Neuroscience.
1977. Vol. 2, pp 751-754
Pcrgamon
Press. Pnnted III Great B&m.
PEPTIDERGIC (VASOACTIVE INTESTINAL PEPTIDE) NERVES IN THE GENITO-URINARY TRACT P. ALM, J. ALUMETS,R. HKKANSON and F. SUND~ Departments of Histology and Pharmacology, University of Lund, Lund, Sweden Abstract-The feline genito-urinary tract receives a rich supply of nerve fibers displaying immunoreactivity like that of vasoactive intestinal peptide. Nerves containing vasoactive intestinal peptide are particularly numerous in the trigonum area of the bladder, around the ureteral openings and in the upper part of the urethra in both sexes, in the epididymis, prostate and vas deferens, and in the uterine cervix. Ganglia located close to or within the wall of the trigonum area and of the upper urethra contain numerous immunoreactive nerve cell bodies, which may be the origin of the fibers containing vasoactive intestinal pcptide that innervate these regions.
adult cats of each sex. The cats were killed by an overdose of sodium pentobarbitone (Nembutal). The specimens were frozen to the temperature of liquid nitrogen in a mixture of propane and propylene. After freeze-drying, the specimens were fixed with diethyl-pyrocarbonate vapour (P~ARSE,POLAK,ADAMS& KENDALL,1974) and embedded in paraffin. Sections were cut at 5 pm, departinized and processed for immunofluorescence using the anti-VIP serum in dilution 1:80 (LARSSONet al., 19766; FAHRENKRUG& SCHAFFALITZKY DE MUCKADELJ_, 1977). For conneuronal peptide in addition to being a hormone, or trols in the immunohistochemical procedure we used a neuronal peptide only (BRYANT, BLOOM, POLAK, antiserum inactivated by the addition of excess antigen ALBUQUERQUE, MODLIN & F’EARSE, 1976; LARSSON, (30 nmol of pure porcine VIP/ml diluted antiserum). EDVINSSON, FAHRENKRUG, HAKANSON, OWMAN, SCHAFFALITZKY DE MUCKADELL& SUNDLER,1976a; RESULTS LARSSON,FAHRENKRUG,SCTHAFFALITZKY DE MUCKAImmunoreactive nerves were occasionally seen in DELL, SUNDLER,H.&KANSON & REHFELD,1976b; SAID & ROSENBERG, 1976; SUNDLER,ALLJMETS, H~KANSON, the stroma of testes and in the smooth muscle of the INGEMANSSON, FAHRENKRUG& SCHAFFALITZKYDE ureters and urinary bladder. VIP-containing nerves were quite numerous in the trigonum area of the MUCKADELL, 19776). So far, VIP-like material has bladder, particularly around the openings of the been detected immunochemically or immunohistochemically in hypothalamic neurons (LARSSONet al., ureters (Fig. l), in the upper part of the urethra and 1976b), in nerves innervating brain vessels (LARSON in the epididymis. The prostate and vas deferens were et al.,1976a), in nerves running in the pancreatic densely innervated (Fig. 2a). Most of the VIP-containparenchyma (LARSON et nl., 1976b; SUNDLER, ing nerves in the bladder, urethra and vas deferens AL~METS, H~ANSON, FAHRENKRUG& SCHAFFA- were distributed within the smooth muscle and submucosal layers. In the female cat, VIP-containing LITZKYDE MUCKADELL,1977u) and in nerves of the gut wall (BRYANTet al., 1976; LARSSONet al., 1976b; nerves were occasionally seen in the ovaries, fallopian tubes, uterine body, and upper part of the vagina. SUNDLEXet al., 1977b). We now wish to report on a rich VIP innervation of the feline genito-urinary of VIP-containing An abundancy nerves was tract, particularly dense in the uterine cervix and vas observed in the uterine cervix (Fig. 2b). Here, the deferens. nerves were predominantly located in the connective
WHEN ‘vasoactive intestinal polypeptide’ (VIP) was isolated from the gut by SAID & M~IT (1970) it was first thought of as a hormone. This contention was based primarily on the chemical similarity between VIP on the one hand and secretin and glucagon on the other Mum, 1972). Recently, however, several groups have independently demonstrated VIP-like immunoreactivity in neuronal elements and have reached the conclusion that VIP is either a
EXPERIMENTAL
PROCEDURES
The investigation was carried out by immunohistochemistry using a previously characterized antiserum against porcine VIP (L-SON et al., 1976b), generously supplied by Drs J. Fahrenkrug and 0. Schaffalitzky de Muckadell, Bispebjerg Hospital, Copenhagen, Denmark. Tissue specimens were collected from the kidneys, ureters, bladder, ovaries, fallopian tubes, uterus, vagina, prostate, vesicula seminalis, epididymis, testes and urethra of three Abbreviation:
VIP, vasoactive
intestinal
peptide.
751
tissue separating the glands. In addition, a dense network of VIP-containing terminals was seen around large blood vessels running close to or within the wall of the cervix, prostate, bladder urethra (Fig. 2~). Numerous VIP-immunoreactive nerve cell bodies were seen in bilateral ganglia located in the trigonum region of the bladder and extending down along the upper urethra (Fig. 2d). Conceivably, the VIP-containing nerves of the genito-urinary tract originate in local ganglia. All immunohistochemical controls were negative.
752
P. ALM. J. ALUMETS. R. HAKANSON and F.
DISCUSSION
The distribution pattern of VIP-containing nerve terminals suggests that they participate in regulating smooth muscle activity and possibly epithelial cell activity as well. Our observations are in agreement with electron microscopic findings of nerve terminals thought to be peptidergic (UNSICKER, 1973; BLJRNSTOCK, 1972). from
adrenergic
of large.
These
dense-core
electron-lucent
terminals
and cholinergic vesicles
vesicles.
and
It should
can
be
majority of the cell bodies in the ganglia do not display VIP immunoreactivity. If they are ‘peptidergic’ too. they probably contain peptides distinct from VIP. The observations made to date indicate that VIP-containing nerves have a ubiquitous occurrence in the body and that VIP may exert a neuronal rather than a hormonal function.
distinguished
ones by their virtual
SUNDLER
lack
be noted
content of small,
Acknowledgements-Grant support from the Swedish Medical Research Council (04X-4499) and the Ford Foun-
that
dation
the
(680-0383 A).
REFERENCES BRYANT M. G., BLWM S. R., POLAI~ J. M.. ALBUQUERQUE R. H.. MODLIN J. & PEARSE A. G. E. (1976) Possible dual role for vasoactive intestinal peptide as gastrointestinal hormone and neurotransmitter substance. Lancer I, 991-993. BURNSTOCK G. (1972) Purinergic nerves. Pharmac. Rev. 24, 509-581. FAHRENKRUG J. & SCHAFFALITZKY DE MUCKADELL 0. B. (1977) Radioimmunoassay of vasoactive intestinal polypeptide (VIP) in plasma. J. lab. clin. Med. To be published. LARSSON L.-I., EDWNSSON L., FAHRENKRUG J., HAKANSON R.. OWMAN C., SCHAFFALITZKY DE MUCKADELL 0. & SUNDLER F. (1976a) Immunohistochemical localization of a vasodilatory polypeptide (VIP) in cerebrovascular nerves. Brain Res. 113, 400-404. LARSSON L.-I., FAHRENKRUG J., SCHAFFALITZKY DE MUCKADELL 0.. SUNDLER F.. HAKAN~~N R. & REHFELD J. F. (19766) Localization of vasoactive intestinal polypeptide (VIP) to central and peripheral neurons. Proc. natn. Acad. Sci.. U.S.A. 73, 3197-3200. PEARSE A. G. E.. POLAK J. M., ADAMS C. & KENDALL P. A. (1974) Diethylpyrocarbonate. a vapour-phase fixative for immunofluorescence studies on polypeptide hormones. Histochem. J. 6, 347-352. SAID S. I. & MUTT V. (1970). Polypeptide with broad biological activity: Isolation from small Intestine. Science, N.Y. 169, 121771218. SAID S. I. & MUTT V. (1972). Isolation from porcine intestinal wall of a vasoactive octacosapeptide related to secretin and to glucagon. Eur. J. Biochem. 28, 199-204. SAID S. I. & ROSENBERG R N. (1976) Vasoactive intestinal polypeptide: Abundant immunoreacttvity in neural cell lines and normal nervous tissue. Science, N.E 192. 907-908. SUNDLER F., ALUMETS J., HKKANSON R., FAHRENKRUG J. & SCHAFFALITZKY DE MUCKADELL 0. (1977a) VIP nerves in the pancreas. Experlentta. To be published. SUNDLER F.. ALUMETS J., HAKANSON R., INGEMANSSONS., FAH~ENKRUG J. & SCHAFFALITZKY DE MUCKADELL 0. (19776) VIP mnervation of the gall-bladder. Gastroenterology 72, 1375-1378. UNSICKER K. (1973) Innervation of the testicular intestitial tissue in reptiles. 2. Ze/lf orsch. mikrosk. Anat. 146, 123-138. (Received 21 April 1977)
753
FIG. 1. VIP-like immunofluorescence. Ureter opening within bladder wall. Immunoreactive running in circular smooth muscle layer. ( x 200.)
nerve fibers
754
FIG. 2. (a) Cross-sectioned vas deferens. Numerous nerve fibres displaying VIP-like immunofluorescence in submucosal and smooth muscle layers. (x 150.)(b) Uterine cervix. Numerous VIP-containing nerves in connective tissue surrounding the glands and around blood vessels. ( x 150.) (c) VIP immunoreactive nerve fibres forming dense plexus around blood vessels located close to the urethral wall. (x 150.) (d) Ganglion situated close to the trigonum area of the bladder. Numerous nerve cell bodies displaying VIP-like immunofluoresence together with immunoreactive nerve fibres within the ganglion. (x 150.)
1977. Vol. 2, pp 751-754
Pcrgamon
Press. Pnnted III Great B&m.
PEPTIDERGIC (VASOACTIVE INTESTINAL PEPTIDE) NERVES IN THE GENITO-URINARY TRACT P. ALM, J. ALUMETS,R. HKKANSON and F. SUND~ Departments of Histology and Pharmacology, University of Lund, Lund, Sweden Abstract-The feline genito-urinary tract receives a rich supply of nerve fibers displaying immunoreactivity like that of vasoactive intestinal peptide. Nerves containing vasoactive intestinal peptide are particularly numerous in the trigonum area of the bladder, around the ureteral openings and in the upper part of the urethra in both sexes, in the epididymis, prostate and vas deferens, and in the uterine cervix. Ganglia located close to or within the wall of the trigonum area and of the upper urethra contain numerous immunoreactive nerve cell bodies, which may be the origin of the fibers containing vasoactive intestinal pcptide that innervate these regions.
