© 1991 S. Karger AG, Basel 0378-7346/91/0311-0030S2.75/0
Gynecol Obstet Invest 1991;31:30-36
Innervation of the Human Uterine Artery and Contractile Responses to Neuropeptides Rickard Ekesbo*, PerAlmb, Per Ekstrôma, Lena-Maria Lundberg0, Mats Âkerlund3 Departments o f aObstetrics and Gynecology and bPathology, University Hospital o f Lund, Sweden
Key Words. Uterine artery • Neutrotransmitters • Neuropeptides • Immunohistochemistry • Contractile effects Abstract. The intrinsic innervation of the human uterine artery was investigated histochemically, and the motor responses to some of the demonstrated peptides and other humoral factors were studied on isolated vascular prep arations. There were nerves with specific immunoreactivities for tyrosine hydroxylase, dopamine (3-hydroxylase, neuropeptide-Y (NPY), vasoactive intestinal peptide (VIP) and peptide histidine methionine, and enzymatic reac tivity for acetylcholine esterase. The most effective stimulator of smooth muscle contractility was arginine vasopres sin followed in order by oxytocin, noradrenaline together with NPY, noradrenaline alone and dopamine. No effect was seen with acetylcholine and tyrosine, and VIP caused inconsistent relaxation of contractile activity induced by PGF 2a- These results suggest that the uterine blood flow is regulated by complex interactions of factors, some occurring in nerve terminals and some being circulating humoral factors.
Uterine blood flow is influenced by the contractile activity of myometrium and smooth muscle of intrinsic arterial walls [1,2]. The myométrial activity is regulated by circulating factors and locally released neurotransmit ters and neuropeptides, but little is known about the influence of these parameters on the human uterine arteries [2], In the present study the main branches of the human uterine artery were investigated for the presence of putative populations of nerves. Furthermore, the con tractile responses to some of the neuronally localized peptides and other humoral factors of possible impor tance in this context were studied.
Material and Methods Subjects From 34 menstruating women, 33-52 years o f age, undergoing hysterectomy on routine gynecological indications, tissue specimens were taken from resected parts o f uterine artery branches at isthmic level.
Histochemical Studies Specimens from 15 o f the women were used for immunohisto chemical studies. Tissue pieces were immersion-fixed in ice-cold 4% formaldehyde in phosphate-buffered saline (PBS, pH 7.4), rinsed several times in ice-cold 15 % sucrose in PBS and frozen in isopen tane at the temperature o f liquid nitrogen. Cryostate sections were made and processed for an indirect immunofluorescence method for the demonstration of neuronal peptides (see below), tyrosine hydroxylase (TH) and dopamine ß-hydroxylase (DBH). For exact details on methodology, see Aim and Lundberg [3]. Antibodies to the following substances, raised in rabbits and diluted in PBS, were used: TH (1:320) and DBH (1:640) (purchased from Eugene Tech, Allendale, USA); neuropeptide-Y (NPY; 1:320), vasoactive intestinal peptide (VIP; 1:200), peptide histidine methi onine (PHM; 1:200), substance P (1:80-1:300), calcitonin-generelated peptide (1:600), met-enkephalin (1:80), ß-endorphin (1:80), dynorphin A (1:40) and dynorphin B/rimorphin (1:40) (all pur chased from Milab AB, Malmö, Sweden); leu-enkephalin (1:500) (purchased from Cambridge Research, Biochemicals, Cambridge, UK); arginine vasopressin (1:50-1:200) and oxytocin (1:50-1:100) (supplied by Ferring AB, Malmö, Sweden). The specificity o f the immunoreactivities for the neuropeptides found was verified by antisera absorbed with excess of antigen, which completely abol ished the immunofluorescence reactions. The specificity o f the TH and DBH immunoreactivities was tested by substituting the TH and DBH antibodies with serum from nonimmunized rabbits at which no immunofluorescence reaction appeared.
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Introduction
Innervation of the Human Uterine Artery and Contractile Responses to Neuropeptides
Motor Responses Main branches o f the uterine arteries for in vitro recordings o f smooth muscle activity were obtained from 19 women. The speci mens were immediately placed in oxygenated Krebs-Ringer solution at 0 °C. Circular preparations o f a length o f 2 -3 mm were dissected and then mounted in an organ bath containing 5 ml o f Krebs-Ringer solution at pH 7.4, at a temperature o f 37 "C and aerated with carbogen (5% CO2, 95% O2). After an adaptation period of 45 -9 0 min isometric contractions at a resting tension o f 4 mN were recorded using a Grass force transducer (model F303). Cumulative dose-response experiments were performed with arginine vasopressin and oxytocin (both from Ferring AB, Malmö, Sweden), dopamine, noradrenaline together with NPY, noradrena line alone, acetylcholine and tyrosine. The contractile response obtained by depolarization with 124 m M KC1 solution was used as standard and the response at each dose level o f test substance expressed as a percentage o f that to K*. For VIP, the inhibition of contractile activity induced by 10~5 Af o f PGF2„ was estimated. Sta tistical comparisons were made by using Student’s t test. The results are expressed as means ± SEM.
