Dennis P. Healy

Localization of immunoreactive glutamyl aminopeptidase in rat brain. II. Distribution and correlation with angiotensin II

Glutamyl aminopeptidase (EAP, EC 3.4.11.7) selectively hydrolyzes N-terminal glutamyl and aspartyl residues from oligopeptides and is present in the brain. (Asp1)Angiotensin II (Ang II) is a substrate for EAP, and increasing evidence suggests that des(Asp1)angiotensin II (Ang III) is an active angiotensin peptide in the brain. To determine whether a relationship exists between EAP and Ang II/III in rat brain, we compared their immunocytochemical distributions. EAP-like immunoreactivity was localized primarily to the adventitial surface of cerebral microvessels throughout the forebrain. Endothelial cells, neurons and glial cells were not labeled. The immunocytochemical staining of microvessel adventitium with EAP antiserum was suggestive of labeling of perivascular pericytes since intravenous horseradish peroxidase resulted in a similar adventitial pattern of staining, in addition to pericyte cell bodies. EAP immunoreactivity was highest within circumventricular organs, areas known to contain high levels of Ang II receptors. Positively stained EAP microvessels were also concentrated in areas containing Ang II/III immunoreactive neurons or nerve terminals, including the hypothalamic paraventricular nucleus and the median eminence. The immunocytochemical localization of EAP suggests that it may be involved in a wide variety of functions within the brain, including: (i) metabolism of circulating peptides in brain areas devoid of a blood-brain barrier, (ii) metabolism of circulating peptides as a component of the blood-brain barrier, (iii) metabolism of intravascularly synthesized peptides, (iv) metabolism of hypothalamic peptides released into the portal circulation, (v) metabolism/conversion of neuronally released Ang II to Ang III in the interstitial space, and (vi) metabolism of neuronally released neuropeptides with vasoactive properties.(ABSTRACT TRUNCATED AT 250 WORDS)

Glutamyl aminopeptidase (aminopeptidase A), the BP-1/6C3 antigen

Summary Glutamyl aminopeptidase (aminopeptidase A, EC 3.4.11.7) was first described in 1961 as a calcium-stimulated aminopeptidase with specificity for N-terminal acidic amino acids. The enzyme was subsequently purified and characterized as a membrane-bound ectoenzyme particularly abundant in the brush border fractions of kidney and intestine. Early interest in this enzyme was stimulated by its conversion of angiotensin II to des-aspartyl angiotensin II (angiotensin III). More recently, glutamyl aminopeptidase has been identified as a pre-B cell differentiation antigen BP-1/6C3. This review focuses on the biochemical properties of this enzyme and its role in the immune and non-immune systems.

Immunocytochemical localization of endopeptidase 24.15 in rat brain

Endopeptidase 24.15 (EC 3.4.24.15; EP 24.15), a zinc-metalloendopeptidase highly active in rat testes, brain and pituitary, converts some prodynorphin- and proenkephalin-derived oligopeptides into the corresponding enkephalins and degrades a variety of bioactive peptides including bradykinin, neurotensin, and both angiotensin I and II. The immunocytochemical localization of the enzyme was studied in rat brain using a polyclonal antibody raised in rabbits against a homogeneous preparation of the enzyme isolated from rat testes. The distribution of EP 24.15 immunoreactivity in the brain was widespread, being present in both neurons and glial cells. Surprisingly, however, staining was predominantly nuclear, and not cytoplasmic as expected based on the biochemical demonstration that EP 24.15 activity is predominantly associated with the soluble protein fraction of brain homogenates. Cytoplasmic staining was detected in large neurons but was less intense than the nuclear staining. The highest density of EP 24.15-staining was detected in nuclei of cerebellar Purkinje cells and in hippocampal dentate gyrus cells. High levels of immunoreactivity were also noted in brain areas which contain peptides known to be substrates of the enzyme in vitro. This localization supports a role for EP 24.15 in neuropeptide metabolism, but also suggests an as yet undefined role in nuclear function.