adult cats of each sex. The cats were killed by an overdose of sodium pentobarbitone (Nembutal). The specimens were frozen to the temperature of liquid nitrogen in a mixture of propane and propylene. After freeze-drying, the specimens were fixed with diethyl-pyrocarbonate vapour (P~ARSE,POLAK,ADAMS& KENDALL,1974) and embedded in paraffin. Sections were cut at 5 pm, departinized and processed for immunofluorescence using the anti-VIP serum in dilution 1:80 (LARSSONet al., 19766; FAHRENKRUG& SCHAFFALITZKY DE MUCKADELJ_, 1977). For conneuronal peptide in addition to being a hormone, or trols in the immunohistochemical procedure we used a neuronal peptide only (BRYANT, BLOOM, POLAK, antiserum inactivated by the addition of excess antigen ALBUQUERQUE, MODLIN & F’EARSE, 1976; LARSSON, (30 nmol of pure porcine VIP/ml diluted antiserum). EDVINSSON, FAHRENKRUG, HAKANSON, OWMAN, SCHAFFALITZKY DE MUCKADELL& SUNDLER,1976a; RESULTS LARSSON,FAHRENKRUG,SCTHAFFALITZKY DE MUCKAImmunoreactive nerves were occasionally seen in DELL, SUNDLER,H.&KANSON & REHFELD,1976b; SAID & ROSENBERG, 1976; SUNDLER,ALLJMETS, H~KANSON, the stroma of testes and in the smooth muscle of the INGEMANSSON, FAHRENKRUG& SCHAFFALITZKYDE ureters and urinary bladder. VIP-containing nerves were quite numerous in the trigonum area of the MUCKADELL, 19776). So far, VIP-like material has bladder, particularly around the openings of the been detected immunochemically or immunohistochemically in hypothalamic neurons (LARSSONet al., ureters (Fig. l), in the upper part of the urethra and 1976b), in nerves innervating brain vessels (LARSON in the epididymis. The prostate and vas deferens were et al.,1976a), in nerves running in the pancreatic densely innervated (Fig. 2a). Most of the VIP-containparenchyma (LARSON et nl., 1976b; SUNDLER, ing nerves in the bladder, urethra and vas deferens AL~METS, H~ANSON, FAHRENKRUG& SCHAFFA- were distributed within the smooth muscle and submucosal layers. In the female cat, VIP-containing LITZKYDE MUCKADELL,1977u) and in nerves of the gut wall (BRYANTet al., 1976; LARSSONet al., 1976b; nerves were occasionally seen in the ovaries, fallopian tubes, uterine body, and upper part of the vagina. SUNDLEXet al., 1977b). We now wish to report on a rich VIP innervation of the feline genito-urinary of VIP-containing An abundancy nerves was tract, particularly dense in the uterine cervix and vas observed in the uterine cervix (Fig. 2b). Here, the deferens. nerves were predominantly located in the connective
WHEN ‘vasoactive intestinal polypeptide’ (VIP) was isolated from the gut by SAID & M~IT (1970) it was first thought of as a hormone. This contention was based primarily on the chemical similarity between VIP on the one hand and secretin and glucagon on the other Mum, 1972). Recently, however, several groups have independently demonstrated VIP-like immunoreactivity in neuronal elements and have reached the conclusion that VIP is either a
EXPERIMENTAL
PROCEDURES
The investigation was carried out by immunohistochemistry using a previously characterized antiserum against porcine VIP (L-SON et al., 1976b), generously supplied by Drs J. Fahrenkrug and 0. Schaffalitzky de Muckadell, Bispebjerg Hospital, Copenhagen, Denmark. Tissue specimens were collected from the kidneys, ureters, bladder, ovaries, fallopian tubes, uterus, vagina, prostate, vesicula seminalis, epididymis, testes and urethra of three Abbreviation:
VIP, vasoactive
intestinal
peptide.
751
tissue separating the glands. In addition, a dense network of VIP-containing terminals was seen around large blood vessels running close to or within the wall of the cervix, prostate, bladder urethra (Fig. 2~). Numerous VIP-immunoreactive nerve cell bodies were seen in bilateral ganglia located in the trigonum region of the bladder and extending down along the upper urethra (Fig. 2d). Conceivably, the VIP-containing nerves of the genito-urinary tract originate in local ganglia. All immunohistochemical controls were negative.
752
P. ALM. J. ALUMETS. R. HAKANSON and F.
DISCUSSION
The distribution pattern of VIP-containing nerve terminals suggests that they participate in regulating smooth muscle activity and possibly epithelial cell activity as well. Our observations are in agreement with electron microscopic findings of nerve terminals thought to be peptidergic (UNSICKER, 1973; BLJRNSTOCK, 1972). from
adrenergic
of large.
