Acta Ph,ysiol Scand 1991, 141, 455467

ADONIS

000167729100069P

Neuropeptide Y (NPY) : a vasoconstrictor in the eye, brain and other tissues in the rabbit S. F. E. N I L S S O N Department of Physiology and Medical Biophysics, University of Uppsala, Uppsala, Sweden

NILSSON, S. F. E. 1991. Neuropeptide Y (NPY): A vasoconstrictor in the eye, brain and other tissues in the rabbit. Acta Physiol Scand 141, 4455467. Received 22 August 1990, accepted 15 November 1990. ISSN 0001-6772. Department of Physiology and Medical Biophysics, University of Uppsala, Uppsala, Sweden. The effect of neuropeptide Y (NPY) on uveal vascular resistance was studied in rabbits by direct determination of uveal blood flow from a cannulated vortex vein. Regional blood flows, in the eye, the brain and several other tissues, were measured, with radioactive microspheres, during neuropeptide Y-infusion in rabbits with and without a-adrenoceptor blockade. Intravenous infusion of increasing doses of neuropeptide Y caused a dose-dependent increase in the total uveal vascular resistance. Maximal effect, a 70% increase, was achieved with 120 pmol kg-l min-'. In the microsphere experiments, this dose rate was given i.v. over 10 minutes and blood flow determinations were made before and at 2 and 10 minutes after the start of the infusion. After 2 minutes of neuropeptide Y, there were marked blood flow reductions in the spleen, kidneys, adrenal glands, gastro-intestinal tract, choroid plexus and pineal and pituitary gland. The effect in the eye was small at 2 minutes, but at 10 minutes local blood flows in the choroid and the ciliary body were decreased by 50% and the iridal blood flow by 30%. Retinal blood flow was not affected by neuropeptide Y. At 10 minutes there were also significant blood flow reductions in the brain, tongue, masseter muscle and several glandular tissues. The effects of neuropeptide Y on local blood flow in rabbits that had been subjected to a-adrenoceptor blockade were very similar to the effects in the animals without a-adrenoceptor blockade. The results show that, in the rabbit, neuropeptide Y has marked effects on local blood flows in several tissues, including the eye, and suggest that neuropeptide Y may significantly contribute to the uveal vasoconstriction during sympathetic nerve stimulation. Key words ; a-adrenoceptor blockade, cerebral blood flow, choroid, ciliary body, iris, neuropeptide Y, ocular blood flow, vasoconstriction.

During the last few years it has become evident that many autonomic nervous functions are dependent on multiple messengers, with neuropeptides playing an important role. I n the sympathetic nervous system, the majority of the postganglionic neurons seem to contain neuropeptide Y (NPY) in addition to noradrenaline Correspondence : Dr Siv Nilsson, Department of Physiology and Medical Biophysics, Box 572, S-751 23 Uppsala, Sweden.

(Wahlestedt 1987, Pernow 1988). NPY isa potent vasoconstrictor in itself in many vascular beds (Lundberg & Tatemoto 1982, Lundberg et al. 1985 a, Hellstrom et al. 1985, Rudehill et al. 1986, 1987), but may also modulate the release and effects of noradrenaline. In vztro, low concentrations of NPY, that are without their own vasoconstrictive effect, potentiate the noradrenaline evoked vasoconstriction (Ekblad et al. 1984, Edvinsson et al.1984 a, Edvinsson 1985, Lundberg et al. 1985 b, Pernow et al. 1986).

455

456

5'. F. E. Nilsson

NPY enhances the contraction elicited by transmural nerve stimulation (Ekblad et al. 1984, Lundberg et al. 1985 b, Pernow et al. 1986), despite the fact that the noradrenaline release is inhibited (Lundberg et al. 1985 b, Pernow et al. 1986, Dahlofet al. 1985 a) or unchanged (Ekblad et al. 1984). Thus, it seems that the postjunctional potentiating effect on noradrenaline evoked contraction dominates over the prejunctional inhibition of noradrenaline release (Lundberg et al. 1985 b). In the pithed rat, NPY has similar modulatory effects as in vztro; NPY enhances the pressor response to preganglionic nerve stimulation and phenylephrine (Dahlof et al. 1985 b) and inhibits the release of noradrenaline (Dahlof et al. 1988). NPY-immunoreactive nerve fibres have been demonstrated within the eye of several species (Terenghi et al. 1983, Bruun et al. 1984, Zhang et al. 1984, Stone et al. 1986, Stone 1985). Although the density of the innervation differs, the distribution pattern seems to be about the same in most species (Stone et al. 1986). The iris dilator muscle has a rich innervation, but there are also a few NPY-fibres to the iris sphincter muscle and to the ciliary muscle. Blood vessels in all parts of the uvea (choroid, iris and ciliary body) are innervated by NPY-immunoreactive nerve fibres and there are also such fibres present in the ciliary processes and the outflow apparatus (Bruun et al. 1984, Stone et al. 1986, Stone 1986). Removal of the superior cervical ganglion reduces the number of NPY-immunoreactive nerve fibres in the eye, suggesting an origin in this ganglion and co-localization with noradrenaline (Terenghi et al. 1983, Bruun et al. 1984, Zhang et al. 1984). The innervation pattern suggests that NPY may have effects on smooth muscle activity, ocular blood flow and aqueous humor dynamics. So far, NPY has been shown to modify autonomic responses in both the iris dilatator and sphincter muscle (Piccone et al. 1987, 1988). The main purpose of the present study was to investigate the effects of NPY on the ocular circulation and to examine if it could modify the effect of a low spontaneous sympathetic nerve activity. The effects of NPY on local blood flows in several other tissues were also studied for comparison. The experiments were done with and without a-adrenoceptor blockade. Preliminary .reports on parts of this investigation have

