The sensory circumventricular organs of the mammalian brain.

The brain's three sensory circumventricular organs, the subfornical organ, organum vasculosum of the lamina terminalis and the area postrema lack a blood brain barrier and are the only regions in the brain in which neurons are exposed to the chemical environment of the systemic circulation. Therefore they are ideally placed to monitor the changes in osmotic, ionic and hormonal composition of the blood. This book describes their. General structure and relationship to the cerebral ventricles Regional subdivisions Vasculature and barrier properties Neurons, glia and ependymal cells Receptors, neurotransmitters, neuropeptides and enzymes Neuroanatomical connections Functions.

Characterization of a specific antibody to the rat angiotensin II AT1 receptor.

We raised a polyclonal antibody against a decapeptide corresponding to the carboxyl terminus of the rat angiotensin II AT1 receptor. This antibody was demonstrated to be specific for the rat receptor according to a number of approaches. These included (a) the ultrastructural localization of immunogold-labeled receptor on the surfaces of zona glomerulosa cells in the adrenal cortex, (b) the specific labeling of Chinese hamster ovarian (CHO) cells transfected with AT1 receptors, (c) the identification of a specific band on Western blots, (d) the immunocytochemical co-localization of angiotensin receptors on neurons in the lamina terminalis of the brain shown to be responsive to circulating angiotensin II, as shown by the expression of c-fos, and (e) the correlation between the expression of the mRNA of the AT1 receptor and AT1 receptor immunoreactivity.(J Histochem Cytochem 47:507-515, 1999)

Reduced glycogen phosphorylase activity in denervated hindlimb muscles of rat is related to muscle atrophy and fibre type.

Changes in the activity of muscle glycogen synthase or phosphorylase (GP) may be responsible for the deregulation of glycogen synthesis and storage which occurs in diabetes mellitus. To clarify the relationship between muscle atrophy, fibre type, insulin-stimulated glucose uptake and GP activity during insulin resistance, we used sciatic nerve severance to induce insulin resistance in rat hindlimb muscles and compared the above parameters in muscles with a range of fibre types. Changes were analysed by comparison with the contralateral hindlimb, which bears more weight due to denervation of the opposing limb, as well as the sham-operated and contralateral limb of a separate rat. Denervation caused a decrease in insulin-stimulated glucose uptake by 1 day after denervation and a decline of GP activity after 7 days in all muscles investigated. GP activity decreased by 73% in soleus, 36% in red gastrocnemius, 35% in tibialis and 13% in white gastrocnemius, which was related to the degree of muscle atrophy and inversely related to the overall GP activity in non-denervated muscles. GP activity in muscles of the contralateral limb from the denervated rat did not differ from either hindlimb of the sham-operated rat. We conclude that the fibre-type related reduction in insulin-stimulated glucose uptake of denervated muscle determines the change in its metabolism and it is this metabolic change which determines the mechanism, rate and degree of muscle atrophy, which is directly related to the decline in GP activity.

Effects of diuretics on renal kallikrein gene expression in rats.

1. To determine the effects of diuresis and changes in electrolyte balance on kallikrein gene expression, renal kallikrein mRNA levels were correlated with urine volumes, urinary electrolyte levels, haematocrit and plasma electrolyte levels in rats treated with substances with a range of diuretic activities. 2. Furosemide and related compounds, benzyl furosemide and isofurosemide, as well as amiloride hydrochloride, chlorothiazide or the vehicle (saline) were administered twice daily for 24 or 72 h to rats housed in metabolic cages. 3. Diuresis occurred after each treatment with furosemide, after the initial treatment with benzyl furosemide and did not occur after isofurosemide, amiloride hydrochloride or chlorothiazide. 4. Kallikrein gene expression in kidney was increased after 72 h treatment with furosemide, after 24 or 72 h treatment with benzyl furosemide or amiloride hydrochloride and was unchanged after 24 or 72 h treatment with isofurosemide or chlorothiazide, compared with vehicle-treated controls. 5. Plasma urea levels were elevated after 72 h treatment with furosemide, benzyl furosemide and chlorothiazide and plasma chloride was decreased after 24 and 72 h benzyl furosemide. Haematocrits were unchanged. There were no changes in urinary electrolyte levels 72 h after treatment with any of the diuretics. 6. Neither diuresis nor measurable changes in plasma or urinary electrolytes correlate with changes in renal kallikrein gene expression after diuretic treatment of rats.

