Neuropeptide Y (NPY)-like immunoreactivity in guinea pig uterus is reduced during pregnancy in parallel with noradrenergic nerves

Summary Neuropeptide Y (NPY) is a recently discovered neuropeptide with vasoconstrictor effects when given in vivo. It occurs in many sympathetic neurons, where it appears to coexist with noradrenaline (NA). It is wellknown that profound changes in the levels of uterine NA occur in many species during pregnancy. Therefore we have investigated the distribution of catecholamine neurons and NPY by immunohistochemistry in the pregnant and nonpregnant guinea pig uterus. In the virgin uterus NPY-like immunoreactivity was present in nerve fibres and terminals in the smooth muscle layers of the uterine horns and around blood vessels. The distribution of NPY fibres was very similar to that of noradrenergic nerves visualized with antibodies against the catecholamine synthesizing enzyme tyrosine hydroxylase (TH). In the pregnant uterus, NPY- and TH-like immunoreactivity disappeared almost completely. In the cervix, a slight decrease of immunoreactivity was observed, whereas in the ovaries no changes were noted between the pregnant and nonpregnant condition. The results indicate that NPY and catecholamines coexists in the adrenergic neurons of the guinea pig uterus, cervix and ovary and that they vary together in the myometrium during pregnancy. We suggest that NPY may be of functional importance for the pregnant uterus.     

Neuropeptide Y (NPY)-like immunoreactivity in guinea pig uterus is reduced during pregnancy in parallel with noradrenergic nerves

Neuropeptide Y (NPY) is a recently discovered neuropeptide with vasoconstrictor effects when given in vivo. It occurs in many sympathetic neurons, where it appears to coexist with noradrenaline (NA). It is wellknown that profound changes in the levels of uterine NA occur in many species during pregnancy. Therefore we have investigated the distribution of catecholamine neurons and NPY by immunohistochemistry in the pregnant and nonpregnant guinea pig uterus. In the virgin uterus NPY-like immunoreactivity was present in nerve fibres and terminals in the smooth muscle layers of the uterine horns and around blood vessels. The distribution of NPY fibres was very similar to that of noradrenergic nerves visualized with antibodies against the catecholamine synthesizing enzyme tyrosine hydroxylase (TH). In the pregnant uterus, NPY- and TH-like immunoreactivity disappeared almost completely. In the cervix, a slight decrease of immunoreactivity was observed, whereas in the ovaries no changes were noted between the pregnant and nonpregnant condition. The results indicate that NPY and catecholamines coexists in the adrenergic neurons of the guinea pig uterus, cervix and ovary and that they vary together in the myometrium during pregnancy. We suggest that NPY may be of functional importance for the pregnant uterus.     

Immunohistochemical localization of calcitonin gene-related peptide and bombesin/gastrin-releasing peptide in nerve fibers of the rat, guinea pig and pig female genital organs

The localization and distribution of calcitonin gene-related peptide (CGRP) and bombesin/gastrin-releasing peptide (GRP) immunoreactivity were studied in the rat, guinea pig and pig female genital organs with indirect immunohistochemical technique. In the rat, guinea pig and pig, CGRP and GRP immunoreactivities were localized in nerve fibers of the uterus, ovary and oviduct. Generally, CGRP-immunoreactive nerve fibers were intensely stained, while GRP-immunoreactive nerve fibers exhibited moderate immunoreactivity. The number of GRP-immunoreactive nerve fibers in these organs was lower in comparison with that of CGRP-immunoreactive nerve fibers. The pattern of distribution of these nerve fibers was very similar in different genital organs of all species studied. In the uterus of rat, guinea pig and pig, CGRP- and GRP-immunoreactive nerve fibers and nerve bundles were observed in the muscular membrane and around blood vessels. Some delicate CGRP- and GRP-immunoreactive nerve fibers were also present in the submucous layer of the uterus. In the oviduct, CGRP- and GRP-immunoreactive nerve fibers were seen in the muscular membrane, around blood vessels and in the submucous layer. In the ovary, CGRP- and GRP-immunoreactive nerve fibers were distributed in medullary stroma, in close contact with blood vessels and between follicles of different stages of development.     

Immunohistochemical localization of calcitonin gene-related peptide and bombesin/gastrin-releasing peptide in nerve fibers of the rat,guinea pig and pig female genital organs

Summary The localization and distribution of calcitonin gene-related peptide (CGRP) and bombesin/gastrin-releasing peptide (GRP) immunoreactivity were studied in the rat, guinea pig and pig female genital organs with indirect immunohistochemical technique. In the rat, guinea pig and pig. CGRP and GRP immunoreactivities were localized in nerve fibers of the uterus, ovary and oviduct. Generally, CGRP-immunoreactive nerve fibers were intensely stained, while GRP-immunoreactive nerve fibers exhibited moderate immunoreactivity. The number of GRP-immunoreactive nerve fibers in these organs was lower in comparison with that of CGRP-immunoreactive nerve fibers. The pattern of distribution of these nerve fibers was very similar in different genital organs of all species studied. In the uterus of rat, guinea pig ang pig, CGRP-and GRP-immunoreactive nerve fibers and nerve bundles were observed in the muscular membrane and around blood vessels. Some delicate CGRP-and GRP-immunoreactive nerve fibers were also present in the submucous layer of the uterus. In the oviduct. CGRP-and GRP-immunoreactive nerve fibers were seen in the muscular membrane, around blood vessels and in the submucous layer. In the ovary, CGRP-and GRP-immunoreactive nerve fibers were distributed in medullary stroma, in close contact with blood vessels and between follicles of different stages of development.     

Levels of neural vasoactive intestinal polypeptide in rat uterus are markedly changed in association with pregnancy as shown by immunocytochemistry and radioimmunoassay

Immunocytochemical studies have shown that the rat uterus is well innervated by nerve fibers containing vasoactive intestinal polypeptide (VIP). The fibers were associated with both vascular and nonvascular smooth muscle cells, and they were somewhat more numerous in the cervix compared to the uterine horns. This was confirmed in radioimmunologic determinations. Pregnancy induced a marked, almost 50% reduction in the total content of VIP in the uterine horns, which was associated with an almost complete disappearance of immunocytochemically visible nerve fibers in this part of the uterus. The innervation normalized within 25 days following delivery. Less marked changes occurred in the VIP innervation of the cervical region, where the concentration of the peptide was reduced mainly as a result of the increased tissue weight during pregnancy.     

Regional difference in the distribution of vasoactive intestinal polypeptide-immunoreactive nerve fibres along the uterus and between myometrial muscle layers in the rat

To investigate a possible regional variation of the vasoactive intestinal polypeptide innervation in the uterus of the cyclic rat, the distribution of vasoactive intestinal polypeptide-containing nerve fibres from the cervix to the oviduct end of the uterine horns was studied using immunohistochemistry. Immunoreactive nerve fibres were most concentrated in the cervix, where they formed a dense plexus in association with the musculature and surrounding blood vessels. In the uterus, a clear regional distribution of the vasoactive intestinal polypeptide innervation was observed. Numerous vascular and non-vascular immunoreactive nerve fibres were present in the lower part of the uterine horns, whereas they were sparse in the median region and absent at the oviduct end. Moreover, non-vascular peptide innervation was mostly concentrated in the circular layer of the myometrium and also occurred in the endometrium. Only a very few immunoreactive nerve fibres were presen t in the longitudinal muscle layer. No change in the peptide innervation pattern was observed during the different stages of the sexual cycle. The marked regional distribution of the peptide innervation in the rat uterus suggests that the regulatory effects of the peptide occur mainly in the lower part of the organ and principally affect the circular muscle layer in the myometrium.     

Changes in phospholipase A2 in myometrium of the guinea pig uterus during pregnancy.