been presented elsewhere (Nilsson 1987, 1988, 1990). MATERIALS AND METHODS A11 experiments were made on albino rabbits (New Zealand White) of either sex, weighing 2.2-3.7 kg. Total uveal blood flow was determined by direct measurement from a cannulated vortex vein in 8 rabbits and regional blood flows were determined with radioactive microspheres in 25 rabbits. Anaesthesia and general operattve procedures. Anaesthesia was induced by i.v. infusion of urethane, 7 ml kg-', of a 25% solution, through a marginal ear vein. Auxiliary dosesofurethane weregiven whenrequired. A servo-controlled heating pad was used to maintain normal body temperature of the animals. All rabbits were tracheotomized, given tubocurarine, 0.5-1 .O mg kg-l (Tubocurana, Nordisk Droge, Copenhagen, Denmark) and artificially ventilated to control the acid-base balance during the experiment. Both femoral veins were cannulated with polyethylene tubing, one for infusion of auxiliary doses of urethane and one for infusion of other drugs. One femoral artery was cannulated for blood pressure registration and the other femoral artery was cannulated for blood sampling. In the microsphere experiments, a catheter, for injection of the microspheres, was inserted into the left heart ventricle, via a brachial artery. Heparin, 500 I U kg-' (HeparinE, KabiVitrum, Stockholm, Sweden), was given i.v. before the start of the experiments. Before and during the experiments, arterial blood samples were collected and the acidbase balance was determined and adjusted to normal values by changing the ventilation and/or administration of a sodium bicarbonate solution. Dzrect determtnatzon of uveal blood flow from a cannulated vortex vezn. After bilateral section of the cervical sympathetic nerves and i.v. administration of indomethacin, 20 mg kg-' (Sigma Chemical Co., St Louis, MO, USA), a vortex vein was cannulated as previously described (Nilsson & Bill 1984). The flow from the vein was determined by an optical dropcounter and the total uveal blood flow (QJ was calculated as four times the flow from the vein. A steel cannula, connected by polyethylene tubing to a pressure transducer, was inserted into the anterior chamber for registration of the intraocular pressure (IOP). This made it possible to calculate the total uveal vascular resistance (R),as the perfusion pressure for the eye can be defined as the difference between the mean arterial blood pressure (MABP) and the intraocular pressure [ R = (MABP-IOP)/QJ. Determination of the total uveal vascular resistance was made before and during i.v. infusion of increasing doses of NPY, 7.5-120 pmol kg-' min-'. Each doselevel was maintained for about 5 min, as this was the

NP Y - Ocular and cerebral vasoconstriction

457

Fig. 1. Increase, in percentage, in uveal vascular resistance caused by intravenous infusion of increasing doses of NPY. Mean values and SE are given ( n = 8).

time required to achieve maximal effect. Synthetic porcine NPY (Sigma Chemical Co., St Louis, MO, USA) dissolved in saline with 0.1% rabbit serum albumin (Sigma Chemical Co.) was used in all experiments. The peptide was kept frozen as a stock solution, which was thawed and diluted immediately before use. Determination of regional bloodflows with radioactive microspheres. Local blood flows and cardiac output were determined with radioactive microspheres (15 pm), according to the reference flow method (Alm & Bill 1972). Microspheres labelled with three different radionuclides, 14'Ce, "3Sn and lo3Ru (NEN, Boston, MA, USA), were used, which made it possible to make three blood flow determinations in each animal. T h e number of injected spheres were between 0.5 x 10' and 2 x 10' per injection. The microspheres were injected into the left heart ventricle over 10-15 s and simultaneously a reference sample was collected from a femoral artery by free flow into pre-weighed plastic tubes during 1 min (10 s per tube). Two different experimental series were made, one without and one with a-adrenoceptor blockade with phenoxybensamine (Dibenylinea, SK&F, Welwyn Garden City, England), 50 mg kg-l. After the blockade, the arterial blood pressure was partly reestablished by intravenous infusion 3 W O ml of a mixture (1 : 1) of saline and Macrodex@ (Pharmacia, Uppsala, Sweden). In both series of experiments, blood flow determinations were made before the start of i.v. infusion of NPY (120 pmol kg-' min-') and at 2 and 10 min after the start of the NPY-infusion. About 30 s before each blood flow determination, the recorder, used for the blood pressure registration, was run at a higher paper speed to allow determination of the heart rate. Immediately after each blood flow determination, an arterial blood sample was collected to determine the acid-base balance.

In rabbits under urethane anaesthesia, sectioning of the cervical sympathetic nerve increases local blood flows in the eye and some other facial tissues (Koskinen & Bill 1984). This suggests that there is a spontaneous sympathetic nerve activity in rabbits under urethane anaesthesia. The cervical sympathetic nerve was therefore sectioned unilaterally in the present experiments to investigate if NPY could modify the spontaneous sympathetic activity and to see if the difference in local blood flow between the sectioned and the intact side persisted after a-adrenoceptor blockade. After the experiments, the animals were killed by injecting a KCI-solution into the left ventricle. The different tissues were dissected and put into preweighed plastic tubes. The tubes containing the reference samples and tissue samples were weighed and then counted in a three-channel gamma-spectrometer. The blood flows were calculated according to the formula : Q, = (CPM,/CPM,) x Ql, where Q, = tissue blood flow, CPM, = radioactivity in the tissue sample, CPM, = radioactivity in the reference sample and Q, = reference flow. CO was calculated as the total injected radioactivity (CPM,,,) divided by the radioactivity in the reference sample (CPM,) multiplied by the reference flow ( Q J [ C O= (CPM,,,/CPM,) x QJ. The blood flow values were expressed in mg min-' for the intraocular tissues, the choroid plexus, the pineal gland and the pituitary gland. All other blood flow values were expressed in gmin-lg-'. The blood flow during the control injection was considered as 100% and the effects of NPY at 2 and 10 min of NPY-infusion were expressed as change from control in percentage. In the experiments with a-adrenoceptor blockade, there was a lower initial blood pressure and a larger rise in mean arterial blood pressure during the NPYinfusion than in the group without a-adrenoceptor

