Prepro-Vasoactive Intestinal Polypeptide-Derived ... - SAGE Journals

Journal of Cerebral Blood Flow and Metabolism 11:932-938 © 1991 The International Society of Cerebral Blood Flow and Metabolism Published by Raven Press, Ltd., New York

Prepro-Vasoactive Intestinal Polypeptide-Derived Peptide Sequences in Cerebral Blood Vessels of Rats: On the Functional Anatomy of Metabolic Autoregulation

En-Tan Zhang, *Jens Darnsgaard Mikkelsen, tJan Fahrenkrug, *Morten Mpller, :j:Dorte Kronborg, and Martin Lauritzen Institute of General Physiology and Biophysics, *Institute of Medical Anatomy, Department B, University of Copenhagen; tDepartment of Clinical Chemistry, Bispebjerg Hospital; and tinstitute of Theoretical Statistics, Copenhagen Business School, Copenhagen, Denmark

Summary: This study describes the distribution of pep tide sequences derived from the prepro-vasoactive intes tinal polypeptide (preproVIP) molecule in perivascular nerves of rat brain arteries and arterioles. The peptides were identified by immunohistochemistry using highly specific antibodies. Five peptide sequences (preproVIP 60-76, peptide histidine isoleucine (PHI), preproVIP 111122, VIP, and preproVIP 15�170) were identified in the perivascular nerves throughout the arterial cerebral cir culation. The density of the immunoreactive fibers was

Dendritic processes of neocortical neurons immunoreac tive for VIP and PHI could be followed towards the brain surface where the processes penetrated into the pial layer, often close to the pial vasculature. Some of the processes were also observed to enter the Virchow-Robin space, close to the arterioles. It is possible that cortical nerve cells containing VIP and PHI release the peptides in the perivascular space during periods of activity and thereby contribute to local vasodilatation associated with changes of neuronal function. Key Words: Vasoactive in

highest in the nerves of the larger extracerebral arteries, declining in smaller branching arteries. All peptide se quences were identified in the nerves of small pial arte rioles overlying the cortical convexity, whereas capillar ies and veins contained no immunoreactive material.

testinal polypeptide (VIP)-Peptide histidine isoleucine VIP precursor-Immunohistochemistry-Radioimmuno assay-Cerebral blood vessels-Innervation-Rat Metabolic autoregulation.

Rat vasoactive intestinal polypeptide (VIP) is

peptide sequences besides the signal peptide con

synthesized from a precursor, preproVIP, consist

tained in the preproVIP molecule are preproVIP

ing of about 170 amino acid residues (Nishizawa et

22-79 (N-terminal flanking peptide) preproVIP 111-

aI., 1985). PreproVIP can be divided functionally

122 (bridging peptide), and preproVIP 156-170 (C

into six domains (Nishizawa et aI., 1985) that in

terminal flanking peptide).

clude the amino acid sequences of VIP and peptide

Nerve fibers containing VIP abound around ce

histidine isoleucine (PHI), which are biologically

rebral blood vessels in a number of species includ

active (e.g. Edvinsson and McCulloch, 1985). Other

ing mouse, gerbil, rat, cat, dog, pig, cow, monkey, and humans (Larsson et aI., 1976; Edvinsson et aI., 1980; Matsuyama et aI., 1983; Gibbins et aI., 1984; Lee et aI., 1984; Edvinsson and McCulloch, 1985;

Received January 17, 1991; revised April 9, 1991; accepted April 23, 1991. Address correspondence and reprint requests to Dr. M. Lau ritzen at Department of General Physiology and Biophysics, Pa num Institute, Blegdamsvej 3c, 2200-DK, Copenhagen N, Den mark.

Hara et aI., 1985; Suzuki et aI., 1988; U emura et al., 1988; Hara et aI., 1989; Miao and Lee, 1990). In rats and dogs, the nerve cell bodies giving rise to the VIPergic fibers of the large cerebral arteries are contained in the ipsilateral sphenopalatine and otic

Abbreviations used: BA, basilar artery; ICA, inferior cerebel

lar artery; PBS, potassium-phosphate-buffered saline; PBS-TX, PBS containing Triton X-IOO; PHI, peptide histidine isoleucine; SCA, superior cerebellar artery; VA, vertebral artery; VIP, va soactive intestinal polypeptide.

ganglia and in the internal carotid miniganglion (Su zuki et aI., 1988; U emura et aI., 1988; Hara et aI., 1989). VIP dilates cerebral blood vessels (Larsson

932

PREPROVIP PEPTIDE SEQUENCES IN RAT BRAIN et ai. , 1976; Wei et ai., 1980; McCulloch and Ed vinsson, 1980; Edvinsson and McCulloch, 1985; Dacey et ai., 1988). In this study, newly developed antibodies were applied to study the distribution of the five afore mentioned peptide sequences of the VIP precursor in the large cerebral blood vessels of rats, in the pial arterioles at the cerebral convexity, and within the cortex itself.

MATERIALS AND METHODS

933

swineserum in PBS, followed by incubation in the pri mary antiserum for 24 h at 4°C. After three lO-min rinses in PBS containing 0.5% Triton X-IOO (PBS-TX), sections were incubated for 60 min in swine antirabbit IgG (No Z0196, Dakopatt, Copenhagen) in dilution 1:50, rinsed three times for 10 min each, in PBS-TX, and incubated in rabbit peroxidase-anti-peroxidase (PAP) conjugates (No ZO113, Dakopatts, Copenhagen) diluted 1:100 for another 60 min. After rinsing in PBS for 10 min and in TRIS/HCl (pH 7.6) for 10 min, sections were incubated in a solution of 0.05% 3,3'-diaminobenzidine containing 0.003% H202 for 15 min, followed by a rinse in distilled water. Subse quently, the sections were placed on gelatinized glass sec tions and air dried. Finally, the vascular preparations and brain sections were coverslipped.

Antisera Rabbit antisera were raised against VIP, PHI, and syn thetic fragments of the VIP precursor. The VIP antiserum was obtained from Cambridge Research Biochemicals (Cambridge, U.K.) and has been shown not to crossreact with somatostatin or PHI. The PHI antiserum (No. 36685) does not show any crossreactivity with VIP or several other regulatory peptides (Fahrenkrug et aI., 1985), but displays about 1 % crossreactivity with corticotropin re leasing factor (H6kfelt et aI., 1987). The synthetic pep tides preproVIP 111-122 and preproVIP 156-170 were ob tained from Peninsula (Burlingame, California, U.S.A.). Solid-phase synthesis was used for the production of pre proVIP 60-76 (Larsen et aI., 1989). Peptides were cou pled to bovine serum albumin with glutaraldehyde and injected subcutaneously in rabbits. The specificity of the antibodies against the latter three peptide sequences has been described recently (Fahrenkrug and Emson, 1989). The following dilutions of the raised antisera were used for immunohistochemistry: VIP, I :400; PHI (code no. 3668-5), 1:1600; preproVIP 60-76 (code no. 8031-1), 1:400; preproVIP 111-122 (code nos. 185B-9 and 9B66-9), 1:400; the preproVIP 156-170 (code no. 7314-5), 1:400.

Animal preparation Adult male Wistar rats (250-350 g) were housed under standard laboratory conditions with free access to food and water. The animals were deeply anesthetized with tribromethanol intraperitoneally (400 mg/kg) and perfused via a cannula inserted in the left ventricle with potassium phosphate-buffered saline (PBS), pH 7.4, containing 15,000 IU heparin per liter for about 2 min. Subsequ�ntly, the rats were perfused with 4% paraformaldehyde III 0.1

M phosphate buffer, pH 7.4, for 15 min. Brains were rapidly removed from the skull, and the major extracere bral arteries on the base of the brain, and the meninges with adherent blood vessels covering the superolateral surface of the cerebral hemispheres were removed by careful dissection under a stereo microscope. The ves sels, the meningeal preparations and the brains were post fixed in the same fixative for 4 h at 4°C. Subsequently the vessels and the meningeal preparations were washed in PBS overnight. The brains were cryoprotected in 20% sucrose in PBS for 3-5 days, cut in a cryostat into 40 j.1m thick coronal sections, and rinsed in PBS.

