Autoradiographic study of 3H-lysine uptake by superior cervical and stellate ganglia in prehypertensive spontaneously hypertensive rats

In order to compare the functional state of sympathetic ganglia in spontaneously hypertensive (SHR) with those in normotensive Wistar Kyoto rats (WKY), protein synthetic activity was examined by light microscopic autoradiography with 3H-lysine. The number of silver grains over the cytoplasm of ganglion cells in the superior cervical and stellate ganglia of newborn and 30-day-old animals were counted on photographic enlargements. In both sympathetic ganglia there were significantly more silver grains over ganglion cells in SHR compared with age-matched WKY at 15, 60, and 120 min after injection of 3H-lysine. The increased incorporation of the label by both sympathetic ganglia was more marked in newborn than in 30-day-old animals. This result shows that protein synthetic activity in these ganglion cells is increased in SHR from the newborn stage. It is suggested that a congenital hyperfunction of sympathetic ganglia occurs in SHR.     

Plasma norepinephrine and dopamine-β-hydroxylase in genetic hypertensive rats

The relationship between blood pressure, plasma norepinephrine (NE), dopamine-β-hydroxylase (DBH) activity and age was investigated in spontaneously hypertensive rat (SHR), a stroke-prone substrain of the SHR and control Wistar-Kyoto rat (WKR). Blood pressure of both SHR strains increased with age and was significantly higher than that of the WKR at all ages tested (3 15 weeks). The blood pressure of stroke-prone SHR was significantly higher than that of the regular SHR after 6 weeks of age. Plasma DBH activity decreased with age in each strain, although the SHR, and especially the stroke-prone SHR, had significantly higher DBH than the controls at an early age. Plasma NE in the WKR did not change with age. Increased plasma NE was observed only in the young SHRs. The highest values were found in the 6 week old stroke-prone SHR. These data suggest that plasma DBH activity is not correlated directly with plasma NE or blood pressure, but that increased sympathetic nerve activity may occur during the development of hypertension in the SHR and the stroke-prone SHR.     

Quantitative analysis of ACTH-immunoreactive cells in the anterior pituitary of young spontaneously hypertensive and normotensive rats

Summary ACTH-immunoreactive cells in the anterior pituitary of 4-week-old spontaneously hypertensive rats (SHR) were studied with immunocytochemical and morphometric techniques. The results were compared with data from age-matched normotensive Wistar-Kyoto rats (WKY). No significant differences were found in volume density and average size of ACTH-immunoreactive cells between these two strains. However, SHR showed a significantly larger anterior lobe (2 P < 0.01) than WKY, indicating that the total number of ACTH-immunoreactive cells in the anterior pituitary is greater in SHR than in WKY. These data are in agreement with radioimmunological determinations showing a significantly elevated content (2 P < 0.01) but only a moderately higher concentration (0.05 < 2 P < 0.10) of ACTH in the anterior pituitary of SHR as compared to WKY. The present results suggest an enhanced availability of ACTH in the anterior pituitary of 4-week-old SHR, a fact which could explain the markedly enhanced stress-induced release of ACTH previously found in these animals. This study further supports the hypothesis that, among other factors, an instability of the hypothalamo-pituitary-adrenal axis may contribute to the development of genetically programmed hypertension.     

Effects of acute and subacute cocaine administration on the CNS dopaminergic system in Wistar-Kyoto and spontaneously hypertensive rats: III. Dopamine uptake

The characteristics of dopamine uptake after acute and subacute cocaine administration were determined in striata from WKY and SHR. In acutely-treated (40 mg/kg, s.c.) rats, significant increases in the V_max of dopamine uptake were observed 30 min after the cocaine injection in both strains, without changes in K_m values. The in vitro IC₅₀ for cocaine was significantly decreased at 30 min in WKY and at 2 h in SHR. However, the in vitro IC₅₀ for GBR-12909 was significantly increased at 30 min and at 2 h in both strains following cocaine administration. In both strains, the density (B_max) of the [³H]GBR-12935 binding site was significantly increased at 30 min and at 2 h with no charges in K_d. In subacutely-treated (20 mg/kg, twice daily for 3 or 7 days) rats, a significant increase in the K_m for dopamine uptake was observed in 7 day treated SHR. The in vitro IC₅₀ for GBR-12909 was significantly increased in 3 day treated WKY. The results suggest that cocaine administration alters dopamine uptake and characteristics of dopamine uptake sites in the rat brain.     

