Angiotensin II AT1 Receptor/Signaling Mechanisms in The Biphasic Effect of The Peptide on Proximal Tubular Na+,K+-ATPase

The present study was designed to determine the cellular signaling mechanisms responsible for mediating the effects of angiotensin II on proximal tubular Na⁺,K⁺-ATPase activity. Angiotensin II produced a biphasic effect on Na⁺,K⁺-ATPase activity: stimulation at 10^-13-10^-10M followed by inhibition at 10^-7-10^-5M of angiotensin II. The stimulatory and inhibitory effects of angiotensin II were antagonized by losartan (1nM) suggesting the involvement of AT, receptor. Angiotensin IT produced inhibition of forskolin-stimulated CAMP accumulation at 10^-13-10^-10M followed by a stimulation in basal CAMP levels at 10^-7-10^-5M. Pretreatment of proximal tubules with losartan (1nM) antagonized both the stimulatory and inhibitory effects of angiotensin II on CAMP accumulation. Pretreatment of the proximal tubules with pertussis toxin (PTx) abolished the stimulation of Na⁺,K⁺-ATPase activity but did not affect the inhibition of Na⁺,K⁺-ATPase activity produced by angiotensin II. Pretreatment of the tubules with cholera toxin did not alter the biphasic effect of angiotensin II on Na⁺,K⁺-ATPase activity. Mepacrine (l0μM), a phospholipase A2 (PLA2) inhibitor, reduced only the inhibitory effect of angiotensin II on Na⁺,K⁺-ATPase activity. These results suggest that the activation of AT₁angiotensin II receptors stimulates Na⁺,K⁺-ATPase activity via a PTx-sensitive G protein-linked inhibition of adenylyl cyclase pathway, whereas the inhibition of Na⁺,K⁺-ATPase activity following AT₁receptor activation involves multiple signaling pathways which may include stimulation of adenylyl cyclase and PLA2     

Angiotensin II-Receptor Antagonist Losartan Does not Prevent Nitroglycerin Tolerance in Patients with Coronary Artery Disease

The study evaluated the effect of Losartan in preventing nitrate tolerance during continuous transdermal nitroglycerin (TD-GTN) therapy in patients with coronary disease. Fifteen subjects with chronic stable ischemia evaluated by exercise test, were randomized to 28 days of TD-GTN 20 mg once a day without free interval plus Losartan 100 mg or Losartan-placebo with a double blind crossover design. Myocardial ischemic parameters during stress test were evaluated after each test period and results of Losartan therapy were compared to those with placebo. Time to onset 1 mm ST-depression was significantly higher after acute TD-GTN 20 mg with respect to placebo run-in, sustained TD-GTN 20 mg plus Losartan 100 mg or Losartan-placebo (p < 0.001). ST-depression at peak exercise and time to recovery of ST segment were markedly lower after acute TD-GTN 20 mg compared to placebo run-in (p < 0.05), sustained TD-GTN 20 mg plus Losartan 100 mg (p < 0.001) or Losartan-placebo (p < 0.05). At 1 mm-ST depression and at peak exercise, systolic blood pressure and rate-pressure product significantly decreased after sustained TD-GTN 20 mg plus Losartan 100 mg (p < 0.001, p < 0.05 respectively) with respect to placebo run-in, acute and sustained TD-GTN 20 mg plus Losartan-placebo. Moreover at peak exercise, these data were also observed after acute TD-GTN 20 mg compared to placebo run-in and sustained TD-GTN 20 mg plus Losartan-placebo (p < 0.001). The AT₁ antagonist Losartan administration does not prevent the development of nitrate tolerance during continuous TD-GTN therapy.     

Identification, Characterization, and Inhibition of the Platelet ADP Receptors

Adenosine diphosphate (ADP) plays a crucial role in hemostasis and thrombosis, and its receptors are potential targets for antithrombotic drugs. Two G-protein-coupled P2 receptors contribute to platelet aggregation: the P2Y1 receptor initiates aggregation through mobilization of calcium stores, whereas the P2Y12 receptor coupled to adenylyl cyclase inhibition is essential for a full aggregation response to ADP and the stabilization of aggregates. The latter is defective in certain patients with a selective congenital deficiency of aggregation to ADP. It is also the target of the antithrombotic drug clopidogrel and of adenosine triphosphate analogues and other compounds currently under evaluation. In addition, the P2X1 ionotropic receptor is present in platelets, but its role is not yet completely known. Studies in P2Y1-knockout mice and experimental thrombosis models using selective P2Y1 antagonists have shown that the P2Y1 receptor, like the P2Y12 receptor, is a potential target for new antithrombotic drugs.     

Angiotensin AT1 receptor inhibition, angiotensin-converting enzyme inhibition, and combination therapy with developing heart failure: Cellular mechanisms of action

Background: Past studies have shown that angiotensin-converting enzyme inhibition (ACEI) alone, angiotensin AT₁ receptor blockade (AT₁ block) alone, and combined treatment have differential effects on left ventricular (LV) function and geometry with developing congestive heart failure (CHF). The purpose of this study was to more carefully examine the cellular basis for these differential effects by using a model of pacing CHF.Methods and Results: Pigs were randomly assigned to five groups: (1) rapid pacing (240 bpm) for 3 weeks (n = 9), (2) concomitant ACEI (benazeprilat, 0.187 mg/kg/day) and pacing (n = 9), (3) concomitant AT₁ block (valsartan, 3 mg/kg/day) and pacing (n = 9), (4) concomitant ACEI and AT₁ receptor blockade (benazeprilat/valsartan, 0.05/3 mg/kg/day, respectively) and pacing (n = 9), and (5) sham controls (n = 10). The dosage protocol was based on obtaining a 50% reduction in angiotensin I and angiotensin II pressor response with no significant effects on mean basal arterial pressure. In the pacing group, LV fractional shortening (LVFS) fell compared with control group (13.4 ± 1.4 v 39.1 ± 1.0%, P < .05). With AT₁ block, LVFS was unchanged from pacing only. ACEI and combined treatment increased LVFS from pacing values (25.2 ± 0.9 v 20.9 ± 1.9%, respectively, P < .05). LV myocyte shortening velocity was reduced with chronic pacing compared with control group (27.2 ± 0.6 v 58.6 ± 1.2 μm/s, P < .05) and remained reduced with AT₁ block (28.0 ± 0.5 μm/s, P < .05). Myocyte shortening velocity increased with ACEI or combination treatment (36.9 ± 0.7 ± 0.7 v 42.3 ± 0.8 μm/s, respectively, P < .05). Concomitant treatment with either ACEI or AT₁ blockade normalized myocyte action potential duration. In the combined ACEI and AT₁ blockade group, all parameters of the myocyte action potential were unchanged from control values.Conclusions: This study showed that combined ACEI and AT₁ receptor blockade produced beneficial effects on myocyte contractility and electrophysiology when compared with either monotherapy alone and therefore may provide unique benefits with CHF.     

Enhanced Angiotensin II-Induced Activation of Na+, K+-ATPase in the Proximal Tubules of Obese Zucker Rats

Renal angiotensin II (AII) is suggested to play a role in the enhanced sodium reabsorption that causes a shift in pressure natriuresis in obesity related hypertension; however, the mechanism is not known. Therefore, to assess the influence of AII on tubular sodium transport, we determined the effect of AII on the Na⁺, K⁺-ATPase activity (NKA), an active transporter regulated by the AT₁ receptor activity, in the isolated proximal tubules of lean and obese Zucker rats. Also, we determined the levels of the tubular AT₁ receptor and associated signal transducing G proteins, as the initial signaling components that mediate the effects of AII on Na⁺, K⁺-ATPase activity. In the isolated proximal tubules, AII produced greater stimulation of the NKA activity in obese compared with lean rats. Determination of the AT₁ receptors by Scatchard analysis of the [¹²⁵I] Sar-Ang II binding and Western blot analysis in the basolateral (BLM) and brush border membrane (BBM) revealed a modest but significant increase (23%) in the AT₁ receptor number mainly in the BLM of obese compared with lean rats. The AII affinity for AT₁ receptors, as determined by IC₅₀ values of AII to displace [¹²⁵I] Sar-Ang II binding in BLM and BBM were similar in lean and obese rats. Western blot analysis revealed significant increases in Giα₁, Giα₂, Giα₃, and Gq/₁₁α in BLM and Giα₁, Giα₃, and Gq/₁₁α in BBM of obese as compared with lean rats. The increase in the levels of the AT₁ receptor and G proteins, mainly in the BLM, may be contributing to the enhanced AII-induced activation of NKA in the proximal tubules of obese rats. This phenomenon, in part, may be responsible for the increased sodium reabsorption and the development of hypertension in obese Zucker rats.     