Results Histochemical Studies Specific neuronal immunoreactivities were found for TH, DBH, NPY, VIP and PHM. TH- and DBH-immunoreactive nerves were found in similar number and distribution patterns (fig. 1). In the outer part of the adventitia, which predominantly con sisted of dense connective tissue, there were coarse axon trunks and less coarse nonvaricose nerve fibers. The lat ter gave off varicose terminals, which surrounded bun dles of smooth muscle cells and small intramural vessels of the adventitia. Most TH- and DBH-immunoreactive nerve structures occurred along the transitional zone between media and adventitia, where varicose terminals ran longitudinally and gave off branches which pro truded into the outer part of the media (fig. 1). AchE-positive nerves were found in higher numbers than the other nerve types (fig. 2). They mainly occurred in the inner part of the adventitia close to the media as plexus-like formations of AchE-positive terminals with branches running longitudinally and transversely into the outer parts of the media. In addition, a few varicose AchE-positive terminals and coarse AchE-positive nerve trunks were found in the outer parts of the adventitia. NPY-immunoreactive fibres were the second most frequent nerve population. In the adventitia there were
single nonvaricose nerve fibers and varicose terminals, which respectively ran freely in connective tissue and along smooth muscle bundles. In comparison, varicose terminals were more frequent and regularly found in the adventitia-media transitional zone (fig. 3). VIP- and PHM-immunoreactive nerves were least frequent (fig. 4,5). They generally occurred as very finegracile varicose terminals which ran along the adventi tia-media transitional zone and gave off branches into the outer part of the media similarly to the TH- and DBH-immunoreactive varicose terminals. There were no overt differences in the distribution and frequency between VIP- and PHM-immunoreactive nerves. Motor Responses Spontaneous contractile activity occurred in about 25% of the preparations. All responded to KC 1 adminis tration and to the test drugs. A typical cumulative dose-response experiment is shown in figure 6, and a summary of all results with the different agents with contractile effects is shown in the diagrams of figures 7-9. Responses were first observed at a concentration of 5 X 10-10 M for arginine vasopres sin, 5 X 10~8 for oxytocin, 5 X 10~7 for noradrenaline together with NPY as well as alone, and 5 X 10-6 M for dopamine. The EC50 and maximum effects for these pep tides are shown in table 1. Arginine vasopressin was the most effective stimulator of smooth muscle contractile activity followed in order by oxytocin, noradrenaline together with NPY, noradrenaline alone and dopamine. Acetylcholine and tyrosine in concentrations up to 10'3 M did not give any reactions at all. VIP in concen trations of 10~6 and 2 X 10-6 M caused a dose-depen dent relaxation of PGF 20-induced contractile activity in two women and had no effect in two (not shown).
Table 1. EC50 and Emax in percent of K’ response for different agonists on uterine artery contractions with the actual concentration within parenthesis Agonist
EC50
Vasopressin Oxytocin Noradrenaline + NPY Noradrenaline Dopamine
1.3 1.4 1.4 3.0 1.0
X X X X X
Emax IO-9 lO-7 10-6 10-6 lO-4
111 94 114 112 64
(3 (3 G (3 (3
X X X X X
10- 7) 10- 5) 10-4) 10- 4) 10- 3)
Means o f results from 5-8 individuals with each substance are indicated.
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From freshly frozen tissue specimens cryostat sections were cut at a thickness of 10 pm and further processed according to the copper-thiocholine method o f Koelle and Friedenwald as modified by Holmstedt to demonstrate acetylcholine esterase (AchE) activity. For details, see Aim et al. [4],
31
3
4
5
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32 Ekesbo/Alm/Ekström/Lundberg/Äkerlund
Innervation of the Human Uterine Artery and Contractile Responses to Neuropeptides
33
10 min
*
t
------ i------ 1---- *
t i
"
*
t t t ! t
K *
t I
w
tK.
% ot K* response
Fig. 6. Typical cumulative dose responses to vasopressin o f an isolated branch o f the human uterine artery. The first arrow in the dose series indicates a concentration in the tissue bath o f 1.2 X 1 0 -10 M and the consecutive ones a trebled concentration up to 3 X 10*5 A/.K* indicates exposure to KC1 solution in a concentration o f 125 mA/and w, washing. Fig. 7. Contractile responses induced by arginine vasopressin (------) and oxytocin (--------) in isolated branches o f the human nonpreg nant uterine artery from 8 individuals.
Fig. 1. Uterine artery, adventitia (A)-media (M) transitional zone. Varicose terminals protruding into the media. X 206. Fig. 2. Uterine artery. AchE-positive varicose terminals running along the adventitia (A)-media (M) transitional zone with branches running into the media (1). Coarse nerve trunks in the outer part of the adventitia (left-bottom). X 64. Fig. 3. Uterine artery. NPY-immunoreactive varicose terminals running along the adventitia (A)-media (M) transitional zone. X 205. Fig. 4. Uterine artery. Fine-gracile (arrow heads) PHM-immunoreactive varicose terminals running along the adventitia (A)-media (M) transitional zone. X 206. Fig. 5. Uterine artery. Fine-gracile VIP-immunoreactive vari cose terminals protruding into the outer part o f the media (M). X 225.
