A randomised trial of home energy efficiency improvement in the homes of elderly COPD patients.

A randomised trial of 178 patients in Aberdeen, UK with a previous hospital admission for chronic obstructive pulmonary disease (COPD) was carried out in order to determine whether improving home energy efficiency improves health-related quality of life in COPD patients. 118 patients were randomised and 60 agreed to monitoring only. Energy efficiency upgrading was carried out in 42% of homes randomised to intervention. Independent energy efficiency action was taken by 15% of control participants and 18% in the monitoring group. The main outcome measures were respiratory and general health status, home energy efficiency and hospital admissions. Intention-to-treat analysis found no difference in outcomes between the two groups. In 45 patients, who had energy efficiency action independent of original randomisation, there were significant improvements in respiratory symptom scores (adjusted mean 9.0, 95% CI 2.5-15.5), decreases in estimated annual fuel costs (- pound65.3, 95% CI - pound31.9- - pound98.7) and improved home energy efficiency rating (1.1, 95% CI 0-1.4). COPD patients are unlikely to take up home energy efficiency upgrading, if offered. Secondary "pragmatic" analysis suggests that those who do take action may achieve clinically significant improvement in respiratory health, which is not associated with an increase in indoor warmth.

Hormone replacement therapy (HRT) or etidronate for osteoporosis in postmenopausal asthmatics on glucocorticoids: a randomised factorial trial.

INTRODUCTION: The study was designed to establish the effects of HRT on osteoporosis and fractures over five years in postmenopausal women with asthma receiving regular glucocorticoids and to compare with etidronate. METHODS: Postmenopausal patients receiving inhaled and/or oral glucocorticoids were randomly assigned to HRT, cyclical etidronate, HRT plus cyclical etidronate or no treatment for five years. The trial was multi-centre and aimed to recruit 750 patients. Outcomes were fractures and changes in bone mineral density (BMD). RESULTS: For reasons detailed in the discussion section of the text, only 50 patients were entered. Three did not fulfil the eligibility criteria and were excluded from the analysis. Among the remaining 47 patients, three (6%) experienced new, symptomatic fractures, one on etidronate and two in the no treatment group. New or worsening morphometric fractures of the thoracolumbar spine occurred in 50% of the 22 patients with spinal radiographs on entry and at five years (one HRT, three etidronate, two HRT plus etidronate and five on no treatment). BMD improved by approximately 1% per annum in those receiving HRT and/or etidronate; comparisons of HRT vs no HRT tended to favour HRT but were only statistically significant at proximal femur. The same trends emerged in the etidronate vs no etidronate comparison, but none reached the 5% level of statistical significance. DISCUSSION: For postmenopausal patients receiving glucocorticoids for asthma, HRT appears as effective as etidronate in preventing loss of BMD over five years and may have a similar effect on fracture prevention.

Mechanism of adrenal angiotensin II receptor changes after nephrectomy in rats.

At 48 h after bilateral nephrectomy in rats there is a two- to threefold increase in the number of adrenal angiotensin II receptors and a decrease in Kd of smooth muscle angiotensin II receptors. These changes have been attributed to the absence of circulating angiotensin II. Serum K+, which increases after nephrectomy may be an important and overlooked modulator. Therefore, the present experiments were designed to assess the role of K+ as a regulator of angiotensin II receptors after nephrectomy. Serum K+ was controlled with Na polystyrene sulfonate (Kayexalate), a resin designed to exchange Na+ for K+ in the gastrointestinal tract. Acutely nephrectomized rats were divided into two groups: experimental animals received Kayexalate resin every 12 h for four doses, and controls received Kayexalate exchanged with KCl in vitro before gavage. There was a significant positive correlation serum K+ and aldosterone (r = 0.78, P less than 0.001). Kayexalate maintained a normal serum K+ of 5.9 +/- 0.2 meq/liter (n = 27), aldosterone 25 +/- 3 ng/dl (n = 27) and adrenal receptor concentration of 934 +/- 156 fmol/mg protein (n = 4). Control animals had significantly higher serum K+ of 10.5 +/- 0.4 meq/liter (n = 23), aldosterone 435 +/- 32 (n = 23), and adrenal receptors of 2726 +/- 235 fmol/mg protein (n = 4). There was a linear relationship between serum K+ and number of adrenal receptors (r = 0.87). No such relationship was present in uterine smooth muscle. Therefore, these studies demonstrate that K+ modulates the number of adrenal but not smooth muscle angiotensin II receptors after nephrectomy. This is the first evidence that potassium modulates angiotensin II receptors independently of changes in angiotensin II blood levels.