Furosemide compounds enhance urinary excretion of active kallikrein independently of their effects on urinary electrolyte excretion.

The chemical and diuretic effects of furosemide on the excretion and activation of urinary prokallikrein were investigated in rats by treatment with furosemide compounds with a range of diuretic activity or amiloride hydrochloride or the vehicle. Diuresis occurred with furosemide and benzyl furosemide, but not with isofurosemide, amiloride hydrochloride, or the vehicle. The urinary excretion rate of active kallikrein was significantly elevated above controls throughout the 72 h of treatment with all of the drugs tested, regardless of the level of diuresis or the rate of urinary electrolyte excretion. In contrast, the urinary excretion rate of total kallikrein (prokallikrein + active kallikrein) was unchanged in all groups. These data indicate that furosemide derivatives increase the activation, but not the excretion, of urinary prokallikrein and that these effects are unrelated to the chemical structure or diuretic activity of the compounds or to overall changes in urinary electrolyte excretion rates.

Redistribution of carbonic anhydrase VI expression from ducts to acini during development of ovine parotid and submandibular glands.

Carbonic anhydrase VI (CA VI) is a secreted enzyme produced predominantly by serous acinar cells of submandibular and parotid glands. We have investigated the developmental pattern of CA VI production by these glands in the sheep, from fetal life to adulthood, using immunohistochemistry. Also, a specific radioimmunoassay for CA VI was used to measure changes in enzyme expression in the parotid gland postnatally. CA VI is detectable by immunohistochemistry in parotid excretory ducts from 106 days gestation (term is 145 days), in striated ducts from 138 days and in acinar cells from 1 day postnatal. The duct cell content of CA VI declined as the acinar cell population increased, a feature also of CA VI immunoreactivity in the submandibular gland. Production of CA VI by submandibular duct cells was detectable initially at 125 days gestation, and acinar production was not seen before 29 days post-natal. Apart from the differing ontogeny of CA VI production in ducts and acini of parotid and submandibular glands, there was a parallel pattern of CA VI expression during the development of these major salivary glands. With the development of the acinar tissues in the postnatal lamb, there was a dramatic increase (about 600-fold) in the level of expression of CA VI in the parotid gland between days 7 and 59 as measured by radioimmunoassay.

Furosemide treatment alters the distribution of kallikrein gene expression in kidneys of mice.

The renal kallikrein gene is normally expressed in convoluted distal and connecting tubules of the renal cortex. After furosemide treatment of mice there was strong expression also in the distal thick ascending limb of the loop of Henle in the renal outer medulla. This expansion of the kallikrein-expressing distal tubule cell population did not occur in mice on a sodium-restricted diet, in sodium-restricted mice given a high potassium diet or in untreated controls. Although the overall level of kallikrein gene expression was significantly greater in kidneys from furosemide-treated mice than in any other group, there was no detectable change in renal kallikrein gene expression in the submandibular glands after any of the treatments.

Renal gene expression.

The advent of cloned DNA probes has revolutionized the study of cells and tissues via the technique of hybridization histochemistry. Because of the intrinsic specificity of the probes the application of the technique to functionally complex and morphologically diverse tissue like the kidney has been especially rewarding. The technology has been advanced enormously by the parallel development of synthesized DNA or oligonucleotides. A specific synthetic probe can be manufactured in a day, purified, labeled and used to determine in which cells a particular gene of interest is being expressed ("switched on"). The applications of this technology are particularly opposite in the renal field. They can provide unique insights into normal physiological processes and will provide new diagnostic approaches of unparalleled specificity. Some specific examples have been chosen over a wide range of institute programs to highlight the potential value of the technique of hybridization histochemistry and to emphasize its potential for studying renal physiology and pathology.

Female-specific genomic PCR sequences from Schistosoma mansoni as probes for hybridization histochemistry.

Rapid methods for sexing single larval or egg stages of schistosomes have been difficult to establish. Such methods would be of value for assessing changes in sex ratios at different stages of the life cycle and studying diseases processes of schistosomiasis. We describe the use of hybridization histochemistry for identification of female eggs of Schistosoma mansoni in sections of infected mouse liver using a highly repetitive genomic DNA sequence generated by the polymerase chain reaction with primers spanning a known female-specific sequence (W1) of S. mansoni. In an initial study, approximately half of the eggs in each liver section from mice infected for 50 days labelled heavily with this probe. Further studies will evaluate the sensitivity and specificity of the probe on clonal populations (either male or female) or larvae in host tissue.

Secretion of glandular kallikrein and renin from the basolateral pole of mouse submandibular duct cells: an immunocytochemical study.

Glandular kallikrein from salivary glands in rats has been measured in the circulation and has been shown to have local vasoactive effects. In mice, renin and epidermal growth factor from the submandibular gland (SM) also reach the circulation, as both have been measured in plasma. The route by which these peptides enter the blood from their site of synthesis in ducts of the SM is unclear. We have investigated by immunocytochemistry the secretory pathways for kallikrein and renin from salivary duct cells in mice. The renal/pancreatic kallikrein-secreting cells of the striated and excretory ducts of the SM were distinguished from the granular convoluted tubule (GCT) cells which secrete other glandular kallikreins on the basis of data obtained in previous studies, in which we used gene-specific oligonucleotide probes to identify the expressing cell types. Renal/pancreatic kallikrein was apparently secreted constitutively from the basolateral surface of striated duct cells and in secretory vesicles from excretory duct cells, whereas apical secretion occurred via the regulated pathway in both cell types. Glandular kallikreins and renin synthesized in GCT cells were secreted from the basolateral surface by dissolution of granules at the cell membrane. There were fenestrated capillaries underlying the duct tree which would enable the secreted products to reach the circulation.

Sites of glandular kallikrein gene expression in fetal mice.

As part of an ongoing study of the cell-specific expression of glandular kallikrein genes in mice, we have investigated cellular sites of expression of the renal/pancreatic kallikrein gene, mGK-6, during fetal life. Expression of alpha I and beta-subunit genes of Na+K+ATPase and bradykinin binding were used as an indication of the functional maturity of the fetal epithelial tubules in which mGK-6 expression was identified. mGK-6 mRNA was first observed at embryonic day 16 (E16) in the submandibular main duct, then at E18 in the sub-lingual main duct, at E19 in renal tubules and at E19 in ducts of the nasal glands. All of these ducts contained detectable epithelial Na+K+ATPase mRNAs from an earlier gestational age than mGK-6 mRNA, suggesting their capacity for electrolyte transport. Bradykinin binding was evident in renal tubules at E18. This study established that renal/pancreatic kallikrein is synthesized in fetal epithelial tubules which are mature functionally.

Sites of expression and induction of glandular kallikrein gene expression in mice.

In order to provide a foundation for comparison across species of glandular kallikrein genes, we have studied the 12 functional mouse genes on the basis of expressing cell types, developmental patterns of expression and gene response to hormonal induction. We have shown expression of the renal kallikrein gene in the female anterior pituitary, the thick ascending limb of renal cortical distal tubules, nasal glands of neonatal mice and at varying levels throughout the duct tree of major salivary glands of immature and adult mice, except for intercalated ducts. This gene did not respond to hormonal induction in salivary glands. The other 11 of the 12 genes are expressed in androgen-responsive cells of granular convoluted tubules of the submandibular salivary gland from 22 days postnatal, when sexual dimorphism of expression first becomes apparent. Expression of these genes is induced prematurely in 22-day-old mice by treatment with testosterone or thyroxine. In the adult female mouse, estrogens also induce elevated levels of expression. One of the glandular kallikrein genes is expressed in Leydig cells of the testis as well as the submandibular gland. This study has extended the basis for cross-species comparison of glandular kallikrein genes.