Kidney Aminopeptidase A and Hypertension, Part II Effects of Angiotensin II

Aminopeptidase A (APA) is the principal enzyme that metabolizes angiotensin II (Ang II) to angiotensin III. Previously, we showed that kidney APA was elevated in spontaneously hypertensive rats and was reduced after angiotensin-converting enzyme inhibition. In the present study, we sought to determine whether kidney APA expression was altered after chronically elevated Ang II, either exogenously delivered via osmotic minipumps or endogenously produced in two-kidney, one clip (2K1C) hypertensive rats. Ang II (200 ng. kg-1. min-1) was infused subcutaneously for 1 or 2 weeks by osmotic minipumps, and 2K1C rats were tested 4 weeks after unilateral renal artery clipping. Blood pressure was not significantly elevated in the Ang II-infused animals but was significantly increased at 3 and 4 weeks in the 2K1C animals. APA was significantly elevated approximately 2-fold in kidney cortical membranes from Ang II-infused animals but was decreased 45% in the clipped kidney and 18% in the nonclipped kidneys from 2K1C animals. Isolated glomeruli from Ang II-infused animals and the nonclipped kidneys from 2K1C animals had markedly higher APA activity and immunoreactivity. Likewise, histochemical and immunohistochemical studies indicated that APA levels were increased in glomeruli from angiotensin-infused animals and in both nonclipped and clipped kidneys from 2K1C animals. In contrast, tubular APA was decreased in tubular elements from 2K1C animals, most markedly in the clipped kidneys. Thus, despite the increase in glomerular APA expression in kidneys from 2K1C animals, the decrease in tubular APA expression is more extensive and accounts for the measured reduction in total APA in cortical homogenates. Because clipped kidneys are not exposed to high blood pressure, these results suggest that glomerular APA expression is positively regulated and tubular APA negatively regulated by Ang II. These results further suggest that changes in kidney APA expression could influence the progression of angiotensin-dependent hypertension.

Aminopeptidase A antiserum inhibits intracerebroventricular angiotensin II-induced dipsogenic and pressor responses.

Angiotensin II increases drinking and blood pressure when administered intracerebroventricularly. Intracerebroventricular injections of antiserum with anticatalytic activity against aminopeptidase A, the principal enzyme that metabolizes angiotensin II to angiotensin III, reduced the drinking and blood pressure responses to 10 pmol angiotensin II by 73% and 59%, respectively. APA antiserum had no effect on responses to angiotensin III administered intracerebroventricularly. A Glu-thiol inhibitor of aminopeptidase A also reduced angiotensin II-induced drinking. These results suggest that metabolism of angiotensin II to angiotensin III is an obligatory activation step for the brain angiotensin system.

Kidney Aminopeptidase A and Hypertension, Part I: Spontaneously Hypertensive Rats

Tissue and plasma levels of aminopeptidase A (APA), the principal enzyme that hydrolyzes angiotensin II (Ang II) to angiotensin III, were measured in spontaneously hypertensive rats (SHR) and their normotensive control strain at 3 different ages corresponding to prehypertensive (4 weeks), developing (8 weeks), and established (16 weeks) phases of hypertension. Plasma APA activity was significantly but modestly elevated in SHR at all 3 ages compared with normotensive Wistar-Kyoto rats. Likewise, levels of APA in brain, heart, and adrenal gland were generally, but again only moderately, elevated in SHR at all ages. However, a large increase in APA activity was seen within the kidney in which APA levels were elevated 41%, 51%, and 68% in SHR at 4, 8, and 16 weeks of age, respectively. Kidney APA levels were also significantly increased in immunoblots from 8- and 16-week-old SHR. Glomeruli isolated from 16-week-old SHR had 57% higher APA activity and increased immunoreactivity compared with Wistar-Kyoto rats. To determine whether the increase in kidney APA activity in SHR was related to Ang II levels, SHR were treated for 2 weeks with the angiotensin-converting enzyme inhibitor captopril. Captopril treatment reduced blood pressure to normotensive values and resulted in a 25% reduction in kidney APA activity. These results suggest that APA expression in the kidney may be regulated by activity of the renin-angiotensin system. If so, this would further suggest that upregulation of APA during conditions in which Ang II levels were elevated would have a protective effect against Ang II-mediated cardiovascular diseases, whereas a decrease in APA expression or a failure to upregulate would exacerbate such conditions.