Relationship between phospholipase C activation and prostaglandin E2 and cyclic adenosine monophosphate production in rabbit tubular epithelial cells. Effects of angiotensin, bradykinin, and arginine vasopressin.

By employing early-passaged rabbit kidney epithelial cells in tissue culture, we demonstrated that angiotensin II (AII) has unique mechanisms of signal transduction. First, unlike its action in other target tissues, micromolar concentrations of AII are required to induce small rises in cytosolic calcium, [Ca2+]i, an action which is not accompanied by the release of inositol phosphates (IP). In contrast, nanomolar bradykinin (BK) mobilizes [Ca2+]i through activation of phospholipase C and release of IP. Neither of these stimulated calcium responses exhibits pertussis toxin (PTx) sensitivity. Secondly, AII and BK at 10(-9) to 10(-7) M stimulate cAMP indirectly through PGE2 production in distal cells. AII- and BK-stimulated PGE2 release is PTx inhibitible, suggestive of the presence of a GTP binding protein mediating the response. By contrast, arginine vasopressin fails to elicit rises in [Ca2+]i but exerts its primary effect on cAMP production in distal cells via direct coupling to a stimulatory GTP binding protein, as evidenced by uncoupling with cholera toxin. Regulation of PGE2 synthesis appears to occur via phospholipase A2, not C, by all three peptides.

Mechanism of decreased vascular reactivity to angiotensin II in conscious, potassium-deficient rats.

Chronic potassium deficiency in the rat results in a decrease in the pressor sensitivity to exogenous angiotensin II (AII). To define the mechanism of this resistance to AII, studies were performed in conscious rats after 14-21 d of dietary potassium deficiency. The pressor response to graded doses of AII was 50% less in potassium-deficient than control animals. In contrast, the pressor response to graded doses of norepinephrine was preserved in potassium-deficient rats; therefore, the decreased response to AII was not due to a generalized defect in vascular reactivity. Pretreatment with either the converting enzyme inhibitor, teprotide, or the prostaglandin synthesis inhibitor, indomethacin, failed to normalize the response to AII. Thus, neither prior receptor occupancy with endogenous AII nor the presence of vasodilatory prostaglandins caused the decreased AII response in potassium deficiency. Since the pressor response to AII involves angiotensin interaction with its vascular receptor, binding studies of mesenteric artery and uterine smooth muscle AII receptors were performed. Scatchard analysis showed that potassium deficiency resulted in a decrease in binding affinity (50% increase in Kd) in both uterine (6.00 vs. 3.82 nM; P less than 0.05) and vascular (1.39 vs. 0.973 nM; P less than 0.005) smooth muscle. Furthermore, despite increased circulating AII, there was an increase in AII receptor number in potassium-deficient uterine (308 vs. 147 fmol/mg protein; P less than 0.005) and vascular (470 vs. 316 fmol/mg protein; 0.05 less than P less than 0.1) smooth muscle. Although potassium deficiency resulted in alterations in receptor-binding parameters, the changes in binding affinity and number were directionally opposite, so that in potassium deficiency there was either no change or an increase in total AII binding. We conclude that the decrease in angiotensin pressor sensitivity in potassium-deficient rats is mediated by a postreceptor defect since it occurs subsequent to the binding of AII to its vascular smooth muscle receptor.

An epoxygenase metabolite of arachidonic acid mediates angiotensin II-induced rises in cytosolic calcium in rabbit proximal tubule epithelial cells.

Previous studies from this and other laboratories have shown that angiotensin II (AII) induces [Ca2+]i transients in proximal tubular epithelium independent of phospholipase C. AII also stimulates formation of 5,6-epoxyeicosatrienoic acid (5,6-EET) from arachidonic acid by a cytochrome P450 epoxygenase and decreases Na+ transport in the same concentration range. Because 5,6-EET mimics AII with regard to Na+ transport, it effects on calcium mobilization were evaluated. [Ca2+]i was measured by video microscopy with the fluorescent indicator fura-2 employing cultured rabbit proximal tubule. AII-induced [Ca2+]i transients were enhanced by arachidonic acid and attenuated by ketoconazole, an inhibitor of cytochrome P450 epoxygenases. Arachidonic acid also elicited a [Ca2+]i transient that was attenuated by ketoconazole. 5,6-EET augmented [Ca2+]i similar to that seen with AII, but was unaffected by ketoconazole. By contrast, the other regioisomers (8,9-, 11,12-, and 14,15-EET) were much less potent. [Ca2+]i transients resulted from influx through verapamil- and nifedipine-sensitive channels. These results suggest a novel mechanism for AII-induced Ca mobilization in proximal tubule involving cytochrome P450-dependent arachidonic acid metabolism and Ca influx through voltage-sensitive channels.

The changing face of infection: a comparative review of admissions to a regional infection unit over three decades.

BACKGROUND AND AIMS: Our group previously published retrospective analyses of 12 months of admissions to the Grampian Regional Infectious Diseases Unit from 1980-81 and from 1991. This study aimed to collect data in 2001 and to compare annual admission numbers, diagnoses, duration of stay and outcome in 1980-81, 1991 and 2001. METHODS: Data on all admissions was collected prospectively throughout 2001. This was compared with the previously published data. RESULTS: Total admissions rose from 605 in 1980-81 to 900 in 1991 and to 1152 in 2001. Sixty one percent of admissions in 1980-81 were confirmed as having infection compared to 72% in 1991 and to 83% in 2001. The most common reason for admission in 2001 was skin and soft tissue infection, but this was only the ninth commonest reason in 1981. Mean length of stay fell from 9.6 days in 1980-81 to 7.4 days in 1991 and to 5.5 days in 2001. The mortality rate fell from 3.1% in 1981 and 1991 to 1.0% in 2001. CONCLUSIONS: This study demonstrates significant changes in type, number and outcome of admissions to a regional infection unit. We discuss possible reasons for these changes.

Calcium reveals different mechanisms of guanylate cyclase activation by atrial natriuretic factor and ATP in rat lung membranes.

CaCl2 inhibited ATP-stimulated guanylate cyclase activity, but had little effect on basal and atrial natriuretic factor-stimulated guanylate cyclase activity in rat lung membranes. LaCl3 had similar effects as CaCl2 on basal and stimulated guanylate cyclase activity. LiCl and other monovalent salts inhibited ATP-stimulated guanylate cyclase activity more than basal enzyme activity. However, atrial natriuretic factor somehow stabilized the enzyme against the inhibitory effect of LiCl. These results suggest that ATP and atrial natriuretic factor activate the enzyme through different mechanisms. Since the effect of calcium on guanylate cyclase activity is different from that of monovalent salts and can be mimicked by lanthanum, it may be mediated by a specific calcium binding site or binding protein.

Drug treatment and obesity.

(1) The initial treatment of obesity must include an attempt to modify previous eating pattern and may involve group therapy or behavioral modification. Drug treatment is not indicated unless this dietary approach is shown to be ineffective. (2) Since anti-obesity drugs do not help to establish a new and permanent eating habit, they should never be prescribed except as part of an overall management plan. (3) The potential for abuse with amphetamine and phenmetrazine is such that their use cannot be justified as anorectic agents. (4) Phenmetrazine, diethylpropion, mazindol and fenfluramine will all produce an additional mean weight loss of approximately 0.2 kg per week. They are contraindicated if there is a history of drug misuse, drug dependence or psychiatric illness. They should always be prescribed with caution. With the exception of fenfluramine, they are best given intermittently on the grounds of efficacy, safety and cost benefit. (5) The individual response to drug therapy is extremely variable and may reflect differences in drug pharmacokinetics, metabolic adaptation or, less frequently, drug tolerance. (6) Following drug withdrawal, weight regain is the rule. It follows that therapy can most easily be justified if there is a short term need for weight loss, e.g. prior to elective surgery. (7) The safety and efficacy of long term drug therapy has yet to be established. It may prove justifiable in patients most at risk from obesity or from obesity associated disorders such as diabetes and hypertension. However, at present the only established indication for prolonged administration of the currently available drugs is the use of metformin in insulin independent diabetics. (8) The indications for the pharmacological treatment of obesity remain poorly defined. A number of new approaches are being evaluated, and the future may lie in the development of drugs which enhance thermogenesis or primarily act upon the gastrointestinal tract.