Tissue-Specific Regulation of Growth Factors and Clusterin by Angiotensin II

Angiotensin II (ANG II) has been implicated in the hypertrophic and fibrotic responses of the heart and kidney to systemic hypertension. To determine whether these actions of ANG II are related to tissue-specific stimulation of growth factors, we infused adult Sprague-Dawley rats with ANG II at 50 ng/min (low dose), 100 ng/min (high dose), or vehicle for 1 week. Rats receiving vehicle or low-dose ANG II were normotensive with normal plasma aldosterone concentration, whereas rats receiving high-dose ANG II were hypertensive with increased plasma aldosterone. Tissue fibrosis was quantified morphometrically, and messenger RNA (mRNA) for transforming growth factor-β₁ (TGF-β₁) and prepro-epidermal growth factor (EGF) was measured in liver, heart, and renal glomeruli and tubules. In addition, mRNA was determined for clusterin, a glycoprotein expressed in response to tissue injury. Compared to vehicle, low-dose ANG II increased TGF-β₁ expression in glomeruli, tubules, and heart, but not in liver, and increased EGF expression in renal tubules only. High-dose ANG II decreased clusterin expression in liver only. Fibrosis was induced by low- and high-dose ANG II in kidney and heart, but not in liver. We conclude that ANG II selectively stimulates TGF-β₁ mRNA in the heart and kidney, which may contribute to cardiac and renal interstitial fibrosis resulting from activation of the renin-angiotensin system independent of hypertension. By stimulating cellular proliferation, selective stimulation by ANG II of EGF in renal tubules may amplify the effects of TGF-β₁. Suppression of clusterin expression in the liver of hypertensive rats may represent a specific response to high levels of circulating ANG II or a response to hypertensive injury.     

Histamine H2 Receptors and Intrinsic Factor Secretion

Intrinsic factor (IF) secretion in healthy male subjects was studied in response to pentagastrin stimulation with and without cimetidine, and to impromidine, a histamine H₂ receptor agonist. Peak and total IF output were reduced by cimetidine, but the concentration was unchanged and the response was unaffected by administration of the compound for 1 week. The IF secretory response to impromidine was similar to that to pentagastrin. It is suggested that the acid and IF components of the parietal cell secretory response are mediated via different intracellular pathways, that histamine H₂ receptors do not fulfill an obligatory role in the secretion of IF and that synthesis of IF is probably not altered by cimetidine.     

Sarcosine1, glycine8 angiotensin II is a functional AT1 angiotensin receptor antagonist

Sarcosine¹, glycine⁸ angiotensin II (SG Ang II) displayed unusual characteristics in early pharmacological studies. It was a potent antagonist of the dipsogenic actions of intracerebroventricularly administered Ang II in the rat, but showed low affinity for bovine cerebellar Ang II receptors. It has recently been shown that SG Ang II binds preferentially to the AT₁ receptor, To determine if SG Ang II is a functional antagonist of the AT₁ receptor-mediated calcium signaling, CHO cells stably transfected with AT₁ receptors were exposed to Ang II in the presence and absence of SG Ang II. At concentrations of 10–100 nM, SG Ang II completely inhibited Ang II-stimulated intracellular Ca²⁺ mobilization in AT_1A and AT_1B receptor-transfected cells. SG Ang II and ¹²⁵I-SG Ang II bound to AT_1A and AT_1B receptor-transfected cells with K _D and K ₁ values of 2–4 nM. In contrast, SG Ang II bound to AT₂ transfected cells with a K ₁ value of 7.86 μM. These results demonstrate that SG Ang II is a selective and functional peptide antagonist of the AT₁ angiotensin II receptor subtype.     

Hormonal Responses to Change in Posture in Hypertensive Man. Evaluation by measurements of Prostaglandin E2, Renin Activity,Angiotensin II,and Norepinephrine in Renal Venous Blood

The hormonal responses to the stimulus of changing from resting supine to sitting upright for 15 minutes were assessed in 20 patients with hypertension, divided into 2 groups. 8 patients had essential hypertension (EH) and 12 unilateral renal artery stenosis (URS). The prostaglandin E₂(PGE₂) concentration, plasma renin activity (PRA), angiotensin II (A-II) concentration, and norepinephrine (NE) concentration were measured in renal vein blood using specific methods. The PGE₂concentration increased after sitting for 15 minutes in all patients (p < 0.001), but the increment was significant only in those with URS. The PRA was lower both at rest and after sitting up in the EH group than in the URS group. After sitting up the A-II concentration increased more in patients with URS than in those with EH (p < 0.05). NE levels rose significantly when all patients were included (p < 0.01), rainly owing tcj changes in the EH group. Supine and sitting PRA and A-I1 were correlated (r = 0.47 and r = 0.52; both p < 0.05), and also sitting PGE2 and A-II (r = 0.46, p < 0.05). The inverse relation between PGE2 and NE for the difference in hormone concentrations between supine and sitting (r = -0.44, p < 0.05) may be explained by an inhibitory effect of PGE2 on renal NE release, earlier observed in experiments in vitro. Similar changes in PGE2 and the measured components of the renin-angiotensin system i n response to change in posture may indicate these factors are .interrelated.     

Alpha1-adrenergic and muscarinic receptors in adult and neonatal rat type II pneumocytes

Binding characteristics of the ₁-adrenergic radioloigand [³H]prazosin, and the muscarinic cholinergic radioligand, [³H]quinuclidinyl benzilate, were determined both in intact cell preparations of rat alveolar type II pneumocytes (TIIPs) and in membrane preparations of rat lung tissue. Binding in adult and neonatal (<24 h postnatal age) rats was also compared. Binding affinities for both receptor classes on TIIPs and whole lung membrane preparations alike did not vary significantly with age. In lung membrane preparations, the concentrations of both receptor classes were higher in neonates than adults. In TIIPs, the ₁-adrenergic receptor concentration was higher in neonates, but muscarinic receptor concentration was higher in adults. To begin investigation of the functional significance of these receptors, the effects of ₁-adrenergic and muscarinic agonists on intracellular calcium ion concentration ([Ca²⁺]_i) were also measured. Both agonists induced consistent increases in [Ca²⁺]_i, which were blocked by respective antagonists. These data indicate the presence of receptors on TIIPs for ₁-adrenergic and muscarinic agonists that may influence cellular function via modulation of [Ca²⁺]_i. Offprint requests to: S. E. Keeney     

Myocardial electrophysiological properties in the presence of an AT1 angiotensin II receptor antagonist

Introduction Blockade of the AT₁ angiotensin II (Ang II) receptor has been shown to provide antihypertensive effects. However, whether AT₁ Ang II receptor antagonists influence myocardial electrophysiological properties remains unclear.Methods and results Accordingly, atrial and ventricular myocardial electrophysiological properties were examined in adult rat (n=13) and guinea pig (n=9) myocardial preparations in the presence of the specific AT₁ Ang II receptor antagonist, valsartan (CGP 48933; 0.5, 5, or 500 mol/L). These concentrations reflect up to 100 fold higher drug concentrations than those observed in clinical trials. Transmembrane potential data were recorded using standard microelectrode techniques at baseline and following superfusion with valsartan. The lower concentrations of valsartan (0.5 and 5 mol/L) had minimal effects on myocardial electrophysiology. In the presence of 500 mol/L of valsartan, resting membrane potential increased from baseline in both rat (–82.3±4.1 vs –76.8±5.8 mV, p<0.05) and guinea pig (–81.6±2.9 vs –76.9±2.0 mV, p<0.05) atrial myocardium. Action potential duration at 90% repolarization was increased in guinea pig atrial (91.7±1.4 vs 80.0±5.6 ms, p<0.05) and ventricular (131.1±8.1 vs 118.7±8.3 ms, p<0.05) myocardium following exposure to 500 mol/L of valsartan. In a separate series of experiments Ang II (1.0 mol/L) had no effect on atrial or ventricular action potential characteristics in either species.Conclusion Thus, the effects of valsartan, which were observed only at concentrations 100 fold higher than those reported in clinical trials, may be due to non-specific drug interactions with the myocyte sarcolemma.     

