Calcium response to adenosine and ATP in rabbit afferent arterioles

The effects of purine compounds in the renal vasculature are almost exclusively restricted to pre-glomerular vessels. Although their physiological role as extracellular messengers is not clear, there are extensive data indicating the importance of adenosine and ATP in the regulation of renal haemodynamics. This study was undertaken to characterize the calcium response of rabbit afferent arteriole to adenosine, ATP and other nucleotides. Experiments were performed in isolated afferent arterioles, microdissected from rabbit kidneys and loaded with fura-2. Intracellular calcium concentration ([Ca²⁺]_i) was measured by video in proximal and distal parts of the afferent arteriole. Application of 100 μM adenosine or ATP increased [Ca²⁺]_i in both arteriolar regions. In all cases the response had two well distinguishable phases: a quick peak increase and a plateau phase that equilibrated at a [Ca²⁺]_i significantly higher than the basal level. UTP (100 μM ) had no effect on the arteriole. Removal of extracellular calcium (2.5 m M EGTA) abolished only the plateau phase in response to adenosine, without significantly changing the peak increase. In contrast, the response to ATP was completely abolished in both arteriolar regions, where [Ca²⁺]_i decreased upon application of the agonist and rapidly increased after restoration of calcium concentration to plasma level. We conclude that P1 and P2X receptors are present along the rabbit afferent arteriole and mediate calcium mobilization, with the same distribution in the proximal and distal segments.     

Intracellular signalling pathways in the vasoconstrictor response of mouse afferent arterioles to adenosine

Aims: Adenosine causes vasoconstriction of afferent arterioles of the mouse kidney through activation of adenosine A₁ receptors and Gi‐mediated stimulation of phospholipase C. In the present study, we further explored the signalling pathways by which adenosine causes arteriolar vasoconstriction. Methods and results: Adenosine (10^?7 m ) significantly increased the intracellular calcium concentration in mouse isolated afferent arterioles measured by fura‐2 fluorescence. Pre‐treatment with thapsigargin (2 μm ) blocked the vasoconstrictor action of adenosine (10^?7 m ) indicating that release of calcium from the sarcoplasmic reticulum (SR), stimulated presumably by IP₃, is involved in the adenosine contraction mechanism of the afferent arteriole. In agreement with this notion is the observation that 2 aminoethoxydiphenyl borate (100 μm ) blocked the adenosine‐induced constriction whereas the protein kinase C inhibitor calphostin C had no effect. The calcium‐activated chloride channel inhibitor IAA‐94 (30 μm ) inhibited the adenosine‐mediated constriction. Patch clamp experiments showed that adenosine treatment induced a depolarizing current in preglomerular smooth muscle cells which was abolished by IAA‐94. Furthermore, the vasoconstriction caused by adenosine was significantly inhibited by 5 μm nifedipine (control 8.3 ± 0.2 μm, ado 3.6 ± 0.6 μm, ado + nifedipine 6.8 ± 0.2 μm) suggesting involvement of voltage‐dependent calcium channels. Conclusion: We conclude that adenosine mediates vasoconstriction of afferent arterioles through an increase in intracellular calcium concentration resulting from release of calcium from the SR followed by activation of Ca²⁺‐activated chloride channels leading to depolarization and influx of calcium through voltage‐dependent calcium channels.     

Age related alterations in the response of the pial arterioles to adenosine in the rat.

To evaluate the effects of aging on vasoreactivity of pial arterioles to adenosine and barium chloride, an hydraulically intact cranial window preparation was developed in the rat. The microvasculature of anesthetized 3- and 24-month-old Fischer-344 rats was studied during superfusion with artificial cerebrospinal fluid with and without test agents and results determined by videomicroscopy techniques. In both cohorts, the response of pial arterioles to adenosine was both dose and vessel size dependent: arteriolar dilation increased with increasing concentrations of adenosine and at any given concentration the percent increase in diameter was greater in the smaller vessels. During adenosine superfusion the absolute changes and percent increase in vessel caliber were greater in the young rats. Arteriolar vasoconstriction due to barium chloride was vessel size dependent but there were no significant differences in response between young and aged rats. The results indicated an attenuated cerebrovascular response in aged rats to adenosine, but not to barium chloride. This may be due to a difference in the mode of action in these two compounds. Venules did not respond to adenosine at any concentration.     

Calcium signaling in ocular arterioles

Local control of blood flow to the photoreceptors and associated neurons in the retina is largely achieved through changes in tone within the choroidal and retinal arterioles. This is primarily achieved through changes in [Ca2+] within the smooth muscle of these vessels, which regulates cell contraction and vascular constriction. Here we review some aspects of the cell physiology involved in these Ca2+-signaling processes, with particular emphasis on the molecular mechanisms involved. Ca2+-influx across the plasma membrane can occur via a variety of Ca2+-channels, including voltage-operated, store-operated, and receptor-operated channels. Ca2+ may also be released from intracellular stores via RyR-, or IP3R-gated channels in the SR membrane. Using high-speed confocal Ca2+-imaging, we have also demonstrated that the resulting signals are far from homogeneous, with spontaneous activity in retinal arterioles being characterized by both localized Ca2+-sparks and more global Ca2+-waves and oscillations. These signals may be specifically and differentially targeted, for example, to Ca2+-sensitive ion channels (stimulus-excitation coupling), or pathways regulating contraction (stimulus-contraction coupling). Exploring the role of changes in such targeting in disease states will provide exciting opportunities for future research.     

