Oestrogenic regulation of brain angiotensinogen.

Oestrogens are now recognized as playing a regulatory role on components of the systemic renin-angiotensin system, such as its precursor, angiotensinogen (AGT). In the brain, this role is poorly understood. The aim of this study was to investigate the influence of oestrogens on brain AGT of female rats at different stages of the oestrous cycle, in pregnancy and following ovariectomy with and without hormone replacement. AGT content of different brain regions was also studied in male rats treated with oestrogens. The brain was divided into five regions: cortex, cerebellum, brainstem, midbrain and thalamus/hypothalamus, and AGT was measured by direct radioimmunoassay using a highly specific AGT antibody. Cyclical fluctuations in AGT content were observed in all regions except the cerebellum over the course of the 4-day oestrous cycle, with peak concentrations at estrus and lowest concentrations at metestrus. Following ovariectomy, brain AGT was significantly decreased in the thalamic/hypothalamic region, an effect that was reversed by oestrogen-replacement. In pregnant rats, AGT contents were elevated in the brainstem region. Oestrogen treatment of male rats induced significant increases in AGT concentrations in all areas except the cortex. In summary, these results show that oestradiol has actions on brain AGT that are region-specific and dependent on the particular physiological and reproductive context. Moreover, the changes in AGT concentrations in the oestrous cycle suggest the involvement of other factors besides oestrogen. Finally, this study supports the view that the brain renin-angiotensin system has a broad role in oestrogen-modulated brain functions beyond those specific to the hypothalamic-pituitary-ovarian axis.

Angiotensinogen localization and secretion in the rat pancreas.

Renin and angiotensinogen have been previously found in the rat pancreas, and angiotensin receptors have been located in the apical domain of duct cells. To evaluate the possibility that angiotensin II could be generated within the duct system, we decided to determine whether angiotensinogen is present in rat pancreatic juice and the angiotensinogen-immunoreactive pancreatic cell types that could be responsible for its production. Angiotensinogen was detected in significant amounts by Western blotting in pancreatic juice collected from several individual rats. Different isoforms between plasma and pancreatic juice angiotensinogens were demonstrated by isoelectric focusing. Immunocytochemical experiments revealed angiotensinogen-immunoreactive cells at the periphery of the islets of Langerhans, and confocal microscopy demonstrated that most angiotensinogen-immunoreactive cells were glucagon-secreting cells. Secretion of angiotensinogen did not follow the regulated secretory pathway since it was absent from the glucagon-containing granules. This was confirmed by electron microscopy immunocytochemistry. Duct and acinar cells did not express angiotensinogen at an immunocytochemical detectable level. The present findings indicated an exocrine secretion of angiotensinogen by glucagon-secreting cells and suggest that one of the final targets of the local pancreatic renin-angiotensin system may be the duct epithelium.

The renin-angiotensin system and male reproduction: new functions for old hormones.

The blood-borne renin-angiotensin system (RAS) is known best for its role in the maintenance of blood pressure and electrolyte and fluid homeostasis. However, numerous tIssues show intrinsic angiotensin-generating systems that cater for specific local needs through actions that add to, or differ from, the circulating RAS. The male reproductive system has several sites of intrinsic RAS activity. Recent focus on the epididymis, by our laboratories and by others, has contributed important details about the local RAS in this tIssue. The RAS components have been localized morphologically and topographically; they have been shown to be responsive to androgens and to hypoxia; and angiotensin has been shown to influence tubular, and consequently, fluid secretion. Components of the RAS have also been found in the testis, vas deferens, prostate and semen. Angiotensin II receptors, type 1 and, to a lesser extent, type 2 are widespread, and angiotensin IV receptors have been localized in the prostate. The roles of the RAS in local processes at these sites are still uncertain and have yet to be fully elucidated, although there is evidence for involvement in tubular contractility, spermatogenesis, sperm maturation, capacitation, acrosomal exocytosis and fertilization. Notwithstanding this evidence for the involvement of the RAS in various important aspects of male reproduction, there has so far been a lack of clinical evidence, demonstrable by changes in fertility, for a crucial role of the RAS in male reproduction. However, it is clear that there are several potential targets for manipulating the activity of the male reproductive system by interfering with the locally generated angiotensin systems.

Role of PGF2alpha and oxytocin in parturition in the brushtail possum (Trichosurus vulpecula).

Maturation of the fetal pituitary and adrenal glands allows the secretion of cortisol, which in turn leads to an increase in prostaglandin and mesotocin production. The production of prostaglandin and mesotocin results in an increase in uterine contractions and initiates birth in marsupials. The major metabolite of PGF(2alpha), 13,14-dihydro-15-keto-prostaglandin F(2alpha) (PGFM), has been found in the plasma of the possum at the time of birth and administration of PGF(2alpha) to female possums induced the adoption of the birth position. Evidence that mesotocin is an integral hormone of birth in the tammar wallaby indicates that both PGF(2alpha) and mesotocin or oxytocin are required for marsupial birth. The presence of PGF(2alpha) receptors in the uterus and corpus luteum of the possum, and the in vitro uterine responsiveness to PGF(2alpha) or oxytocin, were examined. PGF(2alpha) receptors were not observed in possum uteri and the inability of PGF(2alpha) to cause contractions indicates that PGF(2alpha) is not involved directly in contraction of the uterus at parturition. The presence of oxytocin and mesotocin receptors in the uterus of possoms and the ability of oxytocin to induce uterine contraction in vitro supports the view that mesotocin is required for expulsion of the young from the uterus. Low numbers of PGF(2alpha) receptors were found in the possum corpus luteum at birth, indicating an involvement of PGF(2alpha) in regression of the corpus luteum.

Genetic variation in the recognition of Porphyromonas gingivalis antigens in mice.

T-cell cytokine profiles, anti-Porphyromonas gingivalis antibodies and Western blot analysis of antibody responses were examined in BALB/c, CBA/CaH, C57BL6 and DBA/2J mice immunized intraperitoneally with different doses of P. gingivalis outer membrane antigens. Splenic CD4 and CD8 cells were examined for intracytoplasmic interleukin (IL)-4, interferon (IFN)-gamma and IL-10 by FACS analysis and levels of anti-P. gingivalis antibodies in the serum samples determined by enzyme-linked immunosorbent assay. Western blot analysis was performed on the sera from mice immunized with 100 microg of P. gingivalis antigens. The four strains of mice demonstrated varying degrees of T-cell immunity, although the T-cell cytokine profiles exhibited by each strain were not affected by different immunizing doses. While BALB/c and DBA/2J mice exhibited responses that peaked at immunizing doses of 100-200 microg of P. gingivalis antigens, CBA/CaH and C57BL6 demonstrated weak T-cell responsiveness compared with control mice. Like the T-cell responses, serum antibody levels were not dose dependent. DBA/2J exhibited the lowest levels of anti-P. gingivalis antibodies followed by BALB/c with CBA/CaH and C57BL6 mice demonstrating the highest levels. Western blot analysis showed that there were differences in reactivity between the strains to a group of 13 antigens ranging in molecular weight from 15 to 43 kDa. Antibody responses to a number of these bands in BALB/c mice were of low density, whereas CBA/CaH and C57BL6 mice demonstrated high-density bands and DBA/2J mice showed medium to high responses. In conclusion, different immunizing doses of P. gingivalis outer membrane antigens had little effect on the T-cell cytokine responses and serum anti-P. gingivalis antibody levels. Western blot analysis, however, indicated that the four strains of mice exhibited different reactivity to some lower-molecular-weight antigens. Future studies are required to determine the significance of these differences, which may affect the outcome of P. gingivalis infection.

A critical appraisal of the intrinsic pancreatic angiotensin-generating system.

The pancreas is a relative newcomer to the stable of tissues with an intrinsic angiotensin-generating system. The involvement of this system in pancreatic activity will be dependent on the angiotensin-generating paths present in the pancreas and their precise cellular location. Thus far, renin, angiotensin-converting enzyme (ACE), angiotensin II and AT1 and AT2 receptors have been found. These are components of the "classical" renin-angiotensin system. But there is uncertainty as to their location and site of action. Furthermore, it is not known which, if any, alternative enzymes to renin and ACE are present, which angiotensins in addition to angiotensin II are generated and whether or not there are receptors to angiotensin IV and angiotensin-(1-7). Future research should focus on these aspects in order to provide a mechanistic basis to pancreatic physiological functions and to pathological conditions of clinical relevance.

Chronic hypoxia induced down-regulation of angiotensinogen expression in rat epididymis.