458

L:

S. F. E. Nilsson

lo00

T

1.0 0.8

0.6 0.4

400 0.2

200

0.0

1

0

1 -..2

600 400 200

0

Choroid Ciliary Body

Iris

Fig. 2. Effects of cervical sympathetic nerve section on ocular blood flows in experiments without (a) and with (b) a-adrenoceptor blockade. Blood flow on the sectioned side (filled columns) is compared with blood flow on the intact side (open columns). Mean values and SE are given (a; 12 = 12 and b; n = 13). " P < 0.05 denote difference between the two sides.

blockade. The vascular resistance was therefore calculated for some tissues to allow comparison between the two groups. The vascular resistance was calculated as the mean arterial blood pressure divided by the blood flow. (The vascular resistance for the intraocular tissues was also calculated according to this formula, although the perfusion pressure for the eye is equal to the MABP minus the IOP. The IOP was not measured in the microsphere experiments, however.) The vascular resistance during the control injection was considered as 100% and the vascular resistance during the NPY-infusion was expressed as change from control in percentage. Statistical analysis was made by the two-tailed students' t-test, for paired data, when the intact side was compared with the sectioned side, and by ANOVA followed by Dunnets' test, when comparisons between the three blood flow measurements were made. Pvalues less than 0.05 were considered as significant. All values are given as the mean and the SE unless stated otherwise.

Fig. 3. Effects of cervical sympathetic nerve section on local blood flows in experiments without (a) and with (b) a-adrenoceptor blockade. Blood flow on the sectioned side (filled columns) is compared with blood flow on the intact side (open columns). Mean values and SE are given (a; n = 12 and b; n = 13). " P < 0.05, "*P < 0.01 and ""* P < 0.001 denote difference between the two sides.

RESULTS Dose-response curve Intravenous infusion of increasing doses of NPY caused a dose-dependent increase in the uveal vascular resistance (Fig. 1). Near maximal effect, a 70% increase, was achieved with 120 pmol kg-' min-l. This dose was therefore used in the following microsphere experiments.

Effect o f cervical sympathetic nerve section on local blood flows T h e initial blood flow in the choroid and ciliary body were significantly higher on the side with sectioned cervical sympathetic nerve (Fig. 2a). Significantly higher local blood flow on the

NP Y - Ocular and cerebral vasoconstriction

459

Table 1. Mean arterial blood pressure, cardiac output, heart rate and arterial blood gases during the blood flow determinations in the two experimental series.

Without a-adrenoceptor blockade (n = 12)" Control After 2 min NPY-infusion After 10 rnin NPY-infusion With a-adrenoceptor blockade (n = 13) Control After 2 rnin NPY-infusion After 10 min NPY-infusion

MABP (mmHg)

CO (g min-')

HR (min-')

pH

77f4 79 f4 73 +_ 5

415f29 392 f25 347 f27

312k8 308 f8 300 +_ 9

7.47f0.01 4.6f0.1 7.45 f0.02 4.6 k 0.1 7.46 f0.02 4.5 f0.1

11.9f0.5 12.0f0.3 12.5 f0.4

44f2 50 f2 48 k 2

673k32 666 f27 618 f33

331k5 332 f5 330 f5

7.39f0.01 4.7k0.2 738 f0.02 4.6 f0.1 7.39 k 0.02 4.7 f0.2

11.6f0.3 11.7f0.4 11.4f 0.4

Pco,

Po,

(kP4

(kPa)

" n = 11 for the blood gas values

Table 2. Changes in local blood flows caused by intravenous infusion of NPY, 120 pmol kg-' min-', in experiments with and without a-adrenoceptor blockade. Blood flow determinations were made before (control), and at 2 and 10 min after the start of the NPY-infusion. The values are given as change from control in percentage. Mean values and SE are given. Without a-adrenoceptor blockade (n = 12)

Kidney Spleen Adrenal gland Stomach Duodenum Small intestine Large intestine

With a-adrenoceptor blockade (n = 13)

2 min

10 min

2 min

10 min

(Yo)

(70)

(70)

(%I

-41 f3""" - 52 f7"" - 63 f2""" - 56 f5""" -37f4 - 33 f2""" -19f4""

- 52 k4"""t -21 f 17 - 65 k 3""" - 59 k 6"""

-

-1Of26

-

46 Ifr 3"""

- 44 f6"""

33 k 5""" 37 Ifr 3"'" - 26 & 4""" -

- 23 f5"""t

-16f6"

*

33 3"""

- 36 k 8""

-

- 39 f5""" -21 f 10 - 57 f3-t -40 & 6""" - 17 f6"t - 22 f4"""l - 17 f5""

" P < 0.05, "" P < 0.01 and """P < 0.001 denote difference from control. t p < 0.05 and f P < 0.01 denote difference between 2 and 10 min.

sectioned side was also observed in several other facial tissues and the dura (Fig. 3a). Regional cerebral blood flows were not affected by the nerve section (data shown). In the experiments with a-adrenoceptor blockade, the difference in local blood flow between the two sides was statistically significant only for the choroid, Harderian gland and masseter muscle (Figs. 2 b & 3 b).

Effects of i.v. infusion of N P Y on local blood flows in experiments without a-adrenoceptor blockade The NPY-infusion did not affect the MABP, CO or heart rate significantly. There was a tendency towards an initial increase in the MABP, followed by a decrease, however, and the CO tended to decrease during the NPY-

460

S. F . E. Nilsson

Choroid

(*I

t

Ciliary body

2-1

Iris

tt

10 min

-80

-60

1

t

40

-20

0

20

Choroid 10 min

t

40

@)

'8

t

Ciliary body

2min

Iris 10 min -80

-60

40

-20

Change in blood flow

0

20

40

(0%)

Fig. 4. Changes in ocular blood flows caused by intravenous infusion of NPY (120 pmol kg-' min-'), in experiments without (a) and with (b) a-adrenoceptor blockade. Blood flow determinations were made before (control), and at 2 and 10min after the start of the NPYinfusion. The values are given as the change from control in percentage. Open columns = intact cervical sympathetic nerve and filled columns = cervical sympathetic nerve sectioned. Mean values and SE are given (a; n = 12 and b; n = 13). + P < 0.05, ++P < 0.01 and +*+P< 0.001 denote difference from control. t P < 0.05, tt P < 0.01 and ttt P < 0.001 denote difference between 2 and 10 min.

infusion. The arterial pH, Pco, and Po, did not change significantly between the three blood flow determinations (Table 1). Marked effects of NPY on local blood flows were seen in the kidneys, the spleen and the adrenal glands. In these tissues, local blood flows were decreased by 4&60 yoafter 2 min of NPYinfusion, and not much further decreased at 10 min. The decrease in blood flow in the spleen was even less after 10 min of infusion and was no longer statistically significant (Table 2 ) . The NPY-infusion caused blood flow reductions in most parts of the gastro-intestinal tract at 2 min. At 10 min the effect was reduced in the intestine (Table 2). There was only a small effect in the choroid and ciliary body at 2 min, but at 10 min local blood flows were decreased by more than 50% in the choroid and ciliary body. The effect of NPY was similar in both eyes, regardless of

whether the sympathetic nerve supply was intact or not (Fig. 4a). The iridal blood flow was decreased by about 30% at 10 min, but this effect was statistically significant only for the intact side (Fig. 4a). Retinal blood flow was not affected by the NPY-infusion (data not shown). Among other tissues innervated by the cervical sympathetic nerve, NPY caused blood flow reductions in the salivary and lacrimal glands, the tongue, the masseter muscle and the nictitating membrane (Fig. 5a). In all these tissues, except the tongue and the parotid gland, the effect of NPY was significantly less on the side with intact sympathetic nerve than on the sectioned side at 10 min (PGO.05 or less; t-test, paired data). Total cerebral blood flow tended to be decreased at 10 min, but the effect was not statistically significant. On the intact side, total cerebral blood flow was 0.64 f0.04 g min-l g-'

NPY

Submandibular gl.

10min 2 min

Parotid gl. 10 min t

-

Ocular and cerebral vasoconstriction

461

t

... .*.

2min

Lacrimal gl. 10 min

t t t

Harderian gl.

Nictitating mcmbr.

2*j

10 min

tt.

Tongue tt

. 1 '

2

Masnetcr 10 -80

-60

-40

-20

20 -80

0

Change in blood flow ( O h )

-60

-40

-20

0

20

Change in blood flow (%)

Fig. 5. Changes in local blood flows caused by intravenous infusion of NPY (120 pmol kg-' min-'), in experiments without (a) and with (b) a-adrenoceptor blockade. Blood flow determinations were made before (control), and at 2 and 10 min after the start of NPY-infusion. The values are given as the change from control in percentage. Open columns = intact cervical sympathetic nerve and filled columns = cervical sympathetic nerve sectioned. Mean values and SE are given (a; n = 12 and b; n = 13). " P d 0.05, ""P < 0.01 and ""*P< 0.001 denote difference from control. t P d 0.05, tt P < 0.01 and ttt P < 0.001 denote difference between 2 and 10min.

T a b l e 3. Changes in intracranial blood flows caused by intravenous infusion of NPY, 120 pmol kg-' min-', in experiments with and without a-adrenoceptor blockade. Blood flow determinations were made before (control), and at 2 and 10 rnin after the start of the NPY-infusion. The values are given as change from control in percentage. Mean values and SE are given. I = cervical sympathetic nerve intact and S = cervical sympathetic nerve sectioned.

Choroid plexus Pineal gland Anterior pituitary gland Posterior pituitary gland

I S

Without a-adrenoceptor blockade ( n = 12)

With a-adrenoceptor blockade (n = 13)t

2 min

10 min

(%I

(%I

2 min (%)

-47f5""" -38fll"" -18+8" - 58 f5""" -41+5""*

-41 +6""" -45+7"" - 32 f5"" - 59 4""" - 43 5"""

-25+12 - 53 7""" - 34 IfI 9"" - 60 f6""" -42fll""

+

*

*

" P < 0.05, ""P d 0.01 and *""P d 0.001 denote difference from control. t n = 12 for choroid plexus. 1P d 0.01 denote difference between 2 and 10 min.

10 min

(%I -2Of9 - 20 9"I - 36 f7"" - 55 & 5""" -35 f11"

+

462

S. F. E. Nilsson

before the start of the NPY-infusion and 0.65 f0.05 and 0.54 kO.5 g min-' g-' at 2 and 10 min, respectively. The corresponding values for the sectioned side were 0.63 f 0.04, 0.64 f 0.05 and 0.54f0.05 g min-' g-', respectively. Local blood flows in some parts of the brain were significantly reduced at 10 min, however (Fig. 6). In the choroid plexus, pineal gland and the pituitary gland marked vasoconstriction was seen already after 2 min of NPY-infusion, an effect which persisted at 10 rnin (Table 3).