Phospholipase A2 activity has been measured in membrane and cytosolic fractions from non-pregnant and pregnant guinea pig myometrium has been studied. Enzyme activity was measured with 1-stearoyl-2- [3H]arachidonoyl-phosphatidylcholine exhibiting Michaelis-Menton kinetics with Km of 83.8 +/- 21.6 and 53.2 +/- 14.1 for membrane and cytosolic enzymes respectively. Fractionation of the myometrium from non-pregnant guinea pigs suggested that 35% of the activity was membrane associated compared with 20% (P < 0.01) in tissue from pregnant animals. In the presence of 1 mM calcium total activity rose from 3.03 +/- 0.41 to 1737 +/- 368 nmol/h per uterus between non-pregnant and late pregnancy. Calcium activated the membrane enzyme, but the effect was greater late in pregnancy with almost a 6-fold increase in activity at 1 mM calcium compared with a doubling in membrane from non-pregnant guinea pigs. The K0.5 for calcium activation was about 150 microM. Immunoblotting with anti-human-110 KDa phospholipase A2 showed in guinea pig uterus a 34 KDa form of the enzyme that, consistent with changes in activity, showed a fifteen-fold increase in quantity between non-pregnant and late pregnancy. The data are consistent with dramatic increases in the capacity for arachidonic acid release and prostaglandin production in the guinea pig myometrium late in pregnancy.     

Progesterone Receptor Expression Declines in the Guinea Pig Uterus during Functional Progesterone Withdrawal and in Response to Prostaglandins

Progesterone withdrawal is essential for parturition, but the mechanism of this pivotal hormonal change is unclear in women and other mammals that give birth without a pre-labor drop in maternal progesterone levels. One possibility suggested by uterine tissue analyses and cell culture models is that progesterone receptor levels change at term decreasing the progesterone responsiveness of the myometrium, which causes progesterone withdrawal at the functional level and results in estrogen dominance enhancing uterine contractility. In this investigation we have explored whether receptor mediated functional progesterone withdrawal occurs during late pregnancy and labor in vivo. We have also determined whether prostaglandins that induce labor cause functional progesterone withdrawal by altering myometrial progesterone receptor expression. Pregnant guinea pigs were used, since this animal loses progesterone responsiveness at term and gives birth in the presence of high maternal progesterone level similarly to primates. We found that progesterone receptor mRNA and protein A and B expression decreased in the guinea pig uterus during the last third of gestation and in labor. Prostaglandin administration reduced while prostaglandin synthesis inhibitor treatment increased progesterone receptor A protein abundance. Estrogen receptor-1 protein levels remained unchanged during late gestation, in labor and after prostaglandin or prostaglandin synthesis inhibitor administration. Steroid receptor levels were higher in the non-pregnant than in the pregnant uterine horns. We conclude that the decreasing expression of both progesterone receptors A and B is a physiological mechanism of functional progesterone withdrawal in the guinea pig during late pregnancy and in labor. Further, prostaglandins administered exogenously or produced endogenously stimulate labor in part by suppressing uterine progesterone receptor A expression, which may cause functional progesterone withdrawal, promote estrogen dominance and foster myometrial contractions.     

Capsaicin-sensitive structures as potential target sites for neurotensin and bradykinin in guinea pig atria

We tested the influence of capsaicin (CAP) desensitization on the positive chronotropic and inotropic effects of neurotensin (NT), bradykinin (BK), calcitonin gene-related peptide (CGRP) and noradrenaline (NA) in guinea pig isolated atria. The positive chronotropic and inotropic effects of NT and BK were completely inhibited, whereas those elicited by CGRP and NA were either slightly reduced (CGRP) or unaffected (NA), in CAP-desensitized compared to control atria. Cross-desensitization studies using CAP, NT and BK showed that the positive chronotropic and inotropic effects of CAP are slightly affected, whereas those evoked by BK are markedly reduced in NT-desensitized atria. On the other hand, the positive chronotropic and inotropic effects of CAP and NT were similar in BK-desensitized and control atria. The results were interpreted as an indication that NT, BK and CAP produce their excitatory effects in guinea pig atria by interacting with a common population of CAP-sensitive sensory nerve fibers (presumably substance P (SP)- and CGRP-containing nerve fibers). The absence of cross-desensitization between NT or BK and CAP, or between NT and BK, suggests that the activation and desensitization of atrial, CAP-sensitive sensory nerve fibers by the latter agents involve different receptors and/or mechanisms.     