VIP radioimmunoassay For determination of tissue concentrations of VIP, rats were anesthetized as described above, decapitated, and the brains rapidly removed and placed in icecold saline. The basilar artery, the meninges, and the parietal cortices on the two sides were dissected. Subsequently, the spec imens were dried on filter paper, weighed at room tem perature, and stored at - 80°C until extraction and radio immunochemical determination of VIP as previously de scribed (Fahrenkrug and Schaffalitzky de Muckadell, 1977,1978) could be performed.

Estimation of the density of immunoreactive perivascular fibers The density of the nerve fibers exhibiting immunoreac tivity for the peptides in the perivasculature was quanti fied in whole-mount preparations of the basal vasculature belonging to the posterior part of the circle of Willis. The immunoreactive fibers within a unit area (0.2 x 0.2 mm) of a segment of the larger cerebrovascular vessels were counted in the following way: The microscope was fo cussed on the anterior wall of the vessel (closest to the cover slip) and all positively stained nerve fibers were counted within the unit area. A dichotomizing nerve fiber within the unit area was counted as two fibers. In case of two dichotomizing sites the fibers were counted as three fibers, etc. The basilar artery (BA) was examined at three locations (posterior, middle, and anterior), whereas the vertebral artery (VA), the superior cerebellar artery (SCA), and the inferior cerebellar artery (ICA) were ex amined at only one site. For statistical purposes, a general multivariate analysis of variance technique was used to compare the density of perivascular nerve fibers at different sites. Comparisons were performed on a logarithmic scale to fulfil the condi tion of normally distributed variables. Firstly, the four peptide groups (VIP, PHI, preproVIP 22-79 and 111-122) were compared with respect to shifts in the density of nerve fibers at the sites compared by analysis of the in dividual differences. Letting xip denote the density of nerve fibers measured on a logarithmic scale at the i'th site for a given rat in the p'th peptide group, the hypoth esis analyzed was whether the mean of (x1p - xZp' xZp X3 ) could be assumed to be independent of peptides. S ccessively, the density of nerve fibers was compared

tf

Immunohistochemical procedure After rinsing in PBS (pH 7.4) three times for 10 min each, free-floating brain sections, blood vessels, and meningeal preparations were preincubated in 10%

over sites by testing the hypothesis that the mean of the above differences could be assumed to be zero. Wilks lambda was used as test statistic and the corresponding exact F-tests are given as result (Grizzle and Allen, 1969).

J Cereb Blood Flow Metab, Vol. 11, No. 6, 1991

934

E-T ZHANG ET AL. RESULTS

Cell bodies showing immunoreactivity for all five peptide sequences, mainly bipolar neurons in layers II-IV (Figs. 1-6), were abundant in the cortex. VIP and PHI stained particularly well and were consid ered of special interest due to their vasodilatory properties (Larsson et aI., 1976; Edvinsson and Mc Culloch, 1985). Some dendritic processes presum ably arising from intracortical bipolar neurons ex hibiting VIP and PHI immunoreactivity were found to extend into the pial tissue on the brain surface where the processes sometimes dichotomized. Im munoreactive fibers were also observed in the peri vascular pial tissue following the cortical arterioles from the surface into the brain (Virchow-Robin space). Thus, pial arterioles are innervated by ner:v e fibers containing VIP and PHI that appear to ong inate from bipolar neurons within the cerebral cor tex. Well-developed plexuses of perivascular nerve fi bers containing immunoreactive material for each of the five preproVIP derived peptide sequences were also observed in the walls of all major cerebral arteries (Fig. 7A-D), but not around veins or capil-

laries. Table 1 summarizes the density estimates of immunoreactive fibers at various locations through out the posterior portion of the circle of Willis. The shifts in fiber density from the posterior to the mid dle site and from the middle to the anterior site of the BA were identical for the four peptides [F(6,18) =