The present results indicate that the human uterine artery is supplied by a variety of nerve populations such as TH-/DBH-immunoreactive (adrenergic) and AchEpositive (presumably cholinergic). Furthermore, out of the 14 peptides investigated, neuronal immunoreactivity was found for NPY, VIP and PHM. The intramural nerve distribution pattern of the human uterine artery was similar to that of other human arteries, e.g. temporal [5], cerebral [6], omental [7] and hepatic arteries [8], however with differences in types of nerves demon strated. Thus, nerves with substance P and calcitoningene-related peptide immunoreactivity, frequently
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Discussion
34
Ekesbo/Alm/Ekstrôm/Lundberg/Âkerlund
Fig. 8. Contractile responses induced by noradrenaline (-------- ) and noradrena line + NPY (------------) in isolated branches o f the human, nonpregnant uterine artery from 5 individuals.
found in the latter types of arteries, do not occur in the human uterine artery. The aggregation and intimate rela tionship between varicose terminals of the various nerve population in the adventitia-media transitional zone and outer parts of the media suggest an involvement of com plex neurological interactions in the regulation of blood flow through the human uterine artery. The comparatively abundant supply of TH- and DBH-immunoreactive nerves suggests an important role for the adrenergic innervation in the regulation of uter ine blood flow. Furthermore, the pronounced motor responses for noradrenaline, tyrosine and dopamine probably means that catecholamines, whether released locally or circulating, are of importance in the regulation of uterine blood flow.
The adrenergic innervation of the uterus is known to be under special hormonal influence and undergoes structural and functional changes during pregnancy [911], The changes in hormonal concentrations during the human menstrual cycle may also have an influence on this innervation, since the effect of p-adrenergic stimula tion, at least on the myometrial activity, varies accord ingly [12], Studies of the human uterine artery innerva tion under different hormonal conditions are therefore indicated. NPY has been detected in uterine nerve structures in several species and tissues, including the human uterus [3, 13, 14], In many places NPY is localized in adrener gic nerves and co-released with noradrenaline at sympa thetic activation [15]. The present study also disclosed
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Fig. 9. Contractile responses induced by dopamine in isolated branches of the human nonpregnant uterine artery from 5 individuals.
Innervation of the Human Uterine Artery and Contractile Responses to Neuropeptides
tion of smaller branches of arteries and spiral arteries, as well as their motor responses, must also be investi gated. Acknowledgements This study was supported by Lund University, the Royal Physi ological Society in Lund, and the Swedish Medical Research Coun cil (B90-17X-06571). The skillful technical assistance o f Mrs. B. Gustavsson, Mr. M. Petterson and Mrs. L. Thuresson, and the care ful help o f Miss A. Andersson in preparation o f the manuscript are gratefully acknowledged.
References 1 Maigaard S: Contraction and relaxation o f human uterine and placental smooth muscle: Endogenous control and calcium acti vation mechanisms. Acta Obstet Gynecol Scand Suppl 1987;79: 1-39. 2 Akerlund M: Function o f endometrial blood vessels; in WHO Symposium on Contraception and Mechanisms o f Endometrial Bleeding, Geneva, 1988, in press. 3 Aim P, Lundberg LM: Co-existence and origin o f peptidergic and adrenergic nerves in the guinea pig uterus: Retrograde trac ing and immunocytochemistry. Effects o f chemical sympathec tomy, capsaicin treatment and pregnancy. Cell Tissue Res 1988; 254:517-530. 4 Aim P, Lundberg LM, Warthon J, Polak JM: Effects o f preg nancy on the extrinsic innervation o f the guinea-pig uterus. A histochemical, immunohistochemical and ultrastructural study. Histochem J 1988;20:414-426. 5 Jansen I, Uddman R, Hocherman M, Ekman R, Jensen K, Olesen J, Stiemholm P, Edvinsson L: Localization and effects of neuropeptide Y, vasoactive intestinal polypeptide, substance P and calcitonin gene-related peptide in human temporal arteries. Ann Neurol 1986;20:496-501. 6 Edvinsson L, Ekman R, Jansen I, Ottosson A, Uddman R: Pep tide-containing nerve fibers in human cerebral arteries: Immu nocytochemistry, radioimmunoassay, and in vitro pharmacolo gy. Ann Neurol 1987;21:431-437. 7 Edvinsson L, H&kansson R. Steen S, Sundler F, Uddman R. Wahlestedt C: Innervation of human omental arteries and veins and vasomotor responses to noradrenaline, neuropeptide Y, substance P and vasoactive intestinal peptide. Regul Pept 1985; 12:67-79. 8 Carlei F, Lygidakis NJ, Speranza V, Brummelkamp WH, McGurrin JF, Pietroletti R, Lezoche E, Bostwick DG: Neuroen docrine innervation o f the hepatic vessels in the rat and in man. J Surg Res 1988;45:417-426. 9 Thorbert G: Regional changes in structure and functions of adrenergic nerves in guinea-pig uterus during pregnancy. Acta Obstet Gynecol Scand Suppl 1979;79:1-32. 10 Bell C, Malcolm CJ: Observations on the loss of catecholamine fluorescence from intrauterine adrenergic nerves during preg nancy o f the guinea-pig. J Reprod Fertil 1978;53:51-58. 11 Lundberg LM: Growth and degeneration o f uterine innervation. An experimental study in the guinea-pig; thesis Lund, 1988, pp 1-39.