Effects of Z . V . infusion o f N P Y on local blood Jows in experiments with a-adyenoceptor blockade In these experiments, the NPY-infusion caused an increase in the MABP, which was larger and of longer duration than in the experiments without a-adrenoceptor blockade, although it was not statistically significant. There was no significant change in the C O or heart rate (Table 1). The a-adrenoceptor blockade and the accompanying low MABP caused a metabolic acidosis, which despite administration of sodium bicarbonate made the arterial p H slightly lower in the experiments with a-adrenoceptor blockade than in the experiments without a-adrenoceptor blockade. There was no significant difference in arterial p H between the three blood flow determinations, however (Table 1). Also in these experiments, NPY caused marked blood flow reductions in the kidneys, the spleen, the adrenal glands and the gastrointestinal tract (Table 2 ) . In some of these tissues, the effect of NPY appeared to be somewhat less than in the experiments without a-adrenoceptor blockade, but calculation of the change in vascular resistance revealed that there were no significant differences (data not shown). Local blood flows in the uvea and other cephalic tissues were also significantly decreased by NPY (Figs 4 b 8z Sb), but the decrease seemed to be somewhat less than in the experiments without a-adrenoceptor blockade. As this in part could be due to the larger effect on the arterial blood pressure in the experiments with a-adrenoceptor blockade, the changes in vascular resistance were also calculated (Table 4). The choroid and the tongue (sectioned side) were the only tissues in which the effect of NPY

was significantly different between the two groups. In the choroid, the change in vascular resistance caused by NPY was less in the group with a-adrenoceptor blockade, but there was larger change in the vascular resistance in the tongue. There was no significant change in total cerebral blood flow by NPY. Total cerebral blood flow for the intact and sectioned side was 0.92 k0.08 and 0.94 0.07 g min-lg-', respectively, during the control injection. The corresponding values during the NPY-infusion were 0.90 0.07 and 0.94 +_ 0.07 g min-' g-' at 2 rnin and 0.84k0.06 and 0.85 k0.06 g min-' g-' at 10 min. Neither was there any significant change in local cerebral blood flows, except for the frontal cortex (data not shown). Local blood flows in the choroid plexus (sectioned side), pineal gland, anterior and posterior pituitary gland were significantly decreased by NPY also in these experiments, however (Table 3).

DISCUSSION The results of the present investigation show that NPY is a potent vasoconstrictor in all parts of the rabbit uvea. Generally, the effects of the NPY-infusion on regional blood flows in other tissues are in good agreement with a similar microsphere study in pigs (Rudehill et al. 1987). These authors observed large increases in the vascular resistance in the spleen, kidneys, adrenal glands, skeletal muscles and glandular tissues. There are some major differences between the results of the two studies, however. Firstly, cerebral vasoconstriction was not observed by Rudehill et al. (1987) and secondly, the effects in the gastrointestinal tract were different. Although the highest dose used by Rudehill et al. (1987) was about twice that used in the present experiments, their infusion time was only 3 min, which may explain some of the differences. In the present experiments, vasoconstriction in some areas of the brain was observed after 10 min of infusion, but not at 2 min. Species differences are also likely to exist. In the cat for instance, NPY has much less effect on uveal and local cerebral blood flow than in the rabbit (Granstam & Nilsson 1991). I n a recent study (Granstam & Nilsson 1990), we have shown that a large part of the uveal sympathetic vasoconstriction in rabbits is re-

NP Y - Ocular and cerebral vasoconstriction

Occipital cortex White matter Hippocampal region Caudate nucleus Thalamlc region Hypothalamic region Collicles

Pons + Mesencephalon

..

Medulla oblongata Medulla spin& Cerebellum 40

-30 -20 -10 0 10 Change in blood flow (96)

20

Fig. 6. Changes in local cerebral blood flows caused by intravenous infusion of NPY (120 pmol kg-l min-I). Blood flow determinations were made before (control), and at 2 and 10 min after the start of the NPY-infusion. The values, at 10 min, are given as the change from control in percentage. Open columns = intact cervical sympathetic nerve and filled columns = cervical sympathetic nerve sectioned. Mean values and SE are given ( n = 9 except for the cerebellum, for which n = 12). " P < 0.05 and *+P< 0.0 denote difference from control.

sistant to a-adrenoceptor blockade. This observation, together with the uveal vasoconstriction caused by NPY in the present experiments, strongly suggest that NPY may significantly contribute to the sympathetic vasoconstriction in the rabbit uvea. However, NPY seems not to be equally important for the uveal sympathetic vasoconstriction in all species. I n the cat, NPY has only moderate effects on uveal blood flow and the uveal vasoconstriction during sympathetic nerve stimulation is completely blocked by a-adrenergic blockade (Granstam & Nilsson 1991). Preliminary experiments in monkeys indicate that NPY can cause vasoconstriction in all parts of the uvea in this species (Nilsson, unpublished results). After superior cervical ganglionectomy, the eye is completely devoid of adrenergic nerve fibres (Ehinger 1966), while the NPY-denervation is incomplete (Bruun et al. 1984, Terenghi et al. 1983, Zhang et al. 1984), suggesting that some of the NPY-immunoreactive nerve fibres originate elsewhere. In rats, sympathectomy

463

causes a reinnervation of the iris with NPYimmunoreactive fibres from the ciliary ganglion (Bjorklund et al. 1985). Whether NPY-fibres from the ciliary ganglion are normally present in the eye is unclear, however. Many of the cell bodies in the ciliary ganglion are NPY-immunoreactive, strongly suggesting that this could be the case (Stone et al. 1988). Thus, NPY may not only be involved in sympathetic functions in the eye, but may also contribute to parasympathetic effects, such as the iridal vasoconstriction during oculomotor nerve stimulation (Bill et al. 1976, Stjernschantz et al. 1977, Stjernschantz & Bill 1979). The reason for the smaller effect of NPY on choroidal blood flow in the experiments with aadrenoceptor blockade is not clear. One possible explanation could be the lower initial choroidal blood flow in this group (see Fig. 2), which may have caused a lower tissue concentration of NPY. Interestingly, in the tongue, in which initial blood flow was higher with than without a-adrenoceptor blockade (Fig. 3), there was a significantly larger effect of NPY in the experiments with a-adrenoceptor blockade. Cerebral arteries have a dense innervation of NPY-containing nerve fibres (Edvinsson et al. 1983, 1987, Allen et al. 1984) and NPY constricts cerebral arteries in vitro (Edvinsson 1985, Edvinsson et al. 1983, 1984b, 1987), and pial arteries after perivascular injection (Edvinsson et al. 1984b). Allen et al. (1984) reported that intracarotid infusion of NPY (1-2 nmol) in rats decreased cortical blood flow by up to 95 % and suggested that NPY might be involved in cerebral vasospasm. Local blood flow in the rat striatum is decreased by local microinjection (Tuor et al. 1990) as well as by intracarotid injection (Suzuki et al. 1989). One can, of course, argue that it seems strange that a relatively large molecule like NPY could pass the blood-brain barrier in such large amounts so as to achieve a local concentration high enough to cause vasoconstriction. In the present experiments the most marked effects intracranially were observed in those parts lacking the blood brain barrier, that is the choroid plexus, the pineal gland and the pituitary gland. The effect of NPY on local cerebral blood flows need not necessarily be due to a direct effect on the cerebral vasculature. One may speculate that NPY could act on the endothelium to release some other vasoconstrictive agent,