2.31, P

=

0.08] and the density decreased signif

icantly in the posterior to anterior direction [F(2, 12) =

14.73, p

=

0.0006]. Between the VA and the

posterior portion of the BA, the differences in fiber density did not depend on peptide [F(6,20) P

=

=

1.14,

0.38] and the density was significantly lower in

the VA than in the posterior region of the BA [F(2,13)

=

35.80, p � 0.0001]. The fiber density

decreased in the SCA as compared to its site of origin at the anterior part of the BA [F(2,11) 12.45, P

=

=

0.0015], and in the ICA as compared to

the middle part of the BA [F(2,12)

=

74.54, p �

0.0001] without differences between the four pep tides [F(6,16) p

=

=

0.58, p

=

0.75 and F(6,18)

=

0.80,

0.58, respectively]. Thus, the pattern of distri

bution of immunoreactive fibers in the posterior part of the cerebral circulation was similar for the four peptides, though the absolute fiber density dif fered. This could reflect true differences of peptide concentration, differences of antisera avidity for the

FIG. 1. Frontal sections of part of rat neocortex showing peptide histidine isoleucine immunoreactiv� neurons mo�tly of the bipolar type. Processes from the Immunoreactive perikarya can be followed into the molecular layer and �ur ther into the pial tissue (arrow) covering the surface. Original magnification, x 280.


FIG. 2. Frontal section of part of the cingulate cortex show ing peptide histidine isoleucine immunoreactive perikary . � with processes extending into the pial layer (arrows). Origi nal magnification, x 450.

PREPROVIP PEPTIDE SEQUENCES IN RAT BRAIN

935

FIG. 3. A peptide histidine isoleucine immunoreactive pro cess, originating from an intracortical perikaryon extending to a pial arteriole (a). Original magnification, x450.

FIG. 5. Single fibers immunoreactive to vasoactive intestinal polypeptide in the meningeal layer of the superolateral sur face of the rat brain. Whole mount preparation. Original mag nification, x620.

FIG. 4. Nerve fibers, immunoreactive for prepro-vasoactive intestinal polypeptide 111-122 surrounding a meningeal ar teriole. Whole mount preparation. Original magnification,

FIG. 6. Peptide histidine isoleucine immunoreactive fibers surrounding a pial arteriole from the superolateral surface of the rat brain. Whole mount preparation. Original magnifica tion, x245.

x620.


936

E-T ZHANG ET AL.

(B)

FIG. 7. Photomicrographs of whole mount preparations of the basilar arteries reacted with antisera against the following components of the prepro-vasoactive intestinal polypeptide (preproVIP) molecule: vasoactive intestinal polypeptide (VIP; A), peptide histidine isoleucine (PHI; B), preproVIP 111-122 (C), and preproVIP 22-79 (D). The fiber densities of the VIP, PHI, preproVIP 111-122 immunoreactive fibers were similar, whereas the fibers immunoreactive with preproVIP 22-79 were less abundant. A and B, original magnification, x 450. C, original magnification, x 280. 0, original magnification, x 710.

J Cereb Blood Flow Metab, Vol. 11, No.6, 1991

PREPROVIP PEPTIDE SEQUENCES IN RAT BRAIN

937

TABLE 1. Number of perivascular nerves per 0.2 x 0.2 mm in the major cerebral arteries of the posterior portion of the circle of Willis BA

No. of rats

P

M

A

VA

3

23.0 ± 4. 4

18. 7 ± 1.5

14.5 ± 0. 7

17. 2 ± 3. 1

4

20. 3 ± 5. 4

15.3 ± 4. 1

11.3 ± 4. 4

4

10.0 ± 3.2

9. 0 ± 2.7

4.8 ± 1.3

3

26. 3 ± 2.1

25.0 ± 1.7

19.7 ± 0. 6

PHI VIP PreproVIP 60---76 PreproVIP 111-122

SCA

ICA

8. 8 ± 2. 5

7.8 ± 2.1

12. 0 ± 4. 0

7.1 ± 1.9

5. 4 ± 0.9

4.9 ± 1. 6

2. 6 ± 1. 9

3. 8 ± 1.7

18. 2 ± 2. 8

14. 8 ± 3. 2

9. 3 ± 1.8

Values are means ± SD. For statistics see the text. BA, basilar artery; P, posterior; M, middle; A, anterior; VA, vertebral artery; SCA, superior cerebellar artery; ICA, inferior cerebellar artery; PHI, peptide histidine isoleucine; VIP, vasoactive intestinal polypeptide.

peptides, different sensitivity to fixation, or other

(Loren et aI., 1979; Eckenstein and Baughman,

unknown factors.