The vegetative network in the guinea pig and rat sacral spinal cords

The intermediate zones in the sacral segments of guinea pig and rat spinal cords are histochemically investigated for the presence of catecholaminergic and cholinergic structures. The presence of a well-developed noradrenergic network is demonstrated. This network connects the preganglionic vegetative nuclei in horizontal and vertical directions. In addition, only some fibers from the bundles composing this network show acetylcholinesterase activity.     

The vegetative network in the guinea pig and rat sacral spinal cords

Summary The intermediate zones in the sacral segments of guinea pig and rat spinal cords are histochemically investigated for the presence of catecholaminergic and cholinergic structures. The presence of a well-developed noradrenergic network is demonstrated. This network connects the preganglionic vegetative nuclei in horizontal and vertical directions. In addition, only some fibers from the bundles composing this network show acetylcholinesterase activity.     

Intrinsic innervation of the uterus in guinea pig and rat

The pattern of distribution of cholinergic and adrenergic nerves in the uterus of albino rats and guinea pigs was examined histochemically. In the albino rat, the uterus was found well-innervated by both adrenergic and cholinergic nerves with a clear regional variation. Dense innervation was demonstrated at the tubal and cervical ends of the uterus and in the cervix. Cholinergic nerves supplying the glands were more numerous than the adrenergic nerves which were relatively few. In the guinea-pigs, the uterus was richly innervated by adrenergic nerves with a clear regional variation. No cholinesterase-positive nerves or nerve cells were demonstrated.     

Cyclic nucleotide levels during carbachol-induced smooth muscle contractions.

Cyclic nucleotide levels and tension were measured at various times during carbachol-induced smooth muscle contractions. Cyclic GMP levels were markedly increased during contractions of rat vas deferens, guinea pig myometrium and guinea pig taenia coli, but were unchanged during contractions of rat uterus or guinea pig ileum. No significant changes in cyclic GMP levels could be detected in estrogen-primed rat uteri at any of the times or drug concentrations studied. Even in tissues in which large increases in cyclic GMP levels could be detected during carbachol-induced contractions (i.e. guinea pig myometrium and taenia coli) the contractions appeared to precede the cyclic GMP increases by several seconds. No significant changes in cyclic AMP levels were observed during carbachol-induced contractions in any of the smooth muscles studied. Thus, changes in tissue levels of the cyclic nucleotides do not appear to be responsible for the initiation of carbachol-induced smooth muscle contractions.     

Comparative histochemical study of the enzyme changes in the ovary and uterus of mammals with special references to steroidogenesis.

A comparative study of the enzymes delta5-3beta-HSD, cytochrome oxidase and peroxidase has been made in the ovaries and uterus of mammals (mouse, guinea pig, cat and dog) during various reproductive phases. The granulosa cells of developing follicles, hypertrophied interstitial cells of thecal origin and the luteal cells show intense delta5-3beta-HSD and cytochrome oxidase activity. Peroxidases are found to be present in the corpus luteum and the epithelial cords of thecal origin. delta5-3beta-HSD and cytochrome oxidase activity is localized to the endometrium and myometrium of mature and pregnant uterus of mouse and guinea pig, while peroxidase is seen only in the decidua and endometrial glands of pregnant animals. The significance of these enzymes is discussed in relation to the cellular basis of luteinization and steroid hormone synthesis.     

Activity of specific and non-specific cholinesterases in the subcommissural organ of guinea pig and albino rat

Summary The localizations of specific and non-specific cholinesterases were demonstrated by light and electron microscopical methods in the secretory cells of the subcommissural organ of the guinea pig and albino rat.The activity of non-specific cholinesterase at light microscopical level appeared slightly stronger compared to the activity of the specific cholinesterase. No differences in the localizations of the both enzymes could be noticed.In electron microscopic specimens no differences could be observed between the localization or intensity of the specific and non-specific cholinesterase reactions except some nerve fibres round the secretory hypendymal cells in which only a specific cholinesterase reaction product was noticed. The reaction product was found mainly in the cytoplasmic and nuclear membranes, in the rough and smooth surfaced endoplasmic reticulum and around some secretory granules in the ependymal and hypendymal secretory cells of the subcommissural organ in guinea pig and albino rat.The possible role of cholinesterases in the secretory cells of the subcommissural organ is further discussed. Their participation in the metabolism and/or secretion of the hormonal end products is suggested.     