464

S . F. E. Nilsson

Table 4. Changes in regional vascular resistance caused by intravenous infusion of NPY, 120 pmol kg-' min-l, in experiments with and without a-adrenoceptor blockade. Blood flow determinations were made before (control), and at 2 and 10 min after the start of the NPY-infusion. The values are given as change from control, in percentage, at 10 min. Mean values and SE are given. I = cervical sympathetic nerve intact and S = cervical sympathetic nerve sectioned.

Choroid Ciliary body Iris Submandibular gland Parotid gland Lacrimal gland Harderian gland Nictitating membrane Tongue Masseter

I S I S I S I S I S I S I S I S I S I S

Without a-adrenoceptor blockade (%) (n = 12)

With a-adrenoceptor blockade 1%) (n = 13)

146 f42"" 145 f36""" 124f30""" 146f48'" 67 f28" 53 f25" 61 f31" 114f28""" 35f19 219f99" 56 f22" 92f42" 77 f34" 159&79* 12f19 104f54" 36 f24 49 f 19" 43 f 13"" 336+ 115""

42 f9"""t 68 f 13"""t 71 f20'" 167 f32""" 36f21 75 f 19""" 36 f 13" 64f 10""" 38f 15" 59 f 17"" 28f 10 52f 12'" 28 f 10" 47 f 10"'" 33 f 12" 42 f7""" 89 f 1 1""" 100f12"""~ 62f 14"" 183 f22"""

" P < 0.05,"*P < 0.01 and ""'P < 0.001 denote difference from control. tP < 0.05 denote difference between the two groups (t-test, unpaired data). such as for instance endothelin (Yanagisawa et al. 1988). Another possibility is that the effect of N P Y is due to an effect at the larger cerebral arteries outside the blood brain barrier. T h e lack of effect of N P Y on local cerebral blood flows in the experiments with a-adrenoceptor blockade may suggest that the effect of NPY was secondary to release of noradrenaline. There is another very plausible explanation for the lack of effect in the experiments with aadrenoceptor blockade, however. I n these experiments, the initial MABP was very low, 44 mmHg, which is about equal to the lower limit for autoregulation of cerebral blood flow in the rabbit (Linder & Beausang-Linder 1981). As the NPY-infusion caused a rather large increase in MABP in the experiments with a-adrenoceptor blockade, there may have been a dual effect on cerebral blood flow, if the initial MABP was below the autoregulatory range ; increased cerebral blood flow due to increased perfusion pressure and decreased blood flow due to a direct

effect of NPY on the cerebral blood vessels. Thus, these two effects could have counteracted each other. In the present experiments, there were large reductions in local blood flows in most parts of the gut after only 2 min of NPY-infusion. This is in contrast to the study by Rudehill et al. (1987), who found no effect on intestinal blood flow, although the dose used was twice that used in the present study. However, local intraarterial infusion of NPY causes vasoconstriction in the cat colon (Hellstrom et al. 1985). T h e effect of N P Y on intestinal blood flow in the present experiments was less after 10 than after 2 min of infusion, suggesting that an escape phenomenon might have occurred. NPY had marked effects on local blood flows in many cephalic tissues. I n severa; of these tissues, the effect of NPY was clearly less on the side with intact sympathetic nerve supply, which is in contrast to the effect in the eye. Considering the potentiation by NPY on the noradrenaline

NP Y - Ocular and cerebral vasoconstriction and simulation evoked contraction of blood vessels in vitro (Ekblad et al. 1984, Edvinsson et al. 1984a, Edvinsson 1985, Lundberg et al. 1985b, Pernow et al. 1986), one would have expected NPY to have a larger effect on the intact side than on the sectioned side. T h e postjunctional potentiating effect of NPY has mainly been seen at low doses of NPY that are without own vasoconstrictive effect, however. One may suspect that in the present experiments, with a comparatively high NPY-dose, a prejunctional inhibition of noradrenaline release might have dominated over a postjunctional potentiation of noradrenaline mediated vasoconstriction. I f this was the case in the tissues with intact sympathetic nerve supply, the direct postjunctional effect of NPY might have been counteracted by a decreased noradrenaline contraction, due to prejunctional inhibition of noradrenaline release. I n most tissues the difference in local blood flow between the intact and sectioned side was almost abolished in the experiments with aadrenoceptor blockade. T h i s suggests that most of the spontaneous sympathetic nerve activity in rabbits under urethane anaesthesia is mediated by the release of noradrenaline and that it is of a rather low frequency, as noradrenaline is released already at low stimulation frequencies, while NPY is mainly released at higher frequencies (Lundberg et al. 1986). I n the choroid, masseter muscle and Harderian gland there were significant differences in local blood flows between the two sides also in the experiments with aadrenoceptor blockade, however, suggesting that in these tissues NPY might have contributed to the effect. Interestingly, in the choroid and the masseter muscle a large part of the vasoconstriction caused by sympathetic nerve stimulation is resistant to a-adrenoceptor blockade and in the Harderian gland there was a significant non-adrenergic vasoconstriction even at low stimulation frequencies (Granstam & Nilsson 1990). I n conclusion, the results of the present investigation show that NPY has marked effects on regional blood flow in several tissues, including the eye and the brain. T h e ocular vasoconstrictive effect of NPY suggests that in the rabbit NPY may be responsible for the nonadrenergic part of the uveal vasoconstriction caused by sympathetic nerve stimulation (Granstam & Nilsson 1990).