Cellular proliferation in the anterior pituitary of the rat during the postnatal period

Summary Cellular proliferation in the anterior pituitary of 2-, 8-, 15- and 30-day-old rats was examined by injection of bromodeoxyuridine 1 h before autopsy. Bromodeoxyuridine incorporated into DNA was detected immunohistochemically by use of a monoclonal antibody. The highest rate of cell proliferation was found in 2-day-old animals; it decreased thereafter during the postnatal period. Possible toxic effects of colchicine on cellular proliferation were examined. Colchicine treatment (10 mg/kg in 8- and 30-day-old animals) significantly decreased the number of bromodeoxyuridine-labelled cells/mm² in 8-day-old rats. Some sections were doubly immunostained for bromodeoxyuridine and various pituitary hormones. The proportion of doubly-immunostained cells to all proliferating cells was generally low, ranging from 23% at 2 days to 32% at 30 days of age.On leave from the Department of Human Anatomy and Histology, Faculty of Medicine, University of Salamanca, Salamanca, Spain     

The dissociation between the plasma levels of aminopeptidases A and B in spontaneously hypertensive rats

We performed a longitudinal study for 20 weeks on spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKR) to determine the relationship between peptide metabolism and the age-dependent increase in blood pressure. In both SHR and WKR, the plasma level of aminopeptidase A (AP-A) clearly showed an age-dependent decrease. The plasma level of aminopeptidase B paralleled that of AP-A in WKR, but such an age-dependency was not observed in SHR, thus showing a dissociation between the two aminopeptidases. With age in both strains, the level of angiotensin-converting enzyme tended to decrease, while that of kallikrein activity tended to increase. In addition to these findings, a multivariate study testing the relationship of blood pressure to these enzyme activities, as well as to plasma levels of angiotensin I and renin activity, suggested abnormalities in the networks of proteolytic enzymes and in the peptide metabolism surrounding the renin-angiotensin system in SHR. These abnormalities may play some important roles in pathophysiological mechanisms of hypertension in SHR.     

Sensitivity of spontaneously hypertensive and of Wistar Kyoto rats to the antidipsogenic action of eledoisin

This study investigated the sensitivity of spontaneously hypertensive rats (SHR) and of Wistar Kyoto rats (WKR) to the antidipsogenic action of the tachykinin eledoisin (ELE). Drinking was evoked by: (a) intracerebroventricular (i.c.v.) injection of angiotensin II, (b) subcutaneous (s.c.) administration of hypertonic NaCl (1.5 M; 1 ml/100 g b.wt.) or (c) 18 h of water deprivation with free access to food. In accordance with previous studies, the dipsogenic effect of all three treatments was exaggerated in the SHR. And when treated with i.c.v. ELE (12.5-25 ng/rat) they were far less sensitive than WKR to its antidipsogenic action on angiotensin-induced drinking. Smaller differences in strain sensitivity were also observed for the effect of ELE on cell dehydration- and on water deprivation-induced drinking, but only at the dose of 200 and 50 ng/rat, respectively. The different sensitivity of the SHR to the antidipsogenic effect of ELE supports the idea that tachykininergic mechanisms for control of water intake are differently regulated in the SHR than they are in the normotensive WKR.     

HORMONE SECRETION BY CELLS DISSOCIATED FROM RAT ANTERIOR PITUITARIES

A new procedure has been developed for dissociating anterior pituitary tissue and producing a viable suspension of single cells. The procedure involves incubation of small tissue blocks in 1 mg/ml trypsin (15 min), followed by incubation in 8 µg/ml neuraminidase and 1 mM EDTA (15 min), followed by mechanical dispersion. Cell yields are ~55%, based on recovered DNA. By electron microscopy five types of secretory cells (somatotrophs, mammotrophs, thyrotrophs, gonadotrophs, and corticotrophs) plus endothelial and follicular cells can be identified and are morphologically well preserved up to 20 h after dissociation. Throughout this period, the cells incorporate linearly [³H]leucine into protein for up to 4 h at a rate 90% greater than hemipituitaries, and they synthesize, transport intracellularly, and release the two major pituitary secretory products, growth hormone and prolactin. Immediately after dissociation the cells' ability to respond to secretogogues (high K⁺ and dibutyryl cyclic AMP) is impaired, but after a 6–12-h culture period, the cells apparently recover and discharge 24% and 52%, respectively, of their content of prelabeled growth hormone over a 3-h period in response to these two secretogogues. This represents a stimulation of 109% and 470% over that released by cells incubated in control medium. The results demonstrate that function and morphologic integrity are preserved in this cell system. Therefore it is suitable for the study of various aspects of pituitary secretion and its control.     

The effects of prenatal protein deprivation on thyrotroph development

Maternal dietary protein restriction produced by feeding a diet containing 4% casein throughout gestation adversely affects body size and retards development of various organs in the progeny. For the most part, alterations are present in structural or functional entities which evolve during the last trimester of gestation. Fetal thyroid follicle formation and iodine concentrating capacity, which increase rapidly between the 17th gestational day and birth in pups of dams fed the control (24% casein) ration, are retarded in age-matched pups of protein-deficient females. The first immunoreactive thyrotrophs appear in the fetal pituitary on day 17 in both control and prenatally protein-deprived (PPD) young. The total number of thyrotrophs per pituitary was unaffected by maternal protein deficiency, except on day 21 when there were significantly more thyrotrophs per pituitary in fetal control rats. Although pituitary volume was significantly reduced in 18-, 19- and 21-day old fetal PPD rats, as compared with controls, pituitary volume:body weight ratios differed between young in the two dietary groups only on day 21, when the ratio was significantly higher in PPD as compared to control young. Maternal protein deprivation does not affect the morphological maturation of the thyrotrophs of the anterior pituitary of the fetal rat.     

Increased pressor responses to pressor agents in spontaneously hypertensive rats.

Pressor reactivity to a variety of pressor agents after partial ganglionic blockade induced with hexamethonium was investigated in intact, in spinalized, and in chemically sympathectomized, spontaneously hypertensive rats (SHR). Responses of unanaesthetized 6-month-old SHR to noradrenaline, phenylephrine, and angiotensin after hexamethonium administration (32 mg/kg) markedly exceeded those of unanaesthetized, age-matched normotensive Wistar-Kyoto rats (WKR). Responses of anaesthetized SHR to noradrenaline after hexamethonium administration (16 mg/kg) were also increased at the hypertensive stages but not at the prehypertensive stages, when compared with those of anaesthetized normotensive Wistar rats of respective ages. In spinalized and chemically sympathectomized preparations after hexamethonium administration (16 mg/kg), noradrenaline produced equal increases in blood pressure in 6-month-old SHR and WKR. It is suggested that the functional sympathetic nervous system is important for the hyperreactivity of intact SHR.     

Heterogeneity in Arterial Remodeling among Sublines of Spontaneously Hypertensive Rats

Objectives

Spontaneously hypertensive rats (SHR) have been used frequently as a model for human essential hypertension. However, both the SHR and its normotensive control, the Wistar Kyoto rat (WKY), consist of genetically different sublines. We tested the hypothesis that the pathophysiology of vascular remodeling in hypertension differs among rat sublines.