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NPY-immunoreactive nerves in the human uterine ar tery. In human omental arteries, NPY has been proposed to facilitate the effects of noradrenaline, whereas no effect of the peptide itself was found [7], The present finding of a forceful effect of NPY per se on the isolated uterine artery preparations thus indicates regional differ ences in vascular constrictor effects of this peptide, as was suggested previously [15]. The present study confirmed the occurrence of AchEpositive nerve structures in the human uterine artery [16, 17], which is somewhat in contrast to the human myometrium [ 18]. However, the motor effect of this pep tide was insignificant, suggesting a minor role in the reg ulation of blood flow in the human uterine artery. VIP and PHM are formed from the same precursor molecule, which might explain their similar distribution patterns [13, 19]. In the human uterus VIP- and PHMimmunoreactive nerves are concentrated to the vagina and cervix and related to small blood vessels and bun dles of smooth muscle cells [13, 20], In comparison, in the human uterine artery VIP-/PHM-immunoreactive nerves are probably less frequent. In view of the incon sistent motor effect of VIP in the present study, the involvement of this peptide in the regulation of uterine blood flow is uncertain. Oxytocin and vasopressin showed similar, marked potencies in the vasomotor studies, but no immunoreactivity for these peptides was detected morphologically. This is of interest since oxytocin has been ascribed a physiological role in the onset of uterine contractions of labor and a pathophysiological one in preterm labor [21, 22]. Correspondingly, vasopressin is probably an impor tant pathogenetic mechanism causing the uterine hyper activity and ischemia of dysmenorrhea [23, 24], Both the myometrial and vascular effects of this peptide during in vivo studies are marked [25]. However, these substances are apparently not involved in the arterial innervation of the uterus, but exert their effects as humoral factors directed to specific receptors in the myometrium, and possibly also in the smooth muscle of arterial walls. Altogether, the present results suggest a complex in fluence of various neurotransmitters, neuropeptides and other humoral factors on the blood flow through the human uterine artery. In addition to the factors demon strated also come those released from the endothelium of arterial vessels such as endothelin and nitrous oxide [26], as well as prostaglandins and free radicals released from the endometrium [27], Mechanisms responsible for the spiral artery spasm, which initiate menstruation, are still not fully understood and in further studies the innerva
35
12 Àkerlund M, Andersson K-E: Effects o f terbutaline on myomé trial activity and endometrial blood flow during the menstrual cycle. Obstet Gynecol 1976;74:529-536. 13 Blank MA, Allen JM, Huang WM, Yiangou Y, Ch’ng J, Lewis G, Elder MG, Polak JM, Bloom SR: The regional distribution o f NPY-, PHM-, and VIP-containing nerves in the human female genital tract. Int J Fertil 1986;31:218-222. 14 Fried G, Hôkfelt T, Lundberg JM, Terenius L, Hamberger L: Neuropeptide Y and noradrenaline in human uterus and myo metrium during normal and pre-eclamptic pregnancy. Hum Reprod 1986;6:359-364. 15 Lundberg JM, Hokfelt T: Multiple co-existence o f peptides and classical transmitters in peripheral autonomic and sensory neu rons - Functional and pharmacological implications. Progr Brain Res 1986;68:241-262. 16 Bell C: Evidence for dual innervation o f the human extrinsic uterine arteries. J Obstet Gynaecol Br Commonw 1969;76: 1123-1128. 17 Amenta F, Porcelli F, Ferrante F, Cavallotti C: Cholinergic nerves in blood vessels o f the female reproductive system. Acta Histochem 1979;65:133-137. 18 Nakanishi H, Wood C: Cholinergic mechanisms in the human uterus. J Obstet Gynaecol Br Commonw 1971;78:716-723. 19 Itoh N, Obata KJ, Yanaihara N, Okamoto H: Human preprovasoactive intestinal polypeptide contains a novel PHI-27-like peptide, PHM-27. Nature 1983;304:547-549. 20 Aim P, Alumets J, Hâkansson R, Helm G, Owman Ch, Sjoberg NO, Sundler F: VIP nerves in the human female genital tract. Am J Obstet Gynecol 1980;136:349-351. 21 Fuchs AR, Fuchs F, Husslein P, Soloff MS, Femstrom MJ: Oxy tocin receptors and human parturition; a dual role for oxytocin in the initiation of labour. Science 1982;215:1396-1398.
Ekesbo/Alm/Ekstrôm/Lundberg/Àkerlund
22 Àkerlund M, Strômberg P, Hauksson A, Andersson LF, Lyndrup J, Trojnar J, Melin P: Inhibition o f uterine contractions of pre mature labour with an oxytocin analogue. Result from a pilot study. Br J Obstet Gynaecol 1987;94:1040-1044. 23 Àkerlund M, Strômberg P, Forsling ML: Primary dysmenorrhoea and vasopressin. Br J Obstet Gynaecol 1979;86:484487. 24 Àkerlund M: Can primary dysmenorrhoea be alleviated by a vasopressin antagonist? Acta Obstet Gynecol Scand 1987;66: 459-461. 25 Hauksson A, Àkerlund M, Melin P: Uterine blood flow and myométrial activity at menstruation and the influence of vaso pressin and the synthetic antagonist. Br J Obstet Gynaecol 1988; 95:898-904. 26 Casey ML, McDonald PC: By molecular mechanisms in human parturition: Activation o f uterine decidua; in WHO Symposium on Contraception and Mechanisms o f Endometrial Bleeding, Geneva, 1988, in press. 27 Rice-Evans C, Cooke B: Oxygen radicals, bleeding and tissue injury; in WHO Symposium on Contraception and Mechanisms o f Endometrial Bleeding, Geneva, 1988, in press.