The presence of an intrinsic renin-angiotensin system (RAS) in the rat epididymis has been previously established by showing the expression of several key RAS components, and in particular angiotensinogen, the indispensable element for the intracellular generation of angiotensin II. In this study, the possible involvement of this local epididymal RAS in the testicular effects of chronic hypoxia was investigated. Semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR), Western blotting and by in situ hybridization histochemistry of the rat epididymis were used to show changes in localization and expression of angiotensinogen. Results from RT-PCR analysis demonstrated that chronic hypoxia caused a marked decrease (60%) in the expression of angiotensinogen mRNA, when compared with that in the normoxic epididymis. Western blot analysis demonstrated a less decrease (35%) in the expression of angiotensinogen protein. In situ hybridization histochemistry showed that the reduced angiotensinogen mRNA in chronic hypoxia was specifically localized to the epididymal epithelium from the cauda, corpus and caput regions of the epididymis; a distribution similar to that of normoxic rats. It was concluded that chronic hypoxia decreases the transcriptional and translational expression of angiotensinogen, and thus local formation of angiotensin II, in the rat epididymis.

Cardiac and vascular responses in deoxycorticosterone acetate-salt hypertensive rats.

1. Hypertension leads to ventricular hypertrophy and, eventually, to heart failure. The present study has investigated the functional consequences of deoxycorticosterone acetate (DOCA)-salt hypertension in rats by defining the inotropic, chronotropic and vascular responses to noradrenaline (NA; beta1-adrenoceptor agonist), forskolin (adenylate cyclase activator) and theophylline (phosphodiesterase inhibitor). 2. Administration of DOCA (25 mg, s.c., every 4th day) and excess salt (1% NaCl in drinking water) to uninephrectomized rats increased left ventricular wet weight by 35 and 71% after 4 and 8 weeks, respectively. Addition of KCl (0.4%) or CaCl2 (1%) in the drinking water for 4 weeks attenuated blood pressure increases, but not ventricular weight increases (46 and 28%, respectively). 3. Positive inotropic responses in papillary muscles from uninephrectomized rats to NA (-log EC50 6.73+/-0.38; n = 7), forskolin (-log EC50 6.15+/-0.31; n = 7) and CaCl2 (-log EC50 2.40+/-0.02; n = 14) were unchanged in hypertrophied left ventricles of DOCA and DOCA-CaCl2 rats, although maximal responses to NA were decreased in DOCA-KCI rats (1.2+/-0.6 mN, n = 8; DOCA-salt 2.9+/-0.5 mN, n = 6); theophylline was less potent in DOCA-salt rats. Positive chronotropic responses to NA, forskolin and theophylline in right atria and negative inotropic responses to carbachol in papillary muscles were unchanged. 4. Maximal vasoconstrictor responses to NA in thoracic aortic rings were reduced in DOCA-KCI rats to 2.4+/-0.9 mN (n = 5), but were increased in DOCA-CaCl2 rats to 26.6+/-2.2 mN (n = 7; DOCA-salt 7.8+/-2.2 mN, n = 9). Vasorelaxant responses to forskolin and theophylline were unchanged. 5. These results show that cardiac responses are only minimally affected during the development of DOCA-salt hypertension-induced hypertrophy, despite the reported decreases in adenylate cyclase activity, in these rats. This is in contrast with the decreased responses reported in other rat models of cardiac hypertrophy and in the failing human heart. Thus, hypertrophy in hearts of DOCA-salt hypertensive rats does not produce similar changes to the failing human heart.

Angiotensinogen expression by rat epididymis: evidence for an intrinsic, angiotensin-generating system.

Previous studies have suggested the presence of an intrinsic renin-angiotensin system (RAS) in the rat epididymis with functions in epididymal activity and sperm maturation. In the present study, the localization and expression of angiotensinogen, the component of the RAS which is indispensable for intracellular angiotensin generation, were investigated by immunochemistry, hybridization histochemistry and by reverse-transcriptase polymerase chain reaction (RT-PCR). Western blot analysis of protein from the epididymis confirmed the presence of angiotensinogen with the expected molecular mass of about 60 kDa, in agreement with results from other tissues. Immunocytochemistry showed the regional localization of immunoreactivity for angiotensinogen in the rat epididymis. In situ hybridization histochemistry further demonstrated the expression of angiotensinogen mRNA by the epididymal epithelium in a region-specific manner along the length of the rat epididymis. RT-PCR confirmed that the rat epididymis expresses angiotensinogen mRNA. On the other hand, mRNA of renin was not detected in the rat epididymis using Northern blot and RT-PCR analyses. The present study strongly supports the existence of an intrinsic, angiotensin-generating system based on locally formed angiotensinogen as a precursor for angiotensin production. This epididymal RAS may have paracrine or autocrine roles in mediating the epididymal and sperm functions.