Hypertension and diabetes in blacks.

This article summarizes the current state of knowledge on the interrelationship between non-insulin-dependent diabetes mellitus, obesity, insulin resistance, and hypertension in our attempt to explore the pathophysiology of the high incidence of diabetes and hypertension in the Black population in the United States. A central role for hyperinsulinemia is proposed, and questions are raised that are suitable for investigative clinical trials.

Potassium regulates angiotensin II-induced aldosterone biosynthesis in acutely nephrectomized rats.

The studies described herein were designed to determine whether serum potassium modulates the aldosterone secretory response to angiotensin II (Ang II) after nephrectomy. We used isolated adrenal glomerulosa cells harvested from rats maintained with either normal (5.2 meq/liter) or high (6.8 meq/liter) serum potassium for 48 h after bilateral nephrectomy for determining Ang II-aldosterone dose-response relationships. Kayexalate, which exchanges sodium for potassium in the gastrointestinal tract, was used to achieve the desired level of serum potassium. Both groups of cells were responsive to low concentrations of Ang II (10-100 pM). Cells from rats with lower serum potassium levels had lower basal and maximum Ang II-stimulated aldosterone and ED50 values. The importance of variables as contributors to differences between the groups were ranked from most to least important by two group discriminant function analysis and revealed: serum potassium greater than maximum Ang II-stimulated aldosterone greater than ED50 greater than basal aldosterone greater than serum sodium. Stepwise multivariate discriminant analysis demonstrated that all variables except the level of serum sodium contributed to the groups being significantly different at P less than 0.05. These studies demonstrate that adrenal glomerulosa cells obtained from nephrectomized rats maintain sensitive secretory responses to Ang II. Additionally, the level of serum potassium of rats before death directly regulates both the sensitivity and magnitude of the aldosterone secretory response to Ang II in vitro.

Angiotensin II-binding sites in rat and primate isolated renal tubular basolateral membranes.

In the kidney, angiotensin II influences reabsorptive processes by a direct tubular effect(s). The receptors mediating the response may be located on either the luminal (brush border) and/or the contraluminal (basolateral) membranes of the tubular epithelial cells. In the studies reported here, we identify specific [125I]angiotensin II-binding sites in rat and baboon tubular basolateral membranes. Specific binding was saturable, largely reversible, and proportional to membrane protein concentration. Structural specificity was confirmed by the use of angiotensin analogs and structurally unrelated polypeptides. The latter did not compete with radioligand for binding. Scatchard analyses of binding inhibition data indicated a single class of high affinity sites in rat (Kd = 2.2 +/- 0.2 nM; n = 12) and two classes of sites in baboon [Kd = 1.32 (n = 1) and 0.6 +/- 0.1 nM; n = 2) basolateral membranes. The binding site concentrations were 929 +/- 138 fmol/mg protein (rat) and 463 and 439 +/- 120 fmol/mg protein (baboon). Rat binding sites were affected by the addition of cations, as chloride salts, to the incubation medium. Na+ (100-200 mM) decreased the Kd from 4.2 +/- 0.4 nM in the absence of cations to 2.7 +/- 0.3 nM (n = 4). Mg2+ (4 mM) had no effect on Kd, but increased the binding site concentration from 556 +/- 84 to 915 +/- 166 fmol/mg protein. In contrast, 2 mM Ca2+ increased the Kd to 5.3 +/- 0.6 nM, and Mg2+ and Ca2+ added together affected neither Kd nor number of binding sites. Bound (eluted from membranes) and free (in medium) radioactivity, after incubation with membranes to steady state, were 54-68% (n = 3) and 85% (n = 2) intact [125I] angiotensin II, respectively, as determined by rebinding to fresh membranes. These data are inconsistent with binding to a degradative enzyme and indicate the presence of specific [125I] angiotensin II-binding sites in renal tubular basolateral membranes.

Angiotensin II binding sites on isolated rat renal brush border membranes.

There is evidence that angiotensin II has a direct effect on reabsorptive processes of the kidney that are mediated by angiotensin II receptors on the proximal tubules. These receptors have not previously been localized to luminal brush border or basolateral membranes. The present study is the first to characterize the angiotensin II receptor of rat renal brush border membrane vesicles. Results are reported as means +/- SE Specific binding of [125I]iodo-angiotensin II at steady state was significantly increased by 100 mM NaCl or 10-25 mM MgCl2. NaC1 caused a significant decrease in the Kd from 9.5 +/- 1.3 nM (n = 2) to 6.0 to 0.3 nm (n = 3). In contrast, MgCl2 had no effect on Kd but significantly increased the binding site concentration from 162 +/- 76 (n = 2) to 597 +/- 26 fmol/mg protein (n = 3). The effects of these salts were additive. Steady state binding was achieved within 10-15 min at 24 C. Scatchard analyses of binding inhibition data indicated a single class of high affinity sites with Kd similar to that for angiotensin II receptors in known target tissues. Binding was reversible, proportional to membrane protein concentration, and did not consist of uptake into the vesicles. Octapeptide and 2-8 heptapeptide analogs of angiotensin competed for these binding sites with potencies that correlated with their binding inhibition potencies at known extrarenal and renal target tissues. The radioactivity, eluted from membranes with acid after steady state was achieved, was 57 +/- 8% (n= 4) of intact [125I]iodo-angiotensin II, as determined by rebinding to fresh membranes. This observation is inconsistent with binding to a degradative enzyme. These findings indicate the presence of high affinity specific binding sites for [125I]iodo-angiotensin II in renal brush border membranes.

Comparative studies of receptor binding and steroidogenic properties of angiotensins in the rat adrenal glomerulosa.

Angiotensin and analogs were tested in isolated rat adrenal zona glomerulosa cells to find if there was a correlation between receptor affinity and steroidogenic potency. Comparative receptor-binding affinities and corresponding aldosterone-releasing effects obtained for each analog were: [Asp1, Ile5]angiotensin II, 1.0 and 1.0: [Asp1, Val5]angiotensin II, 0.69 and 1.65; [Asn1, Val5]angiotensin II, 1.18 and 0.68; [Sar1]angiotensin II, 2.07 and 1.7; [Me2Gly1]angiotensin II, 0.63 and 0.72; [Ile5]angiotensin III, 0.72 and 0.59; [Val5]angiotensin III, 0.92 and 0.34; [Ile5]angiotensin I, 0.007 and 0.051; des-Asp1-[Ile5]-angiotensin I, 0.004 and 0.03; and [Val5, Ser9]angiotensin I, 0.03 and 0.098. Taken as a group, these agonist analogs demonstrated good correlations between these two variables (r = 0.76; P less than 0.001). There was parallelism between binding inhibition and aldosterone-releasing effect when position 5 was substituted with isoleucine, regardless of the substituent in position 1 of the angiotensins. This parallelism was lost when analogs of angiotensin II or III contained valine in position 5. In addition, angiotensin III was found to be less potent than angiotensin II, regardless of the substituent in position 5 (valine or isoleucine).

Efficacy of octa- and heptapeptide antagonists of angiotensin II as inhibitors of angiotensin III binding in the rat adrenal glomerulosa.

Angiotensin III (Ang III) is a carboxy-terminal 7-amino acid analog of angiotensin II (Ang II) with similar receptor binding affinity and biological activity in adrenal glomerulosa. Specific competitive antagonists have been synthesized for both compounds, and structure-activity studies have demonstrated that Ang II (octapeptide) antagonists compete better for Ang II receptors in adrenal glomerulosa than do Ang III (heptapeptide) antagonists. These differences were observed in spite of only 1 amino acid difference in chain length of antagonist analogs. These earlier observations by our group provided support for the current hypothesis that Ang III binding would be preferentially inhibited by Ang III antagonists compared to Ang II antagonists. To accomplish these studies, we used [125I]Ang III and [125I]Ang II as ligands and 5 pairs of heptapeptide and octapeptide antagonists with identical substituent amino acids in the carboxy-terminal position. [Sar1,Ile8]- and des Asp1 [Ile8]Ang II did not differ in potency as antagonists of Ang II binding, but with 4 other pairs of antagonists, the octapeptide antagonists were more potent than the corresponding hepatapeptide antagonist. Six of 10 antagonists exhibited similar potencies as antagonists of equimolar concentrations of Ang II and Ang III. One heptapeptide antagonist was twice as potent against Ang III, and 3 octapeptide antagonists were more potent against Ang II. In general, the order of potencies of the 10 antagonists as inhibitors of Ang III binding was linearly related to their potencies against Ang II. Hence, our hypothesis of preferential activity of Ang III antagonists (compared to Ang II antagonists) as inhibitors of Ang III binding to adrenal glomerulosa was not borne out by the present studies. When this observation was combined with the finding of similar receptor densities of Ang III and Ang II receptors, we concluded that Ang III and Ang II probably bind to the same receptor site in the adrenal glomerulosa.

