mean values and S.E. are given. The data presented are the summarized results of experimental series A-D. Squares represent changes in blood flow before a-adrenoceptor blockade. Diamonds represent changes in blood flow after a-adrenoceptor blockade. * P ~< 0.05; • * P~<0.01; * * * P~< 0.001.
184
The completeness of the a-adrenoceptor blockade was tested in two different ways. An i.v. bolus injection of noradrenaline, given at the end of some of the experiments with a-adrenoceptor blockade, decreased the arterial blood pressure, indicating that there was a complete a-adrenoceptor blockade. Sympathetic nerve stimulation after a-adrenoceptor blockade did not affect the size of the pupils, also indicating an effective blockade. However, a slight increase in pupillary diameter was observed when the sympathetic nerve was stimulated after a combined a- and /3-adrenoceptor blockade.
4. Discussion
Several different explanations have been suggested for the inability of adrenoceptor blocking agents to completely abolish the effects of sympathetic nerve stimulation. It has been suggested that noradrenaline might be released from nerve endings in such large amounts that the antagonists fail to achieve a competetive blockade of the adrenoceptors (Bevan, 1969). Another explanation is that anti-adrenergic drugs block presynaptic aa-adrenoceptors , which results in an enhanced stimulation-evoked release of noradrenaline (Schoups et al., 1988). This could be sufficient to compensate for the blockade of a-adrenoceptors. It has also been implied that noradrenaline from vascular nerves might act through receptors other than a-adrenoceptors (Hirst and Neild, 1980). During the last few years it has become evident that the perivascular sympathetic nerve fibers in most tissues contain neuropeptide Y (NPY) in addition to noradrenaline (for references see Wahlestedt, 1987; Pernow, 1988). NPY, originally isolated from the porcine brain, consists of 36 amino acids (Tatemoto, 1982) and is a potent hypertensive agent, causing marked vasoconstriction in several tissues (Lundberg and Tatemoto, 1982; Lundberg et al., 1985; Rudehill et al., 1987). NPY is released from sympathetic nerves upon sympathetic nerve stimulation (Lundberg et al., 1986) and has been shown to cause adrenoceptor blockade-resistant vasoconstriction (Lundberg and
Tatemoto, 1982). This makes it a likely mediator of the a-adrenoceptor blockade-resistant part of sympathetic vasoconstriction. In the rabbit eye, Bill (1962) observed that sympathetic nerve stimulation after a-adrenoceptor blockade caused a slight increase in the uveal vascular resistance. Our results are consistent with this observation and show that there is a rather marked uveal vasoconstriction even after a-adrenoceptor blockade when the sympathetic nerve is stimulated at high frequencies. It seems unlikely that this non-adrenergic vasoconstriction should be due to incomplete blockade of a-adrenoceptors; noradrenaline given after the a-adrenoceptor blockade decreased the arterial blood pressure, and the pupillary response to sympathetic nerve stimulation was completely abolished by the aadrenoceptor blockade. In the eye, NPY-like immunoreactive (IR) nerve fibres have been shown to innervate the uveal blood vessels, the ciliary processes and the iris dilator muscle (Bruun et al., 1984; Stone et al., 1986). The distribution of NPY-like IR nerves closely parallels that of sympathetic, noradrenergic nerves (Gibbins and Morris, 1987). Chronic sympathetic denervation has been shown to markedly reduce the content of NPY in the iris/ciliary body (Allen et al., 1983) and to reduce the density of NPY-IR fibres in the iris (Zhang et al., 1984), thereby giving further support to the hypothesis that NPY is co-localized with noradrenaline in ocular sympathetic nerves. The present results suggest that NPY could contribute to the uveal vasoconstriction caused by sympathetic nerve stimulation, especially at high frequencies. This notion is further supported by the marked effect of NPY on uveal blood flow in the rabbit (Nilsson, 1987; 1988). This effect of NPY was resistant to a-adrenoceptor blockade with phenoxybenzamine in doses equivalent to those used in our experiments (Nilsson, unpublished results). NPY does not seem to contribute significantly to the pupillary dilatation observed during sympathetic nerve stimulation as the dilatation was abolished completely by a-adrenoceptor blockade. This is in agreement with the results of earlier experiments with the rabbit dilator muscle in vitro; NPY had no effect of its own but
185 potentiated the effect of phenylephrine (Piccone et al., 1988). In the pig spleen, the release of noradrenaline and NPY is frequency-dependent (Lundberg et al., 1986). Noradrenaline is mainly released at low stimulation frequencies whereas at higher frequencies both noradrenaline and N P Y are released. In the present experiments, high-frequency stimulation at 10 Hz caused a decrease in uveal blood flow which was resistant to a-adrenoceptor blokade whereas the decrease was partly resistant to (4 Hz) or completely abolished by (2 Hz) the a-adrenoceptor blockade when lower stimulation frequencies were used. This suggests that the release of noradrenaline and N P Y is also frequencydependent in the rabbit uvea. It has been suggested that N P Y release is influenced by noradrenaline acting via a negative feedback mechanism involving prejunctional a 2adrenoceptors. Sympathetic nerve stimulation during blockade of the a2-adrenoceptors has been shown to enhance the output of NPY-like immunoreactivity (Dahl6f et al., 1986; Lundberg et al., 1986). A non-competetive a 1- and a2-adrenoceptor blockade was achieved with the adrenoceptor-blocking agent used in this study, phenoxybenzamine. This could result in an enhanced release of N P Y during sympathetic nerve stimulation, leading to an over-estimation of the contribution of the non-adrenergic transmitter to sympathetic vasoconstriction. The blood flow changes in the masseter muscle and lacrimal gland during sympathetic nerve stimulation at different frequencies were very similar to those measured in the uvea. However, in the harderian gland, the adrenoceptor blockaderesistant vasoconstriction was significant even at the lowest stimulation frequency. This indicates that the extent of non-adrenergic sympathetic vasoconstriction during low-frequency stimulation varies between tissues. In the masseter muscle, there was a significant difference between the blood flow on the experimental side and the control side even without stimulation in the first series of experiments. The reason for this is obscure. One possible explanation is that there was insufficient time between the first and the second blood flow determinations to
allow a complete wash-out of transmitter from the extra vascular space. As the masseter muscle has continuous capillaries while the uvea and the salivary glands have fenestrated capillaries, the wash-out of transmitter is likely to be slower in the masseter muscle, especially if the transmitter involved has a high molecular weight as N P Y does. fl-Adrenoceptor-mediated dilatation has been shown to mask a non-adrenergic sympathetic vasoconstriction in the cat submandibular gland (Lundberg and Tatemoto, 1982). Similar results were also obtained in the present experiments. In the salivary glands, a combined fl- and a-adrenoceptor blockade was necessary to unmask a slight non-adrenergic component in the sympathetically induced vasomotor response during stimulation at 10 Hz. Vasodilatation was seen in the salivary glands during low frequency stimulation (2 and 4 Hz) after a-adrenoceptor blockade. In the uvea, however, a clear non-adrenergic vasoconstriction was observed after only a-adrenoceptor blockade. This gives further support to the suggestion that the uveal blood vessels lack /3adrenoceptors; noradrenaline and adrenaline cause uveal vasoconstriction (Aim, 1980; Morgan et al., 1981; 1983), while isoproterenol is without effect on uveal blood flow (Bill, 1962; Morgan et al., 1981). Also c~-adrenoceptor blockade alone was sufficient to reveal a non-adrenergic vasoconstriction in the masseter muscle and the harderian gland. In conclusion, our results show that sympathetic nerve stimulation at higher frequencies decreases the blood flow in the eye and most facial tissues. This decrease is mainly resistant to a-adrenoceptor blockade. This non-adrenergic component of sympathetic vasoconstriction is less pronounced when stimulation is performed at lower frequencies. The most likely mediator of this adrenoceptor blockade-resistant part of sympathetic vasoconstriction seems to be NPY.
Acknowledgements We wish to thank Professor Anders Bill for valuable discussions and Ms. Annsofi Hoist for expert technical assistance. This study was finacially supported by Grant 5 RO1 EY 00475
186 from the National Eye Institute, U.S. Public Health Service and by Grant 85-14X-00147 from the Swedish Medical Research Council.
References Allen, J.M., G.P. McGregor, T.E. Adrian, S.R. Bloom, S.Q. Zhang, K.W. Ennis and W.G. Unger, 1983, Reduction of neuropeptide Y (NPY) in the rabbit iris-ciliary body after chronic sympathectomy, Exp. Eye Res. 37, 213. Alm, A., 1977, The effect of sympathetic stimulation on blood flow through the uvea, retina and optic nerve in monkeys (Macaca irus), Exp. Eye Res. 25, 19. Alm, A., 1980, The effect of topical 1-epinephrine on regional ocular blood flow in monkeys, Invest. Opthalmol. Vis, Sci. 19, 487. Alm, A. and A. Bill, 1972, The oxygen supply to the retina. II. Effects of high intraocular pressure and of increased arterial carbon dioxide tension on uveal and retinal blood flow in cats. A study with radioactively labelled microspheres including flow determinations in brain and some other tissues, Acta Physiol. Scand. 84, 306. Aim, A. and A. Bill, 1973, The effect of stimulation of the cervical sympathetic chain on retinal oxygen tension and on uveal, retinal and cerebral blood flow in cats, Acta Physiol. Scand. 88, 84. Bevan, J.A., 1969, Some structural considerations of the reactivity of vascular smooth muscle, Microvascular Res. 1, 329. Bill, A., 1962, Autonomic nervous control of uveal blood flow, Acta Physiol. Scand. 56, 70. Bill, A., 1984, Circulation in the eye, in: Handbook of Physiology - The Cardiovascular System IV, eds. E.M. Renkin, C.C. Michel and S.R. Geiger (American Physiological Society, Bethesda, MD, U.S.A.) p. 1001. Bruun, A., B. Ehinger, F. Sundler, K. Tornqvist and R. Uddman, 1984, Neuropeptide Y immunoreactive neurons in the guinea-pig uvea and retina, Invest. Ophthalmol. Vis. Sci. 25, 1113. Dahl0f, C., P. Dahl/3f and J.M. Lundberg, 1986, Alpha 2Adrenoceptor-mediated inhibition of nerve stimulationevoked release of neuropeptide Y (NPY)-like immunoreactivity in the pithed guinea-pig, European J. Pharmacol. 131, 279, Gibbins, 1.L. and J.L. Morris, 1987, Co-existence of neuropeptides in sympathetic, cranial autonomic and sensory neurons innervating the iris of the guinea-pig, J. Autonom. Nerv. Syst. 21, 67. Granstam, E. and S.F.E. Nilsson, 1989, Non-adrenergic vasoconstriction in facial tissues, Proc. of the International Union of Physiological Sciences XVII, p. 66. Hillerdal, M., G.O. Sperber and A. Bill, 1987, The microsphere method for measuring low blood flows: theory and computer simulations applied to findings in the rat cochlea, Acta Physiol. Scand. 130, 229. Hirst, G.D.S. and T.O. Neild, 1980, Evidence for two populations of excitatory receptors for noradrenaline on arteriolar smooth muscle, Nature 283, 767.