Reversal of cardiac fibrosis in deoxycorticosterone acetate-salt hypertensive rats by inhibition of the renin-angiotensin system.

Fibrosis impairs cardiac function. This project has determined the expression and deposition of collagens and fibronectin and cardiac function in the deoxycorticosterone acetate (DOCA)-salt hypertensive rat after inhibition of the renin-angiotensin system. DOCA-salt hypertension was induced in 8-wk-old male Wistar rats by uninephrectomy and administration of DOCA (25 mg every fourth day, subcutaneously) and 1% NaCl in the drinking water for 4 wk. Starting 2 wk after surgery, rats were given either oral captopril (100 mg/kg), oral candesartan cilexetil (2 mg/kg), or subcutaneous spironolactone (50 mg/kg) daily for 2 wk (reversal protocol). DOCA-salt rats failed to gain weight with markedly increased water intake and decreased food intake; drug treatment did not alter these parameters. Systolic BP increased from 116+/-5 mmHg in uninephrectomized rats to 179+/-7 mmHg in DOCA-salt rats and was not decreased by treatment (captopril 172+/-1 mmHg; candesartan 187+/-2 mmHg; spironolactone 178+/-3 mmHg). Captopril, candesartan, and spironolactone reversed the increased collagen I mRNA in DOCA-salt rats; only candesartan reversed the increased collagen III mRNA. Collagen IV mRNA was unchanged in DOCA-salt rats and following treatment. Total fibronectin mRNA increased without changing the proportion of fibronectin mRNA as the fetal isoforms EIIIA and EIIIB. Captopril, candesartan, and spironolactone reversed the increased deposition of perivascular and interstitial collagen in DOCA-salt rats; the increased cardiac fibronectin deposition was reversed by candesartan and spironolactone. Captopril, candesartan, and spironolactone also attenuated or reversed the increased diastolic stiffness and the increased dP/dt but not the increased rate-pressure products in DOCA-salt rat hearts. Thus, inhibition of the renin-angiotensin system reverses cardiac fibrosis in DOCA-salt rats and returns some indices of myocardial function to normal.

Expression and localization of the renin-angiotensin system in the rat pancreas.

The possibility of an intrinsic renin-angiotensin system (RAS) in the pancreas has been raised by previous studies in which immunohistochemical examination showed the presence of angiotensin II and its receptor subtypes, type 1 (AT1) and type 2 (AT2). In the present study, gene expression of several key RAS components was investigated by reverse-transcription PCR. mRNA expression for angiotensinogen, renin and angiotensin II receptor subtypes, AT1a, AT1b and AT2 was shown. The presence of angiotensinogen protein, the mandatory component for an intrinsic RAS, was demonstrated by Western blotting and localized by immunohistochemistry to the epithelia and endothelia of pancreatic ducts and blood vessels respectively. Immunoblot analysis detected a predominant protein band of about 60 kDa in the pancreas. This was consistent with the predicted value for angiotensinogen as reported in other tissues. Together with previous findings, the present study shows that the rat pancreas expresses the major RAS component genes, notably angiotensinogen and renin, required for intracellular formation of angiotensin II. The data support the notion of an intrinsic RAS in the rat pancreas which may play a role in the regulation of pancreatic functions.

Cardiac and vascular responses after monocrotaline-induced hypertrophy in rats.

In rats, monocrotaline causes pulmonary vascular damage leading to pulmonary hypertension, right ventricular hypertrophy, and eventually heart failure. This study determined the inotropic and chronotropic responses in isolated cardiac tissues from pulmonary hypertensive rats (single treatment with monocrotaline, 105 mg/kg) to noradrenaline, forskolin, EMD 57033 (calcium sensitizer), and calcium chloride. Further, vasoconstrictor responses to noradrenaline, 5-hydroxytryptamine (5-HT), and KCl were measured in isolated pulmonary artery and thoracic aortic rings. Marked right ventricular hypertrophy was evident 4 weeks after treatment; at 6 weeks, treated rats additionally showed symptoms of severe heart failure. Pulmonary hypertension led to marked increases in pulmonary artery responses to 5-HT and to decreases in positive inotropic responses in right ventricular papillary muscles to all compounds except calcium chloride. The development of heart failure maintained or increased these changes. Positive chronotropic responses were unchanged. In the right ventricle, beta1-adrenoceptor density decreased only in heart failure; beta2-adrenoceptor density was unchanged. The densities of both beta-adrenoceptor subtypes were decreased in the lungs but increased in the liver of pulmonary hypertensive rats. The functional changes in the failing human heart are similar to those in rats with monocrotaline-induced right ventricular hypertrophy. This may be a useful model to define adequate therapy in human right ventricular failure.