Methods and Results

We studied mesenteric resistance arteries of WKY and SHR from three different sources, at 6 weeks and 5 months of age. Sublines of WKY and SHR showed differences in blood pressure, body weight, vascular remodeling, endothelial function, and vessel ultrastructure. Common features in small mesenteric arteries from SHR were an increase in wall thickness, wall-to-lumen ratio, and internal elastic lamina thickness.

Conclusions

Endothelial dysfunction, vascular stiffening, and inward remodeling of small mesenteric arteries are not common features of hypertension, but are subline-dependent. Differences in genetic background associate with different types of vascular remodeling in hypertensive rats.     

Selenium in the anterior pituitary of the rat after a single injection of75Se sodium selenite

In order to investigate the selenite metabolism in the anterior pituitary and compare it with other endocrine organs, rats were injected intraperitoneally with⁷⁵Se sodium selenite (5 mg/kg). The rats were whole body counted shortly after injection and recounted just before sacrifice, which was performed 2, 24, 48 h, and 4, 10, 20, 30, 40, 60, and 80 d after injection. Besides the anterior pituitary, the selenium content was also estimated in the thyroid gland, testis, adrenals, liver, kidney, and blood. The maximum selenium content was observed in all organs 2 h after injection, at which time the anterior pituitary contained 2.9 μg/g wet wt, compared to 13.5 μ/g wet wt in liver and .6 μg/mg wet wt in testis. The excretion of selenite from the anterior pituitary resembled that seen in most other organs investigated, i.e., an initial rapid excretion and a slower secondary phase resembling a first order reaction. Practically all selenium was excreted by 60 d after injection.     

Propranolol-induced alterations in the pathophysiology of spontaneously hypertensive rats

Male and female, spontaneously hypertensive rats (SHR) were treated with propranolol (14 weeks) when they became 1, 4 and 8 months old prior to, during the steep ascent, and when severe high blood pressure becomes established. Propranolol prevented the usual increase in blood pressure at an early age but did not effectively lower blood pressure at all age levels. Propranolol-treated SHR manifested marked alterations in pituitary, adrenal, thymus, heart and gonadal weights. Despite progressively increasing hyperlipidemia and hyperglycemia, there were no significant differences between lipid, glucose, BUN, or serum enzyme levels in treated vs nontreated SHR. Circulating aldosterone and corticosterone levels were reduced in propranolol-treated SHR. Male SHR, treated or untreated, developed severe cardiovascular-renal lesions when they became 8 months old; none of the female SHR manifested any pathologic changes. It is suggested that the anti-hypertensive effects of propranolol were partially mediated by hormonal as well as by hemodynamic mechanisms.     

Developmental changes of Islet-1 and its co-localization with pituitary hormones in the pituitary gland of chick embryo by immunohistochemistry

Although Islet-1 expression in the pituitary gland of early mouse embryo has been previously described, there are no reports concerning the correlation of Islet-1 expression with lineage restrictions in cell types at the later stages of pituitary development. The role of Islet-1 in chickens is also unknown. The purpose of this study was to follow, by using immunohistochemistry, the ontogeny of pituitary Islet-1 and the various cell types that contain Islet-1 throughout chick embryo development. A few Islet-1-immunopositive (Islet-1⁺) cells were first detected in the pituitary primordium in two out of six embryos at embryonic day 5.5 (E5.5), most of the Islet-1⁺ cells being ventrally located. As development progressed, many more Islet-1⁺ cells were observed throughout the pars distalis. The relative percentage of Islet-1⁺ cells amongst the total Rathke’s pouch cells was 4.4% at E6.5. This increased significantly, reaching 11.1% by E10.5, followed by no significant change until hatching. Dual immunohistochemistry showed that adrenocorticotrophs, somatotrophs and lactotrophs did not express Islet-1. The cellular types expressing Islet-1 included luteinizing-hormone-positive (LH⁺) gonadotrophs and thyroid-stimulating-hormone-positive (TSH⁺) thyrotrophs. The cells co-expressing LH and Islet-1 were initially detected at E6.5, the proportion of LH⁺ cells possessing Islet-1 being about 4%; this increased to 63% at E14.5, followed by no significant changes until hatching. TSH and Islet-1 co-localized cells were first observed at E10.5, with about 37% TSH⁺ cell expressing Islet-1; this increased to about 50% by E16.5, after which there was no evident change until hatching. These results suggest that Islet-1 is involved in determining the cell lineages, proliferation, differentiation and maintenance of hormone-secreting functions of pituitary gonadotrophs and thyrotrophs of chick embryo. J. Liu and Y. He contributed equally to this article. This work was supported by grants from Beijing Natural Science Foundation (6042013) and the Natural Science Foundation of China (30471264, 30325034).     