Lundberg, J.M., A. AnggS, rd, J. Pernow and T. Hi3kfelt, 1985, Neuropeptide Y-, substance P- and VIP-immunoreactive nerves in cat spleen in relation to autonomic vascular and volume control, Cell Tissue Res. 239, 9. Lundberg, J.M., A. Rudehill, A. Sollevi, E. TheodorssonNorheim and B. Hamberger, 1986, Frequency- and reserpine-dependent chemical coding of sympathetic transmission: Differential release of noradrenaline and neuropeptide Y from pig spleen, Neurosci. Lett. 63, 96. Lundberg, J.M. and K. Tatemoto, 1982, Pancratic polypeptide family (APP, BPP, NPY, and PYY) in relation to sympathetic vasoconstriction resistant to alpha-adrenoceptor blockade, Acta Physiol. Scand. 116, 393. Morgan, T.R., K. Green and K. Bowman, 1981, Effects of adrenergic agonists upon regional ocular blood flow in normal and ganglionectomized rabbits, Exp. Eye Res. 32, 691. Morgan, T.R., D.J. Mirate, K. Bowman and K. Green, 1983, Topical epinephrine and regional ocular blood flow in aphakic eyes of rabbits, Arch. Ophthalmol. 101, 112. Nilsson, S.F.E., 1986, Studies on ocular blood flow and aqueous humor dynamics: Effects of VIP and PHI related to the effects of facial nerve stimulation, Acta Univ. Ups. 43, 1. Nitsson, S.F.E., 1987, Effects of NPY on ocular blood flow and local blood flow in some other tissues in the rabbit, J. Physiol. 390, 119P. Nilsson, S.F.E., 1988, NPY as a possible mediator of sympathetic vasoconstriction in the rabbit urea, Invest. Ophthalmol. Vis. Sci. 29 (Suppl.) 323. Pernow, J., 1988, Co-release and functional interactions of neuropeptide Y and noradrenaline in peripheral sympathetic vascular control, Acta Physiol. Scand. 133 (Suppl. 568), 1-56. Piccone, M., J. Littzi, T. Krupin, R.A. Stone, M. Davis and M.B. Wax, 1988, Effects of neuropeptide Y on the isolated rabbit iris dilator muscle, Invest. Ophthalmol. Vis. Sci. 29, 330. Rudehill, A., M. Olcrn, A. Sollevi, B. Hamberger and J.M. Lundberg, 1987, Release of neuropeptide Y upon haemorrhagic hypovolaemia in relation to vasoconstrictor effects in the pig, Acta Physiol. Scand. 131,517. Schoups, A.A., V.K. Saxena, K. Tombeur and W.P. De Potter, 1988, Facilitation of the release of noradrenaline and neuropeptide Y by the alpha2-adrenoceptor blocking agents idazoxan and hydergine in the dog spleen, Life Sci. 42, 517. Stone, R.A., A.M. Laties and P.C. Emson, 1986, Neuropeptide Y and the ocular innervation of rat, guinea pig, cat and monkey, Neuroscience 17, 1207. Tatemoto, K., 1982, Neuropeptide Y: Complete amino acid sequence of the brain peptide, Proc. Natl. Acad. Sci. U.S.A. 79, 5485. Wahlestedt, C., 1987, Neuropeptide Y (NPY): Actions and interactions in neurotransmission (Doctoral thesis University of Lund). Zhang, S.Q., G. Terenghi, W.G. Unger, K.W. Ennis and J. Polak, 1984, Changes in substance P- and neuropeptide Y-immunoreactive fibres in rat and guinea-pig irides following unilateral sympathectomy, Exp. Eye Res. 39, 365.