Localization of immunoreactive glutamyl aminopeptidase in rat brain. I: Association with cerebral microvessels

Glutamyl aminopeptidase (aminopeptidase-A, EC 3.4.11.7) is an ectoenzyme that selectively hydrolyses N-terminal glutamyl and aspartyl residues from oligopeptides, including (Asp1) angiotensin II. Here we sought to determine the distribution of glutamyl aminopeptidase (EAP) in rat brain. EAP was purified to homogeneity from rat kidney and polyclonal antiserum raised in rabbits. Immune serum inhibited EAP enzyme activity in kidney homogenates and labeled two major protein bands of M(r) = 136,000 and M(r) = 101,000 in immunoblots of kidney protein. EAP-like immunoreactivity was concentrated on kidney proximal tubule brush borders. Immunocytochemical staining of rat brain indicated that EAP-like immunoreactivity was primarily associated with cerebral microvessels. Positive staining was detected in microvessels ranging in size from capillaries up to vessels approximately 50 microns in diameter. Isolated cerebral microvessels had a 23-fold enrichment in EAP enzyme activity (193.1 +/- 40.4 nmol/mg protein/h) compared to brain homogenates. Finally, immunoblots of isolated cerebral microvessels resulted in a pattern of labeling similar to that seen with kidney homogenates. These results indicate that EAP activity in brain is primarily associated with cerebral microvessels, and suggest that EAP may be involved in the metabolism of circulating or locally formed peptides.

Expression of Aminopeptidase A, an Angiotensinase, in Glomerular Mesangial Cells

Glomerular mesangial cells are known to express angiotensin II type 1 receptors and contract in response to circulating and/or locally produced angiotensin II. In addition, stimulation of mesangial cell matrix protein synthesis by elevated levels of angiotensin II is known to contribute to the development of glomerulosclerosis. Previously, we reported that mesangial cells were positively immunostained with antiserum directed against aminopeptidase A, the principal angiotensinase in the metabolism of angiotensin II. Here we demonstrate directly that aminopeptidase A is expressed in mesangial cells cultured from rat kidney. First, cultured mesangial cells had measurable aminopeptidase A enzymatic activity. Second, immunoblots for aminopeptidase A were positive for isolated glomeruli and mesangial cells, although two bands were seen for mesangial cells (approximately 138 and 144 kD), and only the larger band was seen for isolated glomeruli and kidney. Third, Northern blot hybridizations of total RNA from mesangial cells or kidney were positive and labeled similarly sized bands. Fourth, reverse transcription-polymerase chain reaction amplification of mesangial cell total RNA yielded a partial cDNA of the expected size that was confirmed by sequencing to be identical to rat kidney aminopeptidase A. These results indicate that aminopeptidase A is expressed within mesangial cells. These results further suggest that metabolism of angiotensin II by aminopeptidase A could play a protective role in minimizing the adverse effects of angiotensin II stimulation of mesangial cells.

A68930 is a potent, full agonist at dopamine1 (D1) receptors in renal epithelial LLC-PK1 cells

The selective dopamine1 (D1) receptor agonists SK&F 82526 (fenoldopam) and A68930 and the mixed D1/D2 agonist SK&F 85174 were tested for their ability to stimulate adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) accumulation in the porcine renal epithelial cell line, LLC‐PK1. SK&F 82526 and SK&F 85174 were potent stimulators of cyclic AMP accumulation (EC50s 21.4 and 14.5 nm, respectively), but only partial agonists (intrinsic activities 31% and 46% of dopamine respectively). In contrast, A68930 was a potent, full agonist (EC50 12.7 nm, intrinsic activity 102% of dopamine). The stimulatory effects of A68930 and dopamine on cyclic AMP accumulation were not additive, and the stimulation of cyclic AMP accumulation by A68930 was blocked by the D1‐selective antagonist, SCH 23390. These properties of A68930 suggest that it may be a useful D1‐selective agonist to study renal D1 receptor mechanisms in vitro and in vivo.

Effects of intracerebroventricular captopril on angiotensin-converting enzyme and angiotensinogen mRNA levels in rat brain.

The effects of intracerebroventricular (i.c.v.) infusion of the angiotensin-converting enzyme (ACE) inhibitor captopril on angiotensin-induced drinking, brain ACE activity, and ACE and angiotensinogen (A-ogen) mRNA levels were examined. I.c.v. infusion of captopril at a rate of 1 microgram/microliter per h for 7 days resulted in a 60% reduction in brain ACE activity and an 80% reduction in the drinking response to i.c.v. angiotensin I. Quantitative solution hybridization experiments indicated that brain ACE mRNA levels were decreased by 40%, whereas brain A-ogen mRNA levels were unchanged. These results suggest that ACE and A-ogen mRNA levels are regulated differently in the brain than in the peripheral renin-angiotensin system.