Nucleo-cytoplasmic exchange of non-histone proteins in amphibian embryos

As a basis for understanding the role of non-histone proteins in nuclear differentiation, we have identified one period during embryogenesis when intense accumulation of non-histones occurs in nuclei of Rana pipiens. We then demonstrated, experimentally, the loss of non-histones from nuclei after transplantation into enucleated eggs. ³H-tryptophan or ³H-lysine was injected into blastocoeles of mid-blastulae and into archenterons of late gastrulae; embryos were subsequently studied autoradiographically. Nuclei of animal hemisphere cells from blastulae accumulated only small amounts of ³H-tryptophan within 3 h, whereas a large accumulation occurred in endodermal nuclei of gastrulae as early as 1 h, and after 3 h 95.9% ( ) of the nuclei were densely labelled. Significant accumulation of ³H-lysine occurred in the majority of nuclei of both types within 3 h (blastulae ; gastrulae ). Controls, involving RNase or boiling TCA, demonstrated that the ³H-amino acids have been incorporated mainly into proteins. Endodermal nuclei labelled either with ³H-tryptophan or ³H-lysine after a 3 h incubation were transplanted singly into enucleated eggs. Autoradiograms demonstrated that most non-histones leave the nucleus during its reprogramming in the egg cytoplasm prior to first cleavage; whereas other types of proteins labelled with ³H-lysine remain for the most part in the nucleus. Cytochemical studies indicated that some of the non-histones which leave transplanted nuclei are acidic proteins; whereas some of those proteins which remain in the nucleus are histones.In addition to the above findings, the results of these studies demonstrate the feasibility in the future of studying the nucleocytoplasmic migration of different kinds of macromolecules in a developmental metazoan system and determining their roles in the establishment of nuclear differentiation.     

Expression of P2Y receptors in the rat anterior pituitary

In this study, the distribution patterns of P2Y₁, P2Y₂ P2Y₄, P2Y₆, P2Y₁₂, and P2Y₁₃ receptors in the anterior pituitary cells of rat were studied with double-labeling immunofluorescence and Western blot. The results showed that P2Y receptors were widely expressed in the anterior pituitary. P2Y₁ and P2Y₄ receptors were found to be expressed in the majority of gonadotrophs and thyrotrophs, P2Y₂ receptors were expressed in a small subpopulation of lactotrophs and almost all the folliculo-stellate cells, that were also stained with S100 protein immunoreactivity. P2Y₆ receptors were expressed in macrophages. P2Y₁₃ receptors were expressed in a small subpopulation of cells in the rat anterior pituitary, the identity of which needs to be clarified. P2Y₁ and P2Y₄ receptors are co-expressed in some gonadotrophs and thyrotrophs. Corticotrophs and somatotrophs were found not to express P2Y receptors in this study. FSH and TSH were shown to coexist in the same endocrine cells in rat anterior pituitary. The present data suggests that purines and/or pyrimidines could be involved in regulating the functions of gonadotrophs and thyrotrophs via P2Y₁ and P2Y₄ receptors, some lactotrophs via P2Y₂ receptors, and folliculo-stellate cells via P2Y₂ receptors in the rat anterior pituitary.     