Received: March 30, 1990 Accepted: May 10, 1990 Mats Akerlund, Assoc. Prof. Department o f Obstetrics and Gynecology University Hospital o f Lund Lund (Sweden)
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36
Gynecol Obstet Invest 1991;31:30-36
Innervation of the Human Uterine Artery and Contractile Responses to Neuropeptides Rickard Ekesbo*, PerAlmb, Per Ekstrôma, Lena-Maria Lundberg0, Mats Âkerlund3 Departments o f aObstetrics and Gynecology and bPathology, University Hospital o f Lund, Sweden
Key Words. Uterine artery • Neutrotransmitters • Neuropeptides • Immunohistochemistry • Contractile effects Abstract. The intrinsic innervation of the human uterine artery was investigated histochemically, and the motor responses to some of the demonstrated peptides and other humoral factors were studied on isolated vascular prep arations. There were nerves with specific immunoreactivities for tyrosine hydroxylase, dopamine (3-hydroxylase, neuropeptide-Y (NPY), vasoactive intestinal peptide (VIP) and peptide histidine methionine, and enzymatic reac tivity for acetylcholine esterase. The most effective stimulator of smooth muscle contractility was arginine vasopres sin followed in order by oxytocin, noradrenaline together with NPY, noradrenaline alone and dopamine. No effect was seen with acetylcholine and tyrosine, and VIP caused inconsistent relaxation of contractile activity induced by PGF 2a- These results suggest that the uterine blood flow is regulated by complex interactions of factors, some occurring in nerve terminals and some being circulating humoral factors.
Uterine blood flow is influenced by the contractile activity of myometrium and smooth muscle of intrinsic arterial walls [1,2]. The myométrial activity is regulated by circulating factors and locally released neurotransmit ters and neuropeptides, but little is known about the influence of these parameters on the human uterine arteries [2], In the present study the main branches of the human uterine artery were investigated for the presence of putative populations of nerves. Furthermore, the con tractile responses to some of the neuronally localized peptides and other humoral factors of possible impor tance in this context were studied.
Material and Methods Subjects From 34 menstruating women, 33-52 years o f age, undergoing hysterectomy on routine gynecological indications, tissue specimens were taken from resected parts o f uterine artery branches at isthmic level.
Histochemical Studies Specimens from 15 o f the women were used for immunohisto chemical studies. Tissue pieces were immersion-fixed in ice-cold 4% formaldehyde in phosphate-buffered saline (PBS, pH 7.4), rinsed several times in ice-cold 15 % sucrose in PBS and frozen in isopen tane at the temperature o f liquid nitrogen. Cryostate sections were made and processed for an indirect immunofluorescence method for the demonstration of neuronal peptides (see below), tyrosine hydroxylase (TH) and dopamine ß-hydroxylase (DBH). For exact details on methodology, see Aim and Lundberg [3]. Antibodies to the following substances, raised in rabbits and diluted in PBS, were used: TH (1:320) and DBH (1:640) (purchased from Eugene Tech, Allendale, USA); neuropeptide-Y (NPY; 1:320), vasoactive intestinal peptide (VIP; 1:200), peptide histidine methi onine (PHM; 1:200), substance P (1:80-1:300), calcitonin-generelated peptide (1:600), met-enkephalin (1:80), ß-endorphin (1:80), dynorphin A (1:40) and dynorphin B/rimorphin (1:40) (all pur chased from Milab AB, Malmö, Sweden); leu-enkephalin (1:500) (purchased from Cambridge Research, Biochemicals, Cambridge, UK); arginine vasopressin (1:50-1:200) and oxytocin (1:50-1:100) (supplied by Ferring AB, Malmö, Sweden). The specificity o f the immunoreactivities for the neuropeptides found was verified by antisera absorbed with excess of antigen, which completely abol ished the immunofluorescence reactions. The specificity o f the TH and DBH immunoreactivities was tested by substituting the TH and DBH antibodies with serum from nonimmunized rabbits at which no immunofluorescence reaction appeared.
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Introduction
Innervation of the Human Uterine Artery and Contractile Responses to Neuropeptides
Motor Responses Main branches o f the uterine arteries for in vitro recordings o f smooth muscle activity were obtained from 19 women. The speci mens were immediately placed in oxygenated Krebs-Ringer solution at 0 °C. Circular preparations o f a length o f 2 -3 mm were dissected and then mounted in an organ bath containing 5 ml o f Krebs-Ringer solution at pH 7.4, at a temperature o f 37 "C and aerated with carbogen (5% CO2, 95% O2). After an adaptation period of 45 -9 0 min isometric contractions at a resting tension o f 4 mN were recorded using a Grass force transducer (model F303). Cumulative dose-response experiments were performed with arginine vasopressin and oxytocin (both from Ferring AB, Malmö, Sweden), dopamine, noradrenaline together with NPY, noradrena line alone, acetylcholine and tyrosine. The contractile response obtained by depolarization with 124 m M KC1 solution was used as standard and the response at each dose level o f test substance expressed as a percentage o f that to K*. For VIP, the inhibition of contractile activity induced by 10~5 Af o f PGF2„ was estimated. Sta tistical comparisons were made by using Student’s t test. The results are expressed as means ± SEM.