Distribution, origin and sensitivity to capsaicin of primary afferent substance P-immunoreactive nerves in the heart

This report is intended as an overview of the distribution, origin and sensitivity to capsaicin of substance P-immunoreactive (SP-I) primary afferent cardiac nerves. Immunohistochemical and physiological methods were employed to compare the presence and density of these nerve fibers in the guinea pig and rat hearts. SP-I fibers are numerous in the guinea pig heart including the parietal pericardium, atria, ventricles, valves, coronary arteries and around intrinsic cardiac ganglion cells. The rat heart contains few SP-I fibers. Vagotomy does not influence the number of intensity of immunoreactive fibers in the guinea pig heart. By stimulating the atrium or ventricle and recording from the second or third thoracic dorsal roots Ad1, Ad2 and C fibers were demonstrated in the atria, but only Ad fibers in the guinea pig ventricle; in addition, only Ad fibers were recorded from the vagus nerves. Only Ad1 fibers were demonstrated in the rat heart. Treatment with capsaicin depletes the SP-I and decreases the conduction velocity of C-fibers and some Ad2 fibers in the guinea pig heart. We suggest that SP-I primary afferent nerve fibers are unmyelinated (C-type) or small myelinated (Ad2-type) nerves in the guinea pig heart and that their cell bodies of origin are predominantly in dorsal root ganglia.     

Pituitary adenylate cyclase-activating polypeptide: localization and differential influence on isolated hearts from rats and guinea pigs

This study was done to determine if pituitary adenylate cyclase-activating peptide (PACAP)-immunoreactive nerve fibers occur in cardiac muscle as well as intracardiac ganglia of rats and guinea pigs and to clarify the chronotropic actions of PACAP27 in the same species using isolated heart preparations. PACAP nerve fibers were not detected in atrial or ventricular muscle of rat or guinea pig but a few stained nerve fibers occurred in the atrioventricular bundle of the guinea pig. Stained nerve fibers were prominent in intracardiac ganglia of both species. PACAP27 caused a dose-dependent tachycardia in isolated rat hearts (+39 +/- 3 beats/min with 1 nmol, n = 6). Positive and/or negative chronotropic responses were evoked by PACAP27 in guinea pig heart, depending on dose and prior exposure to the peptide. PACAP27 also caused arrhythmias in several guinea pig hearts. Treatment with atropine eliminated or prevented PACAP-evoked bradycardia and arrhythmias, implicating cholinergic neurons in these responses. Positive chronotropic responses to PACAP were unaffected by beta-adrenergic receptor blockade in either species, suggesting that tachycardia resulted from a direct action on the heart. These observations support the conclusion that endogenous PACAP could have a role in regulating parasympathetic input to the heart but through different mechanisms in rats versus guinea pigs. A direct positive chronotropic influence of endogenous PACAP is unlikely since atrial muscle lacks PACAP-immunoreactive nerve fibers.     

Neurofilament-like and glial fibrillary acidic protein-like immunoreactivities in rat and guinea-pig sympathetic ganglia in situ and after perturbation