Estrogen modulation of angiotensin-stimulated phosphoinositide hydrolysis in slices of rat anterior pituitary

In this study, slices of rat anterior pituitary were prelabeled with [³H]myo-inositol and the ability of angiotensins II and III to stimulate [³H]phosphoinositide hydrolysis was characterized. When using tissue derived from ovariectomized rats, dose-response experiments revealed that angiotensin II significantly increases [³H]inositol monophosphate formation (in the presence of 10 mM LiCI) at concentrations of 10 nM and above. Maximal stimulation by angiotensin II was observed at 1 μM (228% of basal) and 50% maximal stimulation was at 10.8 ± 2.7 nM. Angiotensin III was less potent when compared to angiotensin II (maximal stimulation at 10 μM; 220% of basal: 50% maximal stimulation, 475 ± 159 nM). When using normal female rats, significant stimulation by angiotensin II was not observed until 1 μM angiotensin II. When ovariectomized rats were treated for 7 days with 17β-estradiol, increases in [³H]inositol monophosphate induced by 1 μM angiotensin II were significantly reduced when compared to sesame oil vehicle controls.This study shows that estrogen down-regulates angiotensin receptor coupling in the anterior pituitary. Moreover, it illustrates the influence of the hormonal state of the animal on the regulation of the effects of angiotensins in this tissue.     

Angiotensin-converting enzyme (kinase II) in pituitary gland of spontaneously hypertensive rats

Angiotensin-converting enzyme (ACE) (kinase II; dipeptidyl carboxypeptidase, EC 3.4.15.1) activity was measured in pituitary gland of young (4-week-old) and adult (18-week-old) male, spontaneously hypertensive rats (SHR) and in age-matched normotensive male Wistar-Kyoto (WKY) control rats. In the three lobes of the pituitary gland ACE activity was significantly higher in young than in adult animals, in both SH and WKY rats. In the anterior lobe, ACE activity was lower in SHR when compared to age-matched Wistar-Kyoto controls. In contrast, ACE activity in the intermediate lobe of the pituitary gland was higher in SHR, and in particular in young animals. The observed differences between young WKY and SH rats in both the intermediate and anterior lobes did not appear to be due to a modified affinity of ACE for the substrate hippuryl-His-Leu, but to alterations in ACE maximal velocity or number of available molecules. No differences in ACE activity were detected between SHR and WKY rats in the posterior lobe. Total protein content was higher in the intermediate lobe and lower in the posterior lobe of young SHR when compared to normotensive controls. The present results suggest the possibility for a role of pituitary ACE in spontaneous (genetic) hypertension in rats.     

Increased renal vascular reactivity to norepinephrine in stroke-prone spontaneously hypertensive rat (SHR)

Vascular responses of isolated perfused kidneys to norepinephrine, angiotensin II and KCl were examined in stroke-prone spontaneously hypertensive rats (SHR-SP), stroke-resistant SHR (SHR-SR) and control Wistar Kyoto rats (WKR) at 8 to 10 weeks of age. Average values of systolic blood pressure were 181, 155 and 122 mmHg in the SHR-SP, SHR-SR and WKR, respectively. Basal renal vascular resistance was identical among these rats. In response to norepinephrine, the SHR-SP demonstrated shift in the dose-response curve to the left and lower vasoconstrictor threshold than the controls. The SHR-SR was intermediate between the SHR-SP and the controls in the response to norepinephrine. For angiostein II, the SHR-SP but not SHR-SR displayed a slightly greater response than the controls. However, response to KCl was not significantly different among these three strains of rats. The results suggest that the SHR-SP rats have an intrinsic augmentation of renal vascular reactivity to norepinephrine, which in turn leads to rapid elevation of blood pressure at the early hypertensive stage.