Jennifer D. Penschow

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.

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.

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.

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.

[14] – Location of Gene Expression in Tissue Sections by Hybridization Histochemistry Using Oligodeoxyribonucleotide Probes

Publisher Summary This chapter explores synthesis, labeling, and use of oligodeoxynucleotides in the technique of hybridization histochemistry. The technique of hybridization histochemistry is simple and convenient and has been used for several years for a variety of research topics and myriads of specimens with consistent success. Oligodeoxyribonucleotides are routinely synthesized by the solid-phase method on an Applied Biosystems Inc. 380A DNA synthesizer, using phosphoramidite chemistry. The most convenient method for the routine purification of large numbers of oligonucleotides is polyacrylamide gel electrophoresis (PAGE). The choice of radioisotope used for incorporation into the oligonucleotide depends on the experimental requirements. The temperature for hybridization of sections with oligodeoxynucleotides is related to the probe length, specificity of the probe for the target mRNA, and the formamide and salt concentration of the hybridization buffer. The technique of hybridization histochemistry may be used in conjunction with immunohistochemistry for the investigation of transcriptional regulation of neuroendocrine peptides and identification of the peptide product.

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.

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.

USE OF SYNTHETIC OLIGONUCLEOTIDE AND RECOMBINANT DNA PROBES TO STUDY RENIN GENE EXPRESSION

1. Using hybridization histochemistry renin gene expression has been localized in the juxtaglomerular apparatus (JGA) of the renal cortex in both mouse and sheep kidney.