Results Histochemical Studies Specific neuronal immunoreactivities were found for TH, DBH, NPY, VIP and PHM. TH- and DBH-immunoreactive nerves were found in similar number and distribution patterns (fig. 1). In the outer part of the adventitia, which predominantly con sisted of dense connective tissue, there were coarse axon trunks and less coarse nonvaricose nerve fibers. The lat ter gave off varicose terminals, which surrounded bun dles of smooth muscle cells and small intramural vessels of the adventitia. Most TH- and DBH-immunoreactive nerve structures occurred along the transitional zone between media and adventitia, where varicose terminals ran longitudinally and gave off branches which pro truded into the outer part of the media (fig. 1). AchE-positive nerves were found in higher numbers than the other nerve types (fig. 2). They mainly occurred in the inner part of the adventitia close to the media as plexus-like formations of AchE-positive terminals with branches running longitudinally and transversely into the outer parts of the media. In addition, a few varicose AchE-positive terminals and coarse AchE-positive nerve trunks were found in the outer parts of the adventitia. NPY-immunoreactive fibres were the second most frequent nerve population. In the adventitia there were
single nonvaricose nerve fibers and varicose terminals, which respectively ran freely in connective tissue and along smooth muscle bundles. In comparison, varicose terminals were more frequent and regularly found in the adventitia-media transitional zone (fig. 3). VIP- and PHM-immunoreactive nerves were least frequent (fig. 4,5). They generally occurred as very finegracile varicose terminals which ran along the adventi tia-media transitional zone and gave off branches into the outer part of the media similarly to the TH- and DBH-immunoreactive varicose terminals. There were no overt differences in the distribution and frequency between VIP- and PHM-immunoreactive nerves. Motor Responses Spontaneous contractile activity occurred in about 25% of the preparations. All responded to KC 1 adminis tration and to the test drugs. A typical cumulative dose-response experiment is shown in figure 6, and a summary of all results with the different agents with contractile effects is shown in the diagrams of figures 7-9. Responses were first observed at a concentration of 5 X 10-10 M for arginine vasopres sin, 5 X 10~8 for oxytocin, 5 X 10~7 for noradrenaline together with NPY as well as alone, and 5 X 10-6 M for dopamine. The EC50 and maximum effects for these pep tides are shown in table 1. Arginine vasopressin was the most effective stimulator of smooth muscle contractile activity followed in order by oxytocin, noradrenaline together with NPY, noradrenaline alone and dopamine. Acetylcholine and tyrosine in concentrations up to 10'3 M did not give any reactions at all. VIP in concen trations of 10~6 and 2 X 10-6 M caused a dose-depen dent relaxation of PGF 20-induced contractile activity in two women and had no effect in two (not shown).
Table 1. EC50 and Emax in percent of K’ response for different agonists on uterine artery contractions with the actual concentration within parenthesis Agonist
EC50
Vasopressin Oxytocin Noradrenaline + NPY Noradrenaline Dopamine
1.3 1.4 1.4 3.0 1.0
X X X X X
Emax IO-9 lO-7 10-6 10-6 lO-4
111 94 114 112 64
(3 (3 G (3 (3
X X X X X
10- 7) 10- 5) 10-4) 10- 4) 10- 3)
Means o f results from 5-8 individuals with each substance are indicated.
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From freshly frozen tissue specimens cryostat sections were cut at a thickness of 10 pm and further processed according to the copper-thiocholine method o f Koelle and Friedenwald as modified by Holmstedt to demonstrate acetylcholine esterase (AchE) activity. For details, see Aim et al. [4],
31
3
4
5
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32 Ekesbo/Alm/Ekström/Lundberg/Äkerlund
Innervation of the Human Uterine Artery and Contractile Responses to Neuropeptides
33
10 min
*
t
------ i------ 1---- *
t i
"
*
t t t ! t
K *
t I
w
tK.
% ot K* response
Fig. 6. Typical cumulative dose responses to vasopressin o f an isolated branch o f the human uterine artery. The first arrow in the dose series indicates a concentration in the tissue bath o f 1.2 X 1 0 -10 M and the consecutive ones a trebled concentration up to 3 X 10*5 A/.K* indicates exposure to KC1 solution in a concentration o f 125 mA/and w, washing. Fig. 7. Contractile responses induced by arginine vasopressin (------) and oxytocin (--------) in isolated branches o f the human nonpreg nant uterine artery from 8 individuals.
Fig. 1. Uterine artery, adventitia (A)-media (M) transitional zone. Varicose terminals protruding into the media. X 206. Fig. 2. Uterine artery. AchE-positive varicose terminals running along the adventitia (A)-media (M) transitional zone with branches running into the media (1). Coarse nerve trunks in the outer part of the adventitia (left-bottom). X 64. Fig. 3. Uterine artery. NPY-immunoreactive varicose terminals running along the adventitia (A)-media (M) transitional zone. X 205. Fig. 4. Uterine artery. Fine-gracile (arrow heads) PHM-immunoreactive varicose terminals running along the adventitia (A)-media (M) transitional zone. X 206. Fig. 5. Uterine artery. Fine-gracile VIP-immunoreactive vari cose terminals protruding into the outer part o f the media (M). X 225.
The present results indicate that the human uterine artery is supplied by a variety of nerve populations such as TH-/DBH-immunoreactive (adrenergic) and AchEpositive (presumably cholinergic). Furthermore, out of the 14 peptides investigated, neuronal immunoreactivity was found for NPY, VIP and PHM. The intramural nerve distribution pattern of the human uterine artery was similar to that of other human arteries, e.g. temporal [5], cerebral [6], omental [7] and hepatic arteries [8], however with differences in types of nerves demon strated. Thus, nerves with substance P and calcitoningene-related peptide immunoreactivity, frequently
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Discussion
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Ekesbo/Alm/Ekstrôm/Lundberg/Âkerlund
Fig. 8. Contractile responses induced by noradrenaline (-------- ) and noradrena line + NPY (------------) in isolated branches o f the human, nonpregnant uterine artery from 5 individuals.
found in the latter types of arteries, do not occur in the human uterine artery. The aggregation and intimate rela tionship between varicose terminals of the various nerve population in the adventitia-media transitional zone and outer parts of the media suggest an involvement of com plex neurological interactions in the regulation of blood flow through the human uterine artery. The comparatively abundant supply of TH- and DBH-immunoreactive nerves suggests an important role for the adrenergic innervation in the regulation of uter ine blood flow. Furthermore, the pronounced motor responses for noradrenaline, tyrosine and dopamine probably means that catecholamines, whether released locally or circulating, are of importance in the regulation of uterine blood flow.
The adrenergic innervation of the uterus is known to be under special hormonal influence and undergoes structural and functional changes during pregnancy [911], The changes in hormonal concentrations during the human menstrual cycle may also have an influence on this innervation, since the effect of p-adrenergic stimula tion, at least on the myometrial activity, varies accord ingly [12], Studies of the human uterine artery innerva tion under different hormonal conditions are therefore indicated. NPY has been detected in uterine nerve structures in several species and tissues, including the human uterus [3, 13, 14], In many places NPY is localized in adrener gic nerves and co-released with noradrenaline at sympa thetic activation [15]. The present study also disclosed
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Fig. 9. Contractile responses induced by dopamine in isolated branches of the human nonpregnant uterine artery from 5 individuals.
Innervation of the Human Uterine Artery and Contractile Responses to Neuropeptides
tion of smaller branches of arteries and spiral arteries, as well as their motor responses, must also be investi gated. Acknowledgements This study was supported by Lund University, the Royal Physi ological Society in Lund, and the Swedish Medical Research Coun cil (B90-17X-06571). The skillful technical assistance o f Mrs. B. Gustavsson, Mr. M. Petterson and Mrs. L. Thuresson, and the care ful help o f Miss A. Andersson in preparation o f the manuscript are gratefully acknowledged.
References 1 Maigaard S: Contraction and relaxation o f human uterine and placental smooth muscle: Endogenous control and calcium acti vation mechanisms. Acta Obstet Gynecol Scand Suppl 1987;79: 1-39. 2 Akerlund M: Function o f endometrial blood vessels; in WHO Symposium on Contraception and Mechanisms o f Endometrial Bleeding, Geneva, 1988, in press. 3 Aim P, Lundberg LM: Co-existence and origin o f peptidergic and adrenergic nerves in the guinea pig uterus: Retrograde trac ing and immunocytochemistry. Effects o f chemical sympathec tomy, capsaicin treatment and pregnancy. Cell Tissue Res 1988; 254:517-530. 4 Aim P, Lundberg LM, Warthon J, Polak JM: Effects o f preg nancy on the extrinsic innervation o f the guinea-pig uterus. A histochemical, immunohistochemical and ultrastructural study. Histochem J 1988;20:414-426. 5 Jansen I, Uddman R, Hocherman M, Ekman R, Jensen K, Olesen J, Stiemholm P, Edvinsson L: Localization and effects of neuropeptide Y, vasoactive intestinal polypeptide, substance P and calcitonin gene-related peptide in human temporal arteries. Ann Neurol 1986;20:496-501. 6 Edvinsson L, Ekman R, Jansen I, Ottosson A, Uddman R: Pep tide-containing nerve fibers in human cerebral arteries: Immu nocytochemistry, radioimmunoassay, and in vitro pharmacolo gy. Ann Neurol 1987;21:431-437. 7 Edvinsson L, H&kansson R. Steen S, Sundler F, Uddman R. Wahlestedt C: Innervation of human omental arteries and veins and vasomotor responses to noradrenaline, neuropeptide Y, substance P and vasoactive intestinal peptide. Regul Pept 1985; 12:67-79. 8 Carlei F, Lygidakis NJ, Speranza V, Brummelkamp WH, McGurrin JF, Pietroletti R, Lezoche E, Bostwick DG: Neuroen docrine innervation o f the hepatic vessels in the rat and in man. J Surg Res 1988;45:417-426. 9 Thorbert G: Regional changes in structure and functions of adrenergic nerves in guinea-pig uterus during pregnancy. Acta Obstet Gynecol Scand Suppl 1979;79:1-32. 10 Bell C, Malcolm CJ: Observations on the loss of catecholamine fluorescence from intrauterine adrenergic nerves during preg nancy o f the guinea-pig. J Reprod Fertil 1978;53:51-58. 11 Lundberg LM: Growth and degeneration o f uterine innervation. An experimental study in the guinea-pig; thesis Lund, 1988, pp 1-39.