Growth hormone regulates AT-1a angiotensin receptors in astrocytes.

The hypothesis, based on previous in vivo data, that angiotensin AT1 receptors are regulated by GH or insulin-like growth factor I (IGF-I) has been investigated in this study using primary cultures of rat astrocytes as a model of AT1 receptor expression. At a dose of 1 ng/ml GH, there was an increase in AT1 density within 4 h and a maximum increase of 361 +/- 57% of the control value at 12 h. At 24 h, receptor density was still 176 +/- 23% that in the control. Astrocytes incubated with 1 ng/ml rat IGF-I for 24 h showed no change in AT1 receptor density. Reverse transcriptase-PCR was used to show that astrocytes express both the AT1a receptor subtype and, to a much lesser extent, the AT1b subtype. Treatment with 1 ng/ml recombinant bovine GH for 12 h increased the messenger RNA of the AT1a receptor by 170%, without affecting the AT1b receptor. Inhibition of protein synthesis by cycloheximide and of transcription by the adenosine analog dichlororibofuranosylbenzimidazole both prevented the increase in AT1 receptor density following GH treatment, indicating that the action of GH is transcriptional. In summary, we have shown that GH up-regulates, directly and not via IGF-I, angiotensin receptors of the AT1a subtype in astrocytes by a transcriptional mechanism. The long latency of the response and the dependency on transcription relegate the AT1a gene to the class of GH-regulated genes identified as delayed stable genes. This mechanism of AT1 activation may be one way in which GH activates the renin-angiotensin system and initiates consequential cardiovascular and angiogenic effects.

Tissue-specific changes in angiotensin II receptors in streptozotocin-diabetic rats.

While there have been reports on changes in the renin-angiotensin system and angiotensin II (AT) receptors in diabetes, there is no agreement on the nature of these changes. This study has characterised specific AT receptors in the heart, kidney, liver and adrenal glands of the streptozotocin (STZ)-diabetic rat using radioligand binding studies with the radioligand 125I-[Sar1, Ile8]-angiotensin II. Left ventricular AT receptor density increased by 135% 4 weeks after treatment and by 206% 12 weeks after treatment; in the liver, AT receptor density increased by 476% (4 weeks) and 263% (12 weeks) and in the adrenal gland by 236% (4 weeks) and 109% (12 weeks). In contrast, renal AT receptor density decreased by 49% (4 weeks) and 36% (12 weeks). Competition-displacement assays with losartan, an AT1-selective ligand, showed that the proportion of AT receptor subtypes remained unchanged. STZ treatment decreased plasma angiotensinogen by 72% (4 weeks) and 67% (12 weeks) and increased plasma renin concentration after 12 weeks; plasma renin activity and aldosterone concentrations remained unchanged. Treatment with human insulin (5 U/day) attenuated changes in plasma angiotensinogen and AT receptor density except in the left ventricle. We conclude that there are major changes in AT receptors in the STZ-diabetic rat that are tissue-specific and time-dependent. Plasma angiotensinogen and renin secretion change in directions that result in the maintenance of plasma renin activity and aldosterone concentration.

Novel perspectives on pituitary and brain angiotensinogen.