Subpressor infusions of angiotensin II alter glomerular binding, prostaglandin E2, and cyclic AMP production.

Angiotensin II (ANG II) has been postulated to have pathogenetic role in diminished glomerular function in a number of animal models of acute renal failure. The present studies were designed to test the hypothesis that modest elevations in circulating ANG II potentiate the ability of ANG II to reduce glomerular capillary surface area through an effect on ANG II binding to glomerular mesangial cells and/or influences on other modulators of function. Rat glomeruli isolated by a sieving technique were employed in vitro in an ANG II radioreceptor assay. Subpressor infusion of ANG II for 36 hours in rats increases the affinity and number of ANG II binding sites of isolated glomeruli. The ability of ANG II to influence function was tested by assessing its effect on glomerular surface area in vitro by image-analysis microscopy, a method of measuring mesangial cell contractility. The sensitivity and magnitude of ANG II-induced decrements in glomerular surface area were increased. ANG II infusion diminished glomerular prostaglandin E2 (PGE2) production, increased basal cyclic adenosine 3',5'-monophosphate (cAMP) production, and enhanced ANG II-induced decrements in cAMP production. In control glomeruli, only pharmacological concentrations of ANG II inhibited cAMP, but after ANG II infusion, physiological concentrations of ANG II were capable of inhibiting cAMP by as much as 57% (below basal values). In conclusion, continuous infusion of subpressor concentrations of ANG II in rats enhances the contractile response of the glomerular mesangial cell through effects on the cell's surface receptor for ANG II and on prostaglandin and cAMP production. These actions may be important mediators of the effects of ANG II on glomerular function associated with a number of experimental models of kidney disease.

Angiotensin II type 2 receptor subtype mediates phospholipase A2-dependent signaling in rabbit proximal tubular epithelial cells.

We investigated the ability of angiotensin II (Ang II) or the stable analogue [Sar1]-Ang II to increase intracellular and extracellular free arachidonic acid in primary cultures of rabbit proximal tubular epithelial cells to better characterize the receptor subtype and orientation of phospholipase A2 (PLA2)-mediated signaling. Proximal tubular cells were labeled with [3H]arachidonic acid for 4 hours and then treated with Ang II or [Sar1]-Ang II. Lipids were extracted from labeled cells, separated by thin-layer chromatography, and quantified by liquid scintillation counting. Ang II (10 mumol/L, 1 minute) stimulated an increase in intracellular free [3H]arachidonic acid from 21.0 +/- 2.0 to 32.2 +/- 2.8 disintegrations per minute/microgram protein, an effect that was potentiated by EGTA. [Sar1]-Ang II stimulated a time- and concentration-dependent increase in [3H]arachidonic acid release from labeled cells. Release of [3H]arachidonic acid was maximal at 10 mumol/L [Sar1]-Ang II, with an EC50 of approximately 3 mumol/L. Ang II receptor antagonists caused concentration-dependent inhibition of [Sar1]-Ang II-stimulated [3H]arachidonic acid release with the following order of potency: CGP 42112 = PD 123319 > losartan. Furthermore, in proximal tubular epithelial cells grown on polyester membrane filters, the Ang II receptor that mediated arachidonic acid release was predominantly apical rather than basolateral. These observations are consistent with activation of a Ca(2+)-independent, apical PLA2 isoform in epithelial cells through an Ang II type 2 receptor subtype.

A novel angiotensin receptor subtype in rat mesangium. Coupling to adenylyl cyclase.