Central Imidazoline Receptors and Centrally Acting Anti-Hypertensive Agents

We have examined the location and contribution of imidazoline receptors (IR) in mediating the hypotensive and sympatholytic actions of first and second generation anti-hypertensive agents in rabbits. We found that the hypotension produced by rilmenidine and moxonidine given intravenously (IV) or into the fourth ventricle (4V) was preferentially reversed by the IR antagonists idazoxan and efaroxan (compared to a selective α₂-adrenoceptor antagonist 2-methoxy-idazoxan), suggesting that IR are important in the sympatho-inhibition produced by these agents. Clonidine was not preferentially reversed by the IR antagonists suggesting an action via aadrenoceptors.In anaesthetised rabbits, the rostral ventrolateral medulla(RVLM) was the most potent site for rilmenidine to produce the sympathoinhibition and modulation of sympathetic baroreflexes. a-Methylnoradrenalhe was also sympatholytic suggesting a-adrenoceptors are also present in this site. Microinjection of the IR and a-adrenoceptor antagonists showed that rilmenidine activates IR in the RVLM but that a-adrenoceptors are also activated as a consequence. These studies suggest that rilmenidine acts primarily via IR in the RVLM to reduce sympathetic tone but also imply an important association of a-adrenoceptors and IR in the region.     

Extent of beta1- and beta2-receptor occupancy in plasma assesses the antagonist activity of metoprolol,pindolol, and propranolol in the elderly

Summary We estimated antagonist activity of metoprolol, pindolol, and propranolol in elderly cardiovascular patients by determining the extent to which the drugs occupied rabbit lung beta₁- and rat reticulocyte beta₂-adrenoceptors in plasma samples during drug treatment. The randomized, double-blind, crossover study was carried out by administering twice daily 100 mg metoprolol, 5 mg pindolol, and 80 mg propranolol for 7 days to 20 hypertensive subjects with a mean age of about 70 years. A 2-week interval was kept between administration of the different regimens. Receptor occupancy was measured at 1 hour before and 2 hours after administration of the last dose of each regimen by adding rabbit lung beta₁- and rat reticulocyte beta₂-receptors to plasma samples and by labeling the receptors with a radiolabeled beta-antagonist, (–)-[³H]CGP-12177. The results and conclusions were the following: (a) The extent to which metoprolol, pindolol, and propranolol occupied rabbit lung beta₁-and rat reticulocyte beta₂-adrenoceptors in plasma samples estimated accurately the intensity of beta-receptor antagonism in the patients who did not tolerate physiological and pharmacological tests measuring the degree of beta₁- and beta₂-adrenoceptor blockade. (b) The mean beta₁- and beta₂-receptor occupancy of pindolol and propranolol varied between 76% and 99% during the treatments. The mean beta₁-receptor occupancy of the metoprolol regimen varied between 54% and 92%, and its beta₂-receptor occupancy varied between 6% and 38%. Thus the antagonist activity of the metoprolol regimen differed significantly from that of the other regimens (ANOVA for repeated measures, p<0.05 and 0.001, for the beta₁- and beta₂-occupancy, respectively). (c) The extent of beta₁- and beta₂-receptor occupancy in plasma samples was in conformity with the literature on the intensity, selectivity, and duration of beta-blockade after similar drug doses. (d) The data on the receptor occupancy of beta-blocking drugs in plasma samples appear to be valuable in analyzing their effects, and it may be a method for optimizing drug therapy for aged cardiovascular patients.     

Angiotensin II-receptor subtypes in human atria and evidence for alterations in patients with cardiac dysfunction

Angiotensin II (All) has been implicated as an important factorin the pathophysiology of heart diseases. Following the recentidentification of two subtypes of the All receptor in cardiactissue of animals, we investigated the possible occurrence ofthese, or similar, subtypes in human atrial tissue. In right-atrialtissue from patients undergoing heart surgery, we determinedthe All-receptor profile in receptor binding studies, using[¹²⁵I]-angiotensin as radioligand and All as well as two compoundsselective for the receptor subtypes to identify and quantifyAll-receptor subpopulations. In 35 patients (23 requiring coronarybypasses, 10 vaivular surgery and two combined coronary andvalvular surgery), the left-ventricular ejection fraction wasdetermined in the preoperative phase, and right- and left-atrialpressure during surgery. In membranes of human right atria,All receptors are present in high density (median: B_max= 294fmol. mg^–1 protein, range: 111-2073) and two differentsubtypes can be distinguished. Type-1 receptors (AT₁) accountedfor 33 ± l0% of the population whereas type-2 receptors(AT₂) made up 67 ± 10% of the population. There was nocorrelation between any of the measured cardiac functions andtotal All-receptor density or receptor affinity. However, thepercentage of AT₁ receptors was higher in the atria of patientswith normal right-atrial pressure; left-ventricular ejectionfraction was positively and right-atrial pressure inverselycorrelated with the percentage of AT₁ receptors (r=0·740and -0·901, respectively; P<0·001, for both).Moreover, the percentage of AT receptors was directly correlatedwith the levels of left-atrial pressure (r=0·853; P<0·001).It is concluded that the ratio of AT₁ to AT₂ receptors correlateswell with right-atrial pressure and left-ventricular function.This is a first indication of a possible involvement of All-receptorsubtypes in the pathophysiology of cardiac dysfunctions.     