All the angiotensin peptides originate from angiotensinogen, a glycoprotein synthesized by several tissues, including the brain and the anterior pituitary. In the rat, immunohistochemistry has been used to localize angiotensinogen in gonadotropes and in uncharacterized cells surrounding sinusoids. Both cell types are capable of secreting angiotensinogen in cell culture; only the gonadotropes contain angiotensin II (AngII) and are capable of secreting it in culture. It has been asserted that the perisinusoidal cells are the only source of angiotensinogen for the generation of AngII by gonadotropes. Our current data favor the existence of a complete intracellular renin-angiotensin system (RAS) in gonadotropes and a separate extracellular system which utilizes the high concentration of angiotensinogen from perisinusoidal cells. Furthermore, we postulate that gonadotrope AngII serves mainly reproductive functions, while the proximity of angiotensinogen-secreting cells to folliculostellate cells, and their access to the intercellular sinusoidal and follicular spaces, places the extracellular RAS in a strategic position to affect pituitary growth and the mediation of acute-phase immune responses. In the rat brain, angiotensinogen is expressed by the 16-18th day of fetal life and by areas generally concerned with vasopressor, electrolyte, and fluid homeostasis. Antisense deoxyoligonucleotides to angiotensinogen mRNA lower blood pressure in hypertensive rats and inhibit in vitro growth of neuroblastoma cells, indicating a significant role for angiotensinogen in mitogenic and homeostatic functions. It is commonly agreed that astrocytes express angiotensinogen. Neuronal angiotensinogen has also been demonstrated by immunohistochemistry, as a secretion from neuronal cell cultures, and by reverse-transcriptase polymerase chain reaction. The fate of secreted astrocytic and neuronal angiotensinogen remains obscure. Angiotensinogen is regulated in a tissue-specific manner with smaller or absent responses observed for brain tissue. By using astrocyte and neuronal cultures the actions on angiotensinogen production of growth hormone, IGF-1, inflammatory lipopolysaccharide, and phorbol ester have been examined. Recent observations show that angiotensinogen is regulated positively or negatively by glucocorticoids and that a positive synergism between cAMP and glucocorticoids exists. On the basis of analogous systems for other proteins, a scheme involving glucocorticoid receptors, CREB, and AP-1 transcription factors is formulated to explain glucocorticoid-cAMP interactions. These transcriptional interactions may form a significant functional link between the RAS and adrenergic mechanisms.

Antisense inhibition of angiotensinogen in hepatoma cell culture is enhanced by cationic liposome delivery.

Abnormalities in expression of renin angiotensin system components, including angiotensinogen, have been implicated in the development and maintenance of hypertension in the spontaneously hypertensive rat model of hypertension. Antisense compounds are being used as physiological tools to provide information on cardiovascular function and hypertension and also show great potential for development as therapeutic agents. We have previously shown that peripheral administration of antisense oligonucleotides to angiotensinogen in vivo decreases hypertensive blood pressures with concomitant changes in angiotensinogen protein and angiotensin II. However, studies using naked phosphorothioated oligonucleotide targeted to the same region did not produce changes in angiotensinogen mRNA in vivo or in cell culture. We now provide data which show that enhanced oligonucleotide delivery utilizing cationic liposomes significantly increases the attenuation of angiotensinogen protein and decreases mRNA in a dose dependent manner. These data provide an understanding of the mechanism of action of the antisense oligonucleotide and also establish optimal conditions and doses for further studies.

Ontogeny of thyroid hormone receptors in the brushtail possum (Trichosurus vulpecula).

Newborn marsupials do not have a thyroid gland at birth. The gland develops while the young marsupial is in the mother's pouch. The young brushtail possum initiates secretion of thyroid hormones from its own thyroid at about Day 65 post partum. However, during the first three weeks of pouch life thyroxine is passed from the mother to the young via the milk. To determine if this maternal thyroxine can effect organ development in the young possum before it initiates secretion of thyroxine from its own thyroid, the ontogeny of thyroid hormone receptors was determined in nuclear extracts of lung, liver and kidney by radioreceptor assay, using (125)I-labelled tri-iodothyronine as ligand. Receptor density was calculated for tissues removed from young possums at Days 25 (n = 5), 50 (n = 4), 100 (n = 3) and 150 (n = 4) and from adults (n = 5). Receptors were found in possums of all age groups, including the small 25-day pouch young. Significant differences were not found in the receptor density between different tissues or at various ages. The association constant Ka (4.0+/-2.6 L nmol[-1] for lung) was similar in different tissues and at the various ages examined. The passage of thyroid hormones from the mother to the developing marsupial via the milk may have a role in the slow development of organ systems early in pouch life by acting on thyroid receptors in the pouch young. However, the functional maturation of the thyroid gland of the young possum, not an increase in receptors, appears to coincide with the rapid increase in the rate of growth and development which occurs in later pouch life.

Angiotensin receptors in cardiac and renal hypertrophy in rats.