These
dense-core
electron-lucent
terminals
and cholinergic vesicles
vesicles.
and
It should
can
be
majority of the cell bodies in the ganglia do not display VIP immunoreactivity. If they are ‘peptidergic’ too. they probably contain peptides distinct from VIP. The observations made to date indicate that VIP-containing nerves have a ubiquitous occurrence in the body and that VIP may exert a neuronal rather than a hormonal function.
distinguished
ones by their virtual
SUNDLER
lack
be noted
content of small,
Acknowledgements-Grant support from the Swedish Medical Research Council (04X-4499) and the Ford Foun-
that
dation
the
(680-0383 A).
REFERENCES BRYANT M. G., BLWM S. R., POLAI~ J. M.. ALBUQUERQUE R. H.. MODLIN J. & PEARSE A. G. E. (1976) Possible dual role for vasoactive intestinal peptide as gastrointestinal hormone and neurotransmitter substance. Lancer I, 991-993. BURNSTOCK G. (1972) Purinergic nerves. Pharmac. Rev. 24, 509-581. FAHRENKRUG J. & SCHAFFALITZKY DE MUCKADELL 0. B. (1977) Radioimmunoassay of vasoactive intestinal polypeptide (VIP) in plasma. J. lab. clin. Med. To be published. LARSSON L.-I., EDWNSSON L., FAHRENKRUG J., HAKANSON R.. OWMAN C., SCHAFFALITZKY DE MUCKADELL 0. & SUNDLER F. (1976a) Immunohistochemical localization of a vasodilatory polypeptide (VIP) in cerebrovascular nerves. Brain Res. 113, 400-404. LARSSON L.-I., FAHRENKRUG J., SCHAFFALITZKY DE MUCKADELL 0.. SUNDLER F.. HAKAN~~N R. & REHFELD J. F. (19766) Localization of vasoactive intestinal polypeptide (VIP) to central and peripheral neurons. Proc. natn. Acad. Sci.. U.S.A. 73, 3197-3200. PEARSE A. G. E.. POLAK J. M., ADAMS C. & KENDALL P. A. (1974) Diethylpyrocarbonate. a vapour-phase fixative for immunofluorescence studies on polypeptide hormones. Histochem. J. 6, 347-352. SAID S. I. & MUTT V. (1970). Polypeptide with broad biological activity: Isolation from small Intestine. Science, N.Y. 169, 121771218. SAID S. I. & MUTT V. (1972). Isolation from porcine intestinal wall of a vasoactive octacosapeptide related to secretin and to glucagon. Eur. J. Biochem. 28, 199-204. SAID S. I. & ROSENBERG R N. (1976) Vasoactive intestinal polypeptide: Abundant immunoreacttvity in neural cell lines and normal nervous tissue. Science, N.E 192. 907-908. SUNDLER F., ALUMETS J., HKKANSON R., FAHRENKRUG J. & SCHAFFALITZKY DE MUCKADELL 0. (1977a) VIP nerves in the pancreas. Experlentta. To be published. SUNDLER F.. ALUMETS J., HAKANSON R., INGEMANSSONS., FAH~ENKRUG J. & SCHAFFALITZKY DE MUCKADELL 0. (19776) VIP mnervation of the gall-bladder. Gastroenterology 72, 1375-1378. UNSICKER K. (1973) Innervation of the testicular intestitial tissue in reptiles. 2. Ze/lf orsch. mikrosk. Anat. 146, 123-138. (Received 21 April 1977)
753
FIG. 1. VIP-like immunofluorescence. Ureter opening within bladder wall. Immunoreactive running in circular smooth muscle layer. ( x 200.)
nerve fibers
754
FIG. 2. (a) Cross-sectioned vas deferens. Numerous nerve fibres displaying VIP-like immunofluorescence in submucosal and smooth muscle layers. (x 150.)(b) Uterine cervix. Numerous VIP-containing nerves in connective tissue surrounding the glands and around blood vessels. ( x 150.) (c) VIP immunoreactive nerve fibres forming dense plexus around blood vessels located close to the urethral wall. (x 150.) (d) Ganglion situated close to the trigonum area of the bladder. Numerous nerve cell bodies displaying VIP-like immunofluoresence together with immunoreactive nerve fibres within the ganglion. (x 150.)