1984), but the source for the vascular innervation

Figures 4-6 show the innervation of the pial ar

has remained unclear. In the present study it was

terioles adherent to the dissected meninges. The fi

shown that intracortical processes, apparently con

ber density was much lower here than in the larger

tinuous with cell bodies immunoreactive for VIP

arteries. This correlates with a lower concentration

and PHI, penetrate the cortical surface to enter the

of VIP in the meningeal preparation as compared

pial membrane. The dichotomization of the same

with the BA (Table 2).

immunoreactive nerve fibers in the pia indicates in nervation from cortical neurons. Other processes obtained close contact with the intracortical pial ar

DISCUSSION

The present study demonstrated that all five pep tide sequences derived from the prepro VIP mole cule were represented in the cerebral arteries and arterioles of rats. The immunoreactive fibers had a similar, but uneven distribution throughout the pos terior part of the cerebral circulation in accordance with previous observations in cats, rats, and dogs (Larsson et aI., 1976; Edvinsson et aI., 1980; Mat suyama et al., 1983; Gibbins et al., 1984; Hara et al., 1985; Uemura et aI., 1988). The physiological im portance of the uneven regional fiber density is un clear, but may reflect that the peptidergic fibers originate from different ganglia (Gibbins et aI., 1984; Hara et aI., 1985). The similar distribution of the five peptides suggests that they are colocalized. It is yet unknown whether the peptides are stored in nerve terminals as parts of the precursor molecule or as cleaved fragments, and where the precursor

terioles within the Virchow-Robins space. It is pos sible that increased activity of neurons containing VIP or PHI causes release of the two peptides that in turn trigger vasodilatation within the same re gion, serving as the coupling agent between nervous activity and blood flow (Ingvar and Lassen, 1975). Thus, peptides derived from the preproVIP mole cule (including VIP and PHI) may participate in lo cal blood flow regulation as suggested previously for VIP (Eckenstein and Baughman, 1984; Lee et aI., 1984). The biological effect of preproVIP 22-79, preproVIP 111-122, and preproVIP 156-170 in the peripheral and central nervous system remain to be elucidated. Acknowledgment: This work was supported by the Medical Research Council (Denmark), The Danish Mi graine Society, and the Foundation for Experimental Re search in Neurology.

molecule undergoes posttranslational modifica tions: in the ganglionic cell body, during axonal transport, or at the perivascular nerve terminal. Previous studies have shown that VIP immuno reactive fibers from the cortex on some occasions make intimate contacts with cerebral blood vessels TABLE 2. Vasoactive intestinal polypeptide concentrations determined by radioimmunoassay

artery

Leptomeninges

Cortex

48.3 ± 40.8

9.8 ± 4. 5

52.0 ± 16.8

=

(n

5)

Values are means

±

=

14)

SD in pmol g-l wet weight.

(n

=

Dacey RO, Bassett JE, Takayasu M (1988) Vasomotor responses of intracerebral arterioles to vasoactive intestinal peptide, substance P, neuropeptide Y, and bradykinin. J Cereb Blood Flow Metab 8:254-261 Eckenstein F, Baughman RW (1984) Two types of cholinergic innervation in cortex, one co-localized with vasoactive in testinal polypeptide. Nature 309:153-155 Edvinsson L, Fahrenkrug J, Hanko J, Owman C, Sundler F, Uddman R (1980) VIP (vasoactive intestinal polypeptide)

Basilar

(n

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E-T ZHANG ET AL.

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