Angiotensin II mediates its effects through activation of specific angiotensin (AT) receptors which can be regulated during cardiovascular disease. This study has investigated whether an increased cardiac and renal AT receptor density is important in the development of left ventricular and renal hypertrophy in three rat models of hypertension [spontaneous hypertensive (SHR), deoxycorticosterone acetate (DOCA)-salt and 2K1C renal hypertensive rats]. Although all hypertensive rats developed left ventricular and renal hypertrophy, AT receptor density increased only in the left ventricle and kidney of SHR during the development of hypertension. Thus, cardiac and renal hypertrophy per se do not increase AT receptor density. AT receptors were increased in the liver of DOCA-salt rats, 2K1C rats and 52-week-old SHR and in adrenal glands of DOCA-salt rats and SHR. A plausible explanation for tissue-dependent AT receptor regulation involves tissue-selective control of local renin-angiotensin systems independent of circulating hormone levels, combined with disease-induced cell damage.

Identification and characterization of angiotensinIV binding sites in rat neurone and astrocyte cell cultures.

This study demonstrates the existence of the putative receptor for the hexapeptide (3-8) fragment of angiotensin II (AngIV) on rat astrocytes and neurons grown in cell culture. Binding of 125I-AngIV was saturable and distinct from that of the AngII receptor subtypes. Equilibrium binding was attained in 15 min in astrocytes and 75 min in neurons at 22 degrees C. The bound peptide was confirmed by HPLC to be intact AngIV while the bound peptide was substantially degraded, even in the presence of peptidase inhibitors. Scatchard analysis of equilibrium binding was consistent with a two binding site model, revealing a high affinity and a low affinity binding site in both cell types. In neurons, the respective association constants (Ka) were 2.72 +/- 0.23 nM-1 and 727 +/- 354 nM-1, with associated receptor densities of 109.30 +/- 58.87 and 1723 +/- 1167 fmol/mg protein. Similar analyses in astrocytes gave Kas of 5.71 +/- 2.85 nM-1 and 277 +/- 205 nM-1, and respective densities of 191.1 +/- 90.1 and 1425 +/- 1250 fmol/mg protein. However, the quantitative reliability of these binding isotherms may be influenced by the degration of unbound peptide. Competitive binding analysis was used to determine the specificity of the receptor site, with the relative order of affinities being AngIV > AngIII > AngII(4-8), and no displacement by AngII, Iosartan and PD123319 in either neurons or astrocytes. Autoradiography with 125I-AngIV performed on neuronal cultures demonstrated that binding was confined to a subpopulation of the total cells. These data support the existence of a specific binding site for AngIV in both neurons and astrocytes, consistent with the properties of binding reported previously in the brain, and distinguish this site from the AngII receptor subtypes.

Effects of pituitary adenylate cyclase-activating polypeptide and vasoactive intestinal polypeptide on cyclic AMP accumulation in sheep pituitary cells in vitro.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are known to stimulate adenylate cyclase activity in rat pituitary cells but no direct effects have been reported on sheep pituitary cells. In this study we determined whether either peptide could stimulate intracellular cAMP accumulation in dispersed sheep pituitary cells in primary culture. Time course studies with PACAP showed that tachyphylaxis developed rapidly and so a short incubation time (5 min) was used to define the dose-response relationship. PACAP dose-dependently stimulated intracellular cAMP levels with a half-maximum response at 2.9 +/- 0.2 nmol/l (n = 4). In contrast, VIP only caused a small increase in intracellular cAMP levels at the highest dose tested (1 mumol/l). The VIP antagonist [4Cl-D-Phe6,Leu17]VIP had no effect on the cAMP response to either PACAP or VIP while the peptide PACAP(6-38), a putative PACAP antagonist, blocked the cAMP response to PACAP. The desensitisation to PACAP was further investigated by pretreating cells with PACAP for 30 min. After a further 15 min in culture medium alone, these cells showed no cAMP response to subsequent treatment with PACAP but could respond to forskolin. When a longer incubation period of 240 min was used between the first and second treatment with PACAP, a partial return in responsiveness to PACAP was observed. In summary, these results show that PACAP activates adenylate cyclase in sheep pituitary cells but that there is rapid development of tachyphylaxis. Experiments with the antagonists suggest that the response to PACAP is via the PACAP type I receptor. In contrast, physiological doses of VIP do not stimulate cAMP accumulation in sheep pituitary cells.