Location of glandular kallikrein mRNAs in mouse submandibular gland at the cellular and ultrastructural level by hybridization histochemistry using 32P- and 3H-labeled oligodeoxyribonucleotide probes.

We investigated the location of expression of mouse glandular kallikrein genes in the submandibular gland of adult male mice at the ultrastructural level by hybridization histochemistry, using 32P- and 3H-labeled oligodeoxyribonucleotide probes. Vibratome slices were hybridized, flat-embedded, sectioned, and autoradiographs prepared. The 32P-labeled probe, which was specific for a region common to all mouse glandular kallikrein mRNAs, provided resolution at the cellular and subcellular level, demonstrating mRNA transcripts encoded by the majority of the 12 mouse glandular kallikrein genes in the perinuclear area of granular convoluted tubule cells (GCT) associated with rough endoplasmic reticulum (RER). The 3H-labeled probe was specific for mRNA transcripts of mGK-6, the renal kallikrein gene, which is also expressed in salivary glands. Occasional morphologically distinct granulated cells within GCTs, as well as striated duct cells, were found to express this gene. Resolution obtained with this 3H-labeled probe showed mGK-6 mRNA in striated duct cells to be located on RER and in the nucleus and perinuclear area of the cell. There was also an apparent mitochondrial association with regions of RER that labeled with this probe. The location of hybrids was confirmed by simultaneous assessment of sites of silver grains in serial sections. There are therefore at least two types of mGK-6-expressing cells in ducts of the submandibular gland, which are distinct from those expressing other kallikrein genes. In striated duct cells, there is evidence of a close mitochondrial association with RER that contains labeled mGK-6 transcripts, which is unexplained.

Human secreted carbonic anhydrase: cDNA cloning, nucleotide sequence, and hybridization histochemistry.

Complementary DNA clones coding for the human secreted carbonic anhydrase isozyme (CA VI) have been isolated and their nucleotide sequences determined. These clones identify a 1.45-kb mRNA that is present in high levels in parotid submandibular salivary glands but absent in other tissues such as the sublingual gland, kidney, liver, and prostate gland. Hybridization histochemistry of human salivary glands shows mRNA for CA VI located in the acinar cells of these glands. The cDNA clones encode a protein of 308 amino acids that includes a 17 amino acid leader sequence typical of secreted proteins. The mature protein has 291 amino acids compared to 259 or 260 for the cytoplasmic isozymes, with most of the extra amino acids present as a carboxyl terminal extension. In comparison, sheep CA VI has a 45 amino acid extension [Fernley, R. T., Wright, R. D., & Coghlan, J. P. (1988b) Biochemistry 27, 2815]. Overall the human CA VI protein has a sequence identity of 35% with human CA II, while residues involved in the active site of the enzymes have been conserved. The human sheep secreted carbonic anhydrases have a sequence identity of 72%. This includes the two cysteine residues that are known to be involved in an intramolecular disulfide bond in the sheep CA VI. The enzyme is known to be glycosylated and three potential N-glycosylation sites (Asn-X-Thr/Ser) have been identified. Two of these are known to be glycosylated in sheep CA VI. Southern analysis of human DNA indicates that there is only one gene coding for CA VI.

Differential expression of inhibin alpha and beta A subunit genes in rat and mouse ovarian follicles during pregnancy.

Relative levels of rat ovarian alpha inhibin (alpha I) and beta A inhibin (beta AI) mRNAs were measured during pregnancy by dot-blot hybridization of ovarian poly(A+) RNA. Follicular patterns of alpha I and beta AI expression in contralateral ovaries from the same rats were also studied by hybridization histochemistry. Oligodeoxynucleotide probes specific for porcine alpha I and beta AI were synthesized, 32P end-labelled and used as hybridization probes on dot-blots of ovarian RNA and frozen sections of ovarian tissue from pregnant rats. During pregnancy, levels of alpha I and beta AI mRNAs remained fairly constant from day 7 after mating until parturition and then fell within 16 h post partum. In all ovaries observed, expression of inhibin genes was located in granulosa cells of healthy antral follicles. In general, the strongest signals for alpha I and beta AI mRNAs were obtained in large follicles, with weaker signals in smaller follicles. Follicular patterns of alpha I and beta AI expression during pregnancy were often dissimilar when alpha I and beta AI were compared over a range of follicles. Considerable alpha I mRNA was detectable in some follicles in which beta AI was reduced or undetectable, despite strong signals for both alpha I and beta AI in an adjacent follicle. Essentially, alpha I mRNA levels were relatively consistent between groups of follicles, whereas beta AI levels varied considerably. beta AI mRNA was never observed in a follicle in the absence of alpha I mRNA, indicating that activin production in any follicle occurs in the presence of alpha I mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin-like growth factor II may play a local role in the regulation of ocular size.