Sartan-AT1 receptor interactions: in vitro evidence for insurmountable antagonism and inverse agonism

Sartans are non-peptide AT(1) receptor antagonists used to treat hypertension and related pathologies. Their effects on the G protein-dependent responses of angiotensin II (Ang II) were the same in vascular tissues and in isolated cell systems. All are competitive but, when pre-incubated, they act surmountably (only rightward shift of the Ang II concentration-response curve) or insurmountably (also decreasing the maximal response). Insurmountable behaviour reflects the formation of tight sartan-receptor complexes; it is often partial due to the co-existence of tight and loose complexes. Their ratio positively correlates with the dissociation half-life of the tight complexes and depends on the sartan: i.e. candesartan>olmesartan>telmisartan approximately equal EXP3174>valsartan>irbesartan>losartan. When AT(1) receptors display sufficient basal activity (in case of receptor over-expression, mutation and, especially, tissue stretching) sartans may also act as inverse agonists. This rather affects long-term, G protein-independent hypertrophic responses leading to cardiovascular remodelling.     

veratridine,Angiotensin Receptors and Aldosteronogenesis in Bovine Adrenal Glomerulosa Cells

Veratridine inhibited angiotensin binding to its receptors in bovine adrenal glomerulosa cells and vascular smooth muscle. Fifty percent inhibition of adrenal receptors required about 2x10^-5 M veratridine. Receptors from vascular tissue were less sensitive. Graphic analysis showed that inhibition resulted primarily from a reduction of receptor number. Angiotensin stimulation of phosphatidylinositol turnover and of aldosterone production was inhibited by veratridine. Analogues of veratridine varied in their receptor inhibitory potency, but these variations did not correlate with the potencies of analogues as hypotensive agents or inhibitors of aldosteronogenesis. Very low extracellular sodium concentrations inhibited the adrenal effects of angiotensin. Neither veratridine nor grayanotoxin, both of which open sodium channels in excitable tissues, had a detectable effect on sodium fluxes in adrenal cells. Inhibition of aldosteronogenesis by veratridine is more likely the result of receptor and post-receptor effects than an alteration of the sodium channel     

Angiotensin II inhibits the electrogenic Na/HCO3 cotransport of cat cardiac myocytes

The Na⁺/HCO₃⁻ cotransporter (NBC) plays an important role in intracellular pH (pH_i) regulation in the heart. In the myocardium co-exist the electrogenic (eNBC) and electroneutral (nNBC) isoforms of NBC. We have recently reported that angiotensin II (Ang II) stimulated total NBC activity during the recovery from intracellular acidosis through a reactive oxygen species (ROS) and ERK-dependent pathway. In the present work we focus our attention on eNBC. In order to study the activity of the eNBC in isolation, we induced a membrane potential depolarization by increasing extracellular K⁺ [K⁺]_o from 4.5 to 45 mM (K⁺ pulse). This experimental protocol enhanced eNBC driving force leading to intracellular alkalization (0.19 ± 0.008, n = 6; data expressed as an increase of pH_i units after 14 min of applying the K⁺ pulse). This alkalization was completely abrogated by the NBC blocker S0859 (− 0.004 ± 0.016*, n = 5; * indicates p < 0.05 vs control) but not by the Na⁺/H⁺ exchanger blocker HOE642 (0.185 ± 0.04, n = 4), indicating that we are exclusively measuring eNBC. The K⁺ pulse induced alkalization was canceled by 100 nM Ang II (− 0.008 ± 0.018*; n = 5). This inhibitory effect was prevented when the myocytes were incubated with losartan (AT₁ receptor blocker, 0.18 ± 0.02; n = 4) or SB202190 (p38 MAP kinase inhibitor, 0.25 ± 0.06; n = 5). Neither chelerythrine (PKC inhibitor, − 0.06 ± 0.04*; n = 4), nor U0126 (ERK inhibitor, − 0.07 ± 0.04*; n = 4) nor MPG (ROS scavenger, − 0.02 ± 0.05*; n = 8) affected the Ang II-induced inhibition of eNBC. The inhibitory action of Ang II on eNBC was corroborated with perforated patch-clamp experiments, since no impact of the current produced by eNBC on action potential repolarization was observed in the presence of Ang II. In conclusion, we propose that Ang II, binding to AT₁ receptors, exerts an inhibitory effect on eNBC activity in a p38 kinase-dependent manner.     