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NPY-immunoreactive nerves in the human uterine ar tery. In human omental arteries, NPY has been proposed to facilitate the effects of noradrenaline, whereas no effect of the peptide itself was found [7], The present finding of a forceful effect of NPY per se on the isolated uterine artery preparations thus indicates regional differ ences in vascular constrictor effects of this peptide, as was suggested previously [15]. The present study confirmed the occurrence of AchEpositive nerve structures in the human uterine artery [16, 17], which is somewhat in contrast to the human myometrium [ 18]. However, the motor effect of this pep tide was insignificant, suggesting a minor role in the reg ulation of blood flow in the human uterine artery. VIP and PHM are formed from the same precursor molecule, which might explain their similar distribution patterns [13, 19]. In the human uterus VIP- and PHMimmunoreactive nerves are concentrated to the vagina and cervix and related to small blood vessels and bun dles of smooth muscle cells [13, 20], In comparison, in the human uterine artery VIP-/PHM-immunoreactive nerves are probably less frequent. In view of the incon sistent motor effect of VIP in the present study, the involvement of this peptide in the regulation of uterine blood flow is uncertain. Oxytocin and vasopressin showed similar, marked potencies in the vasomotor studies, but no immunoreactivity for these peptides was detected morphologically. This is of interest since oxytocin has been ascribed a physiological role in the onset of uterine contractions of labor and a pathophysiological one in preterm labor [21, 22]. Correspondingly, vasopressin is probably an impor tant pathogenetic mechanism causing the uterine hyper activity and ischemia of dysmenorrhea [23, 24], Both the myometrial and vascular effects of this peptide during in vivo studies are marked [25]. However, these substances are apparently not involved in the arterial innervation of the uterus, but exert their effects as humoral factors directed to specific receptors in the myometrium, and possibly also in the smooth muscle of arterial walls. Altogether, the present results suggest a complex in fluence of various neurotransmitters, neuropeptides and other humoral factors on the blood flow through the human uterine artery. In addition to the factors demon strated also come those released from the endothelium of arterial vessels such as endothelin and nitrous oxide [26], as well as prostaglandins and free radicals released from the endometrium [27], Mechanisms responsible for the spiral artery spasm, which initiate menstruation, are still not fully understood and in further studies the innerva
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12 Àkerlund M, Andersson K-E: Effects o f terbutaline on myomé trial activity and endometrial blood flow during the menstrual cycle. Obstet Gynecol 1976;74:529-536. 13 Blank MA, Allen JM, Huang WM, Yiangou Y, Ch’ng J, Lewis G, Elder MG, Polak JM, Bloom SR: The regional distribution o f NPY-, PHM-, and VIP-containing nerves in the human female genital tract. Int J Fertil 1986;31:218-222. 14 Fried G, Hôkfelt T, Lundberg JM, Terenius L, Hamberger L: Neuropeptide Y and noradrenaline in human uterus and myo metrium during normal and pre-eclamptic pregnancy. Hum Reprod 1986;6:359-364. 15 Lundberg JM, Hokfelt T: Multiple co-existence o f peptides and classical transmitters in peripheral autonomic and sensory neu rons - Functional and pharmacological implications. Progr Brain Res 1986;68:241-262. 16 Bell C: Evidence for dual innervation o f the human extrinsic uterine arteries. J Obstet Gynaecol Br Commonw 1969;76: 1123-1128. 17 Amenta F, Porcelli F, Ferrante F, Cavallotti C: Cholinergic nerves in blood vessels o f the female reproductive system. Acta Histochem 1979;65:133-137. 18 Nakanishi H, Wood C: Cholinergic mechanisms in the human uterus. J Obstet Gynaecol Br Commonw 1971;78:716-723. 19 Itoh N, Obata KJ, Yanaihara N, Okamoto H: Human preprovasoactive intestinal polypeptide contains a novel PHI-27-like peptide, PHM-27. Nature 1983;304:547-549. 20 Aim P, Alumets J, Hâkansson R, Helm G, Owman Ch, Sjoberg NO, Sundler F: VIP nerves in the human female genital tract. Am J Obstet Gynecol 1980;136:349-351. 21 Fuchs AR, Fuchs F, Husslein P, Soloff MS, Femstrom MJ: Oxy tocin receptors and human parturition; a dual role for oxytocin in the initiation of labour. Science 1982;215:1396-1398.
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22 Àkerlund M, Strômberg P, Hauksson A, Andersson LF, Lyndrup J, Trojnar J, Melin P: Inhibition o f uterine contractions of pre mature labour with an oxytocin analogue. Result from a pilot study. Br J Obstet Gynaecol 1987;94:1040-1044. 23 Àkerlund M, Strômberg P, Forsling ML: Primary dysmenorrhoea and vasopressin. Br J Obstet Gynaecol 1979;86:484487. 24 Àkerlund M: Can primary dysmenorrhoea be alleviated by a vasopressin antagonist? Acta Obstet Gynecol Scand 1987;66: 459-461. 25 Hauksson A, Àkerlund M, Melin P: Uterine blood flow and myométrial activity at menstruation and the influence of vaso pressin and the synthetic antagonist. Br J Obstet Gynaecol 1988; 95:898-904. 26 Casey ML, McDonald PC: By molecular mechanisms in human parturition: Activation o f uterine decidua; in WHO Symposium on Contraception and Mechanisms o f Endometrial Bleeding, Geneva, 1988, in press. 27 Rice-Evans C, Cooke B: Oxygen radicals, bleeding and tissue injury; in WHO Symposium on Contraception and Mechanisms o f Endometrial Bleeding, Geneva, 1988, in press.
Received: March 30, 1990 Accepted: May 10, 1990 Mats Akerlund, Assoc. Prof. Department o f Obstetrics and Gynecology University Hospital o f Lund Lund (Sweden)
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