The ultimate size and shape of the eye has a profound influence on its refraction and function. However, the role of growth factors in normal ocular development is poorly understood. Insulin-like growth factors IGF-I and -II have major effects on cell growth and differentiation in tissue culture. Recently their importance for in vivo development has been studied; IGF-II is predominant prenatally, with a probable local role in the differentiation of some mesodermally derived tissues. Ocular development and size is partially dictated by the condensation of the outer collagenous scleral coat (the 'white') of the eye from orbital mesoderm. We investigated IGF-II expression and IGF-II receptor distribution during normal ocular development in the mouse fetus using in situ hybridization and immunohistochemistry. IGF-II mRNA was expressed by the loose mesenchymal orbital tissue as it differentiated to form the sclera, but not in the compact mature sclera or cornea, or in the ectodermally derived retina or skin. IGF-II gene expression was seen in the orbit at E14, reached a peak just before parturition and then declined to background levels after birth. Similarly, type 2 IGF receptors were shown with immunohistochemistry to be present on developing scleral cells and to be modulated in parallel with IGF-II mRNA expression. We suggest the IGF-II expression by differentiating cells that compact to form the collagenous ocular coat plays a local role in determining the ultimate shape and size of the developing eye.

Immunohistochemical study of peripolar cells of the sheep.

The proposal that the cytoplasmic granules of glomerular peripolar cells (PPCs) of the adult sheep and lamb contain a kallikrein-like substance has been re-examined. After extensive characterisation and purification of anti-kallikrein antibodies, it was concluded that PPCs do not contain immunohistochemically detectable amounts of urinary kallikrein; nor do such cells appear to synthesise kallikrein as shown by hybridisation histochemistry studies. They are also not renin-immunopositive. On the other hand, a proportion of them exhibit neuron-specific enolase (NSE) immunoreactivity, unlike the apparently closely related visceral and parietal epithelial cells.

Tissue distribution and levels of gene expression of three steroid hydroxylases in ovine fetal adrenal glands.

Northern blotting and hybridization histochemistry were used to evaluate the ontogeny and cellular distribution of the mRNAs of the cytochrome P-450 enzymes: cholesterol side-chain cleavage (P-450scc), 17 alpha-hydroxylase (P-450(17 alpha] and 21-hydroxylase (P-450c21) in 40 ovine fetal adrenals from 42 days of gestation until term (151 days). The genes for P-450(17 alpha) and P-450scc were expressed strongly in tissue from young (40-60 days) and old fetuses (120 days to term), but to a very minor degree in 90-120 day fetuses. P-450c21 showed a steady increase throughout gestation. In the morphologically immature an unzoned adrenal of the 40-50 day fetus there was some differentiation in gene expression, all cells containing P-450scc and P-450c21 but a few lacking P-450(17 alpha). Once morphological zonation had occurred (80 days), P-450(17 alpha) was confined to the fasciculata. After 120 days there was a radial maturation pattern of the fasciculata cells morphologically, adult-type cells first appearing at the medullary border. However, P-450(17 alpha) and P-450scc mRNAs were equally well expressed in all sections of the fasciculata. The conclusions were: 1) the previously demonstrated triphasic cortisol biosynthetic capacity of ovine fetal adrenals was correlated with the presence, absence, and reappearance of mRNAs P-450(17 alpha) and P-450scc; 2) morphological appearance of fetal adrenocortical cells and expression of three major steroidogenic enzyme genes were not correlated.