Dynamics and Patterning of 5-Hydroxytryptamine 2 Subtype Receptors in JC Polyomavirus Entry

The organization and dynamics of plasma membrane receptors are a critical link in virus-receptor interactions, which finetune signaling efficiency and determine cellular responses during infection. Characterizing the mechanisms responsible for the active rearrangement and clustering of receptors may aid in developing novel strategies for the therapeutic treatment of viruses. Virus-receptor interactions are poorly understood at the nanoscale, yet they present an attractive target for the design of drugs and for the illumination of viral infection and pathogenesis. This study utilizes super-resolution microscopy and related techniques, which surpass traditional microscopy resolution limitations, to provide both a spatial and temporal assessment of the interactions of human JC polyomavirus (JCPyV) with 5-hydroxytrypamine 2 receptors (5-HT₂Rs) subtypes during viral entry. JCPyV causes asymptomatic kidney infection in the majority of the population and can cause fatal brain disease, and progressive multifocal leukoencephalopathy (PML), in immunocompromised individuals. Using Fluorescence Photoactivation Localization Microscopy (FPALM), the colocalization of JCPyV with 5-HT₂ receptor subtypes (5-HT_2A, 5-HT_2B, and 5-HT_2C) during viral attachment and viral entry was analyzed. JCPyV was found to significantly enhance the clustering of 5-HT₂ receptors during entry. Cluster analysis of infected cells reveals changes in 5-HT₂ receptor cluster attributes, and radial distribution function (RDF) analyses suggest a significant increase in the aggregation of JCPyV particles colocalized with 5-HT₂ receptor clusters in JCPyV-infected samples. These findings provide novel insights into receptor patterning during viral entry and highlight improved technologies for the future development of therapies for JCPyV infection as well as therapies for diseases involving 5-HT₂ receptors.     

Evidence for a local angiotensin-generating system and dose-dependent inhibition of glucose-stimulated insulin release by angiotensin II in isolated pancreatic islets

Aims/hypothesis A local angiotensin-generating system has been found in the exocrine pancreas. This study aimed, primarily, to investigate the existence of a local angiotensin-generating system in the pancreatic islets and, secondly, to elucidate its role in regulating insulin secretion.Methods Real-time RT-PCR and western blot were used to investigate if angiotensin-generating components are present in the mouse pancreatic islets, which are subject to regulation by islet transplantation. The localisation of AT₁-receptors in islets was investigated by immunohistochemistry. Batch-type incubations of isolated islets were applied for studying the influence of angiotensin II on the glucose-stimulated insulin release, glucose oxidation and (pro)insulin, and total protein biosynthesis.Results Major components, namely angiotensinogen, ACE, AT ₁ - and AT ₂ -receptors, were expressed in endogenous islets. AT₁-receptors were localised to pancreatic beta cells. Exposure of the isolated islets to angiotensin II induced a dose-dependent inhibition of glucose-stimulated insulin release and inhibited (pro)insulin biosynthesis. This inhibitory action was fully preventable by pretreatment of the islets with losartan, an AT₁-receptor antagonist. We also investigated if the expression of these components was changed after islet transplantation. Notably, a markedly increased expression of mRNA for the AT ₁ -receptor was observed in islets retrieved from 4-week-old syngeneic islet transplants, a finding that was confirmed at the protein level.Conclusion/interpretation These data indicate the existence of an islet angiotensin-generating system of potential importance in the physiological regulation of glucose-induced insulin secretion, thus diabetes mellitus. The increased expression of the AT₁-receptor in islet transplants could have relevance to islet-graft function.Abbreviations Ang II Angiotensin II - AT₁ angiotensin II receptor type 1 - AT₂ angiotensin II receptor type 2 - Ao angiotensinogen - RAS Renin-angiotensin system - KRBB Krebs-Ringer bicarbonate buffer