465

I wish to express my deep gratitude to Ms Annsofi Holst, for expert technical assistance and to Professor Anders Bill for valuable advice and discussions. This work was financially supported by grant 5 R01 EY 00475 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., TODD, N., CROCKARD H.A., SCHON,F., YEATS,J.C. & BLOOM,S.R. 1984. Presence of neuropeptide Y in human circle of Willis and its possible role in cerebral vasospasm. Lancet ii, 550-552. ALM, A. & BILL, A. 1972. The oxygen supply to the retina. 11. Effects of high intraocular pressure and of increased arterial carbon dioxide tension on weal 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, 306-319. J. & ALM, A. 1976. Effects BILL,A., STJERNSCHANTZ, of hexamethonium, biperiden and phentolamine on the vasoconstrictive effects of oculomotor nerve stimulation in rabbits. Exp Eye Res 23, 615462. BJORKLUND,H., HOKFELT,T., GOLDSTEIN,M., TERENIUS, L. & OLSON,L. 1985. Appearance of the noradrenergic markers tyrosine hydroxylase and neuropeptide Y in cholinergic nerves of the iris following sympathectomy. 3.Neurosci 5,1633-1643. A., EHINGER, B., SUNDLER, F., TORNQVIST, K. BRUUN, & UDDMAN, R. 1984. Neuropeptide Y immunoreactive neurons in the guinea-pig uvea and retina. Invest Ophthalmol Vis Sci 25, 1113-1 123. DAHLOF,C., DAHLOF,P., TATEMOTO, K. & LUNDBERG, J.M. 1985a. Neuropeptide Y (NPY) reduces field stimulation-evoked release of noradrenaline and enhances force of contraction in the rat portal vein. Naunyn-Schmiedeb. Arch Pharmacol 328, 327-330. DAHLOF,C., D A H L ~ F P., & LUNDBERG, J.M. 1985b. Neuropeptide Y (NPY): Enhancement of blood pressure increase upon a-adrenoceptor activation and direct pressor effects in pithed rats. European 3 Pharmacol 109, 289-292. DAHLOF, P., PERSON,K., LUNDBERG, J.M. & DAHLOF, C. 1988. Neuropeptide Y (NPY) induced inhibition of preganglionic nerve stimulation evoked release of adrenaline and noradrenaline in the pithed rat. Acta Physiol Scand 132, 51-57. EDVINSSON, L. 1985. Characterization of the contractile effects of neuropeptide Y in feline cerebral arteries. Acta Physiol Scand 125, 3341. EDVINSSON, L., COPELAND, J.R., EMSON,P.C., McCULLOCH,J. & UDDMAN, R. 1987. Nerve fibres containing neuropeptide Y in the cerebrovascular bed : immunocytochemistry, radioimmunoassay, and vasomotor effects.3Cerebral Blood Flow Met 7, 45-57.