The diversity of angiotensin II (Ang II) actions implies multiple receptor subtypes. To characterize these subtypes in rat mesangial cells, we used the angiotensin subtype 1A (AT1A) antagonist losartan (DuP 753), the subtype 2/1B (AT2/AT1B) antagonist PD 123319, and the AT2 antagonist CGP 42112A in radioreceptor and adenylyl cyclase assays. In radioligand binding competition experiments, approximately 25% of the specific binding sites labeled by 125I-[Sar1]Ang II were inhibited by low concentrations of PD 123319 (0.1 to 10 nM), whereas the AT2 antagonist CGP 42112A was inactive at concentrations less than 0.1 microM. Conversely, losartan inhibited 75% of the binding at low concentrations (0.1 nM to 0.1 microM), but higher concentrations (up to 10 microM) were required to inhibit the second component of 125I-[Sar1]Ang II binding. The effects of the different antagonists on the inhibition by Ang II of forskolin-stimulated cyclic AMP production were also analyzed. Ang II inhibited forskolin-stimulated adenylyl cyclase in a concentration-dependent fashion (IC50, 35 +/- 7 nM), and the maximal inhibition of adenylyl cyclase was 44 +/- 2%. In the radioligand binding experiments, both losartan and PD 123319 antagonized the inhibition of adenylyl cyclase elicited by 0.1 microM Ang II (IC50, 0.5 +/- 0.2 and 1.2 +/- 0.4 microM, respectively), whereas CGP 42112A was less potent (IC50, 5.7 +/- 1.6 microM). Comparison of binding affinities at AT1B receptor sites with antagonist potencies in the adenylyl cyclase assay show good agreement for losartan and CGP 42112A, whereas PD 123319 is less potent than expected from membrane binding assays, possibly because of partial agonist properties.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II-induced changes in guanine nucleotide binding and regulatory proteins.

Systemic infusion of angiotensin II, a potent agonist, using doses that are initially subpressor, eventually produces sustained blood pressure elevation and reductions in glomerular capillary ultrafiltration coefficient characterized by enhanced signal transduction to angiotensin II and other agonists. In this setting, there is a significant increased affinity of angiotensin II binding to smooth muscle and glomerular mesangial receptors and enhanced sensitivity and magnitude of angiotensin II-induced decrements in cyclic AMP. Since G proteins are important modulators of binding and signal transduction, the present studies were designed to test the hypothesis that differences in the relative amounts of G proteins may be present and have accounted for differences observed. G proteins were identified and quantitated by isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis, radiolabeling in the presence of activated toxins with [gamma-32P]NAD+, immunoprecipitation, and immunoblotting. A 168% and 465% increase in pertussis toxin-catalyzed ADP ribosylation of alpha 40-41 was found in angiotensin II-treated groups over control groups for glomerular and mesenteric membranes, respectively. Immunoblotting revealed a 250% and 35% increase in the levels of the Gi isoforms alpha i-2 and alpha i-3, respectively, and a decrease of 53% in alpha i-1 from the angiotensin II-treated group. No differences were observed in cholera toxin labeling or immunoblotting of Gs. These results demonstrate multiple mechanisms whereby angiotensin-induced signal transduction can be modulated involving both the receptors and G proteins. These observed differences in G proteins in systemic and renal vasculature accompanying angiotensin II infusion suggest the possibility of a regulatory role in the pathophysiology of angiotensin II-induced hypertension and renal disease.

Influence of cations on kinetics of angiotensin II binding to adrenal, renal, and smooth muscle receptors.

A number of biological responses to angiotensin II have been demonstrated to be modulated acutely by cations, but the exact mechanism has not been elucidated. We have utilized a radioreceptor assay for angiotensin II to determine whether this acute regulatory mechanism could be related to a change in either number or affinity of angiotensin II binding to receptors. Three target tissues were used (adrenal glomerulosa, glomeruli, and uterine smooth muscle). Both mono- and divalent cations influence the kinetics of angiotensin II binding in a similar manner in all tissues. Divalent cations increase the number of receptor sites to a greater extent than did monovalent cations, while monovalent cations changed binding affinity to a greater extent than did divalent cations. These studies demonstrate that both the number and affinity to a greater extent than did monovalent cations, while monovalent cations changed binding affinity to a greater extent than did divalent cations. These studies demonstrate that both the number and affinity of angiotensin receptors can be rapidly modulated by a variety of cations.