Summary The presence of neurofilament (NF)-like and glial fibrillary acidic protein (GFAP)-like immunoreactivities was studied in sympathetic ganglia of adult rats and guinea pigs during normal conditions and after perturbation. In the superior cervical ganglion (SCG) of normal rats, many ganglion cells and nerve fibers show NF immunoreactivity. Some of these nerve fibers disappear after preganglionic decentralization of SCG; this indicates the presence of a mixture of preand postganglionic NF-positive nerves in the ganglion. Cuts in both preand postganglionic nerves result in a marked increase in GFAP immunoreactivity in SCG, whereas NF immunoreactivity increases in nerve cell bodies after preganglionic cuts. Only a few ganglion cells show NF immunoreactivity in the normal SCG of guinea pig. All intraganglionic NF-positive nerves are of preganglionic origin; decentralization abolishes NF immunoreactivity in these nerve fibers. The inferior mesenteric ganglion, the hypogastric nerves and colonic nerves in guinea pigs contain large numbers of strongly NF-immunoreactive nerve fibers.When the SCG of adult rat is grafted to the anterior eye chamber of adult rat recipients, both ganglionic cell bodies and nerve fibers, forming on the host iris from the grafted ganglion, are NF-positive. As only the perikarya of these neurons normally exhibit NF immunoreactivity, and the terminal iris arborizations are NF-negative, it appears that the grafting procedure causes NF immunoreactivity to become more widespread in growing SCG neurons.     

Uterine adrenergic and cholinesterase-positive nerves and myometrial catecholamine concentrations during pregnancy in sheep

Uterine adrenergic and cholinesterase (AChE)-positive innervation of the sheep uterus during anestrus and at 4 stages of pregnancy were examined by histochemical methods. In addition, uterine and cervical myometrium concentrations of norepinephrine (NE) and dopamine (DA) were determined using high-performance liquid chromatography. During anestrus, adrenergic and AChE-positive nerve fibers in the uterine myometrium and endometrium were primarily associated with the vasculature. Innervation of myometrial smooth muscle was almost exclusively by adrenergic fibers. In the endometrium, fibers of both types were observed closely associated with endometrial glands, and adrenergic fibers were observed in the connective tissue beneath the luminal epithelium. Density of uterine innervation decreased by day 65 of pregnancy with an additional decrease by day 105. Myometrial NE concentrations were higher in the cervix than the uterus. Uterine NE concentrations generally were not affected by pregnancy. Although cervical NE per gram of tissue decreased during pregnancy, this effect of pregnancy was not detected when NE was expressed per microgram of DNA. Myometrial DA concentrations were higher in uterine segments than in the cervix. DA concentrations decreased during pregnancy in all tissues except the posterior uterine segment. The DA to NE ratio in the uterus was greater than that for the cervix and was not generally affected by the stage of pregnancy. These results demonstrate that cholinergic and adrenergic nerves supply the sheep uterus. Decreasing fiber density during pregnancy suggests that a majority of the innervation to the sheep uterus is supplied by 'short' nerve fibers whose activity is regulated by steroids of pregnancy. The possible role of DA as a neurotransmitter in the sheep uterus is discussed.     

Pharmacological studies on the venomous spotted butterfish (Scatophagus argus Linn) sting extract on experimental animals

A sting of the fish S. argus, a venomous edible spotted butterfish, produces tremendous local pain, severe swelling, rise of body temperature, throbbing sensation etc. To establish the pharmacological activities of S. argus sting extract, the present investigation, was carried out on experimental animals. The LD50 of extract was found to be 9.3 mg/kg (iv) in male albino mice. The extract showed loss of sensation, urination and salivation in mice. It potentiated pentobarbitone induced sleeping time in male albino mice and produced hypothermia. Extract produced a fall of cat and guinea pig blood pressure, which was completely abolished by mepyramine. It produced a transient reduction of respiratory rate in rat, but decreased respiratory amplitude in cat, which was abolished after vagotomy. On isolated toad heart, the extract increased both the amplitude and rate of contraction. On isolated guinea pig heart, the sting extract decreased both the rate and amplitude of contraction leading to cardiac arrest, but it had no effect on isolated guinea pig auricle. The extract produced a reversible blockade of electrically induced twitch response of isolated chick biventer cervices preparation, but it had no effect on the isolated rat phrenic nerve diaphragm preparation. It produced a slow contractile response on isolated guinea pig ileum, rat uterus and rat fundal strip preparations but produced slow relaxation on isolated rat duodenum preparation. The contractile response on isolated guinea pig ileum and rat fundal strip was antagonised by SC19220. It did not produce any significant cutaneous haemorrhage in mice and did not produce any haemolysis on saline washed erythrocytes. The sting extract significantly increased capillary permeability of guinea pig dorsal flank and produced oedema in mice hind paw.