466

S.F.E. Nilsson

EDVINSSON, L., EKBLAD, E., HAKANSON, R. & WAHLEcoding of sympathetic transmission: differential release of noradrenaline and neuropeptide Y from STEDT, C. 1984a. Neuropeptide Y potentiates the pig spleen. Neurosci Lett 63, 9 6 1 0 0 . effect of various vasoconstrictor agents on rabbit NILSSON,S.F.E. 1987. Effects of NPY on ocular blood blood vessels. B r 3 Pharmacol 83, 519-525. EDVINSSON, L., EMSON,P., MCCULLOCH, J., TATE- flow and local blood flow in some other tissues in the rabbit. 3 Physiol 390, 119P. MOTO, K. & UDDMAN, R. 1983. Neuropeptide Y: cerebrovascular innervation and vasomotor effects NILSSON,S.F.E. 1988. NPY as a possible mediator of sympathetic vasoconstriction in the rabbit uvea. in the cat. Neurosci Lett 43, 79-84. EDVINSSON, L., EMSON,P., MCCULLOCH, J., TATE- Invest Ophthalmol Vis Sci 29, Suppl., p. 323. MOTO, K. & UDDMAN, R. 1984b. Neuropeptide Y: NILSSON,S.F.E. 1990. NPY causes non-adrenergic vasoconstriction in the eye, brain and some other immunocytochemical localization to and effect upon tissues in rabbits. Annals NAS (in press). feline pial arteries and veins in vitro and in situ. NILSSON, S.F.E. &BILL,A. 1984. Vasoactive intestinal Acta Physiol Scand 122, 155-163. polypeptide (VIP): Effects in the eye and on EHINGER,B. 1966. Ocular and orbital vegetative regional blood flows. Acta Physiol Scand 121, nerves. Acta Physiol Scand 67, Suppl. 268, 1-35. 385-392. EKBLAD,E., EDVINSSON,L., WAHLESTEDT,C., J. 1988. Co-release and functional interUDDMAN, R., HAKANSON, R. & SUNDLER, F. 1984. PERNOW, actions of neuropeptide Y and noradrenaline in Neuropeptide Y co-exists and co-operates with peripheral sympathetic vascular control. Acta noradrenaline in perivascular nerve fibres. Regul Physiol Scand 133, Suppl. 568, 1-56. Peptides 8, 225-235. A. & LUNDBERG, J.M. 1986. GRANSTAM, E. & NILSSON, S.F.E. 1990. Non- PERNOW,J., SARIA, Mechanisms underlying preand postjunctional adrenergic vasoconstriction in the eye and some effects of neuropeptide Y in sympathetic vascular other facial tissues in the rabbit. European 3 control. Acta Physiol Scand 126, 239-249. Pharmacol 175, 175-186. R., DAVIS,M. & GRANSTAM, E. & NILSSON,S.F.E. 1991. Effects of PICCONE,M., KRUPINT., STONE, WAX, M. 1987. Effects of neuropeptide Y on cervical sympathetic nerve stimulation and neuisolated rabbit iris sphincter muscle. Invest Ophthropeptide Y (NPY) on cranial blood flow in the cat. almol Vis Sci 28, Suppl., p. 67. Acta Physiol Scand (in press). PICCONE, M., LITTZI,J., KRUPIN,T., STONE, R.A., HELLSTROM, P.M., OLERUP,0. & TATEMOTO, K. DAVIS,M. & WAX,M.B. 1988. Effects of neuro1985. Neuropeptide Y may mediate effects of peptide Y on the isolated rabbit iris dilator sympathetic nerve stimulation on colonic motility muscle. Incest Ophthalmol Vis Sci 29, 330-332. and blood flow in the cat. Acta Physiol Scand 124, RUDEHILL, A., SOLLEVI, A,, FRANCO-CERECEDA, A. & 613-624. LUNDBERG, J.M. 1986. Neuropeptide Y (NPY) and KOSKINEN,L.-0. & BILL, A. 1984. Thyrotropinthe pig heart : Release and coronary vasoconstrictor releasing hormone (TRH) causes sympathetic effects. Peptides 7, 821-826. activation and cerebral vasodilation in the rabbit. RUDEHILL, A,, O L C ~ NM., , SOLLEVI, A,, HAMBERGER, Acta Physiol Scand 122, 127-136. B. & LUNDBERG, J.M. 1987. Release of neuropeptide LINDER, J. & BEAUSANG-LINDER, M. 1981. Autonomic Y upon haemorrhagic hypovolaemia in relation to influence on cerebral and ocular blood flow. Acta vasoconstrictor effects in the pig. Acta Physiol Univ Ups 406, 1-51. Scand 131, 517-523. LUNDBERG, J.M. & TATEMOTO, K. 1982. Pancreatic STJERNSCHANTZ, J. & BILL, A. 1979. Effects of polypeptide family (APP, BPP, NPY and PYY) in intracranial stimulation of the oculomotor nerve on relation to sympathetic vasoconstriction resistant to ocular blood flow in the monkey, cat, and rabbit. a-adrenoceptor blockade. Acta Physiol Scand 116, Invest Ophthalmol Vis Sci 18, 99-103. 393402. STJERNSCHANTZ, J., ALM, A. & BILL, A. 1977. LUNDBERG, J.M., ANGGARD,A,, PERNOW,J. & Cholinergic and aminergic control of uveal blood HOKFELT, T . 1985a. Neuropeptide Y- substance Pflow in rabbits. Bib1 Anat 16, 4 2 4 6 . and VIP-immunoreactive nerves in cat spleen in STONE,R.A. 1986. Neuropeptide Y and the innerrelation to autonomic vascular and volume control. vation of the human eye. Exp Eye Res 42, Cell Tissue Res 239, 9-18. 349-3 5 5. LUNDBERG, J.M., PERNOW,J., TATEMOTO, K. & STONE,R.A., LATIES,A.M. & EMSON,P.C. 1986. DAHLOF,C. 1985 b. Pre- and postjunctional effects Neuropeptide Y and the ocular innervation of rat, of NPY on sympathetic control of rat femoral guinea pig, cat and monkey. Neurosci 17,1207-1216. artery. Acta Physiol Sand 123, 511-513. STONE, R.A., MCGLINN,A.M., KUWAYAMA, Y. & LUNDBERG, J.M., RUDEHILL, A,, SOLLEVI, A,, THEO- GRIMES,P.A. 1988. Peptide immunoreactivity of DORSSON-NORHEIM, E. & HAMBERGER, B. 1986. ciliary ganglion and its accessory cells in the rat. Frequency- and reserpine-dependent chemical Brain Res 475, 389-392.

NP Y - Ocular and cerebral vasoconstriction

467

SUZUKI, Y., SATOH, S., IKEGAKI, I., OKADA,T., WAHLESTEDT, C. 1987. Neuropeptide .Y (NPY): SHIBUYA, M., SUGITA,K. & ASANO, T . 1989. actions and interactions in neurotransmission. Doctoral thesis, University of Lund, pp. 1-77. Effects of neuropeptide Y and calcitonin geneM., KURIHARA, H., KIMURA,S., related peptide on local cerebral blood flow in the YANAGISAWA, TOMOBE, Y., KOBAYASHI, M., MITSUI, Y., YAZAKI, rat striatum. 3 Cereb Blood Flow Metab 9, 268-270. TERENGHI, G., POLAK,J.M., ALLEN,J.M., ZHANG, Y., GOTO, K. & MASAKI,T. 1988. A novel vasoconstrictor peptide produced by vascular endoS.Q, UNGER, W.G. & BLOOM, S.R. 1983. Neurothelial cells. Nature 332, 411415. peptide Y-immunoreactive nerves in the uvea G., UNGER,W.G., ENNIS, ZHANG,S.Q, TERENGHI, of guinea pig and rat. Neurosci Lett 42, 33-38. J. 1984. Changes in substance PK.W. & POLAK, L. & TUOR, U.I., KELLY, P.A.T., EDVINSSON, and neuropeptide Y-immunoreactive fibres in rat MCCULLOCH, J. 1990. Neuropeptide Y and the and guinea-pig irides following unilateral sympacerebral circulation. 3 Cereb Blood Flow Metab 10, thectorny. Exp Eye Res 39, 365-372. 591-601.