Angiotensin II enhances the afferent arteriolar response to adenosine through increases in cytosolic calcium

Aims: Angiotensin II (Ang II) is a strong renal vasoconstrictor and modulates the tubuloglomerular feedback (TGF). We hypothesized that Ang II at low concentrations enhances the vasoconstrictor effect of adenosine (Ado), the mediator of TGF. Methods: Afferent arterioles of mice were isolated and perfused, and both isotonic contractions and cytosolic calcium transients were measured. Results: Bolus application of Ang II (10^?12 and 10^?10 m ) induced negligible vasoconstrictions, while Ang II at 10^?8 m reduced diameters by 35%. Ang II at 10^?12, 10^?10 and 10^?8 m clearly enhanced the arteriolar response to cumulative applications of Ado (10^?11 to 10^?4 m ). Ado application increased the cytosolic calcium concentrations in the vascular smooth muscle, which were higher at 10^?5 m than at 10^?8 m . Ang II (10^?11 to 10^?6 m ) also induced concentration‐dependent calcium transients, which were attenuated by AT₁ receptor inhibition. Simultaneously applied Ang II (10^?10 m ) additively enhanced the calcium transients induced by 10^?8 and 10^?5 m Ado. The transients were partly inhibited by AT₁ or A₁ receptor antagonists, but not significantly by A₂ receptor antagonists. Conclusion: A low dose of Ang II enhances Ado‐induced constrictions, partly via AT₁ receptor‐mediated calcium increase. Ado increases intracellular calcium by acting on A₁ but not A₂ receptors. The potentiating effect of Ang II on Ado‐induced arteriolar vasoconstrictions may involve calcium sensitization of the contractile machinery, as Ang II only additively increased cytosolic calcium concentrations, while its effect on the arteriolar constriction was more than additive. The potentiating effect of Ang II might contribute to the resetting of TGF.     

Effects of stimulation and inhibition of protein kinase C on the cytosolic calcium concentration in rabbit afferent arterioles

The effect of the protein kinase C (PKC) inhibitor chelerytrine (Ch) and the PKC activator 12–0-tetradecanoyl-phorbol-13-acetate (TPA) on the cytosolic calcium concentration ([ Ca²⁺]_i) in isolated intact rabbit afferent arterioles was investigated. [Ca²⁺]_i was measured in the proximal and distal parts of the arteriole. Administration of 1 μM Ch gave rise to a peak followed by an elevated level of [Ca²⁺]_i in both these parts. Neither the peak nor the elevated level of [Ca²⁺]_i was significantly reduced by 1 μM nifedipine. The relative peak increase in [Ca²⁺]_i in response to 1 μM noradrenaline (NA) or to 10 nM angiotensin II (AII) was significantly blunted in both parts after preincubation with 1 μM Ch. Depolarization with 25 mM K⁺ increased [Ca²⁺]_i in both parts. Preincubation with Ch did not affect the increase in [Ca²⁺]_i induced by 25 mM K⁺. TPA (10 and 100 nM ) did not significantly affect the basal [Ca²⁺]_i in the afferent arteriole. The [Ca²⁺]_i response to NA or 25 mM K⁺ was not affected by TPA. We conclude that blockade of PKC increases [Ca²⁺]_i in afferent arteriolar smooth muscle by a mechanism independent of L-type voltage-sensitive calcium channels. Inhibition of PKC blunts the relative increase in [Ca²⁺]_i in response to AII and, to a lesser extent, that induced by NA. We conclude that PKC might be important in modulating the calcium changes that occur in response to these vasoconstrictors.     

Structure of renal afferent arterioles in the pathogenesis of hypertension

Renal vascular resistance is increased in essential hypertension, as in genetic models of hypertension. Here we review the evidence that this is at least in part due to structural changes in the afferent arterioles. Rat studies show that the renal afferent arteriole is structurally narrowed in young and adult spontaneously hypertensive rats (SHR). Furthermore, in the second generation of crossbred SHRs/normotensive rats (SHR/WKY F(2)-hybrids), a narrowed afferent arteriole lumen diameter at 7 weeks is a predictor of later development of high blood pressure. The reduced lumen diameter of resistance vessels is accompanied by a decrease in media cross-sectional area in SHR and could therefore be due to inhibited growth. Evidence from a primate model of hypertension has shown a negative correlation between left ventricular hypertrophy and afferent arteriole diameter, but apparently no relation to blood pressure. In SHR, the antihypertensive effect of angiotensin converting enzyme (ACE) inhibitors is mediated through renal vascular mechanisms, while ACE inhibitors (like AT(1) antagonists) have a more persistent effect on blood pressure after treatment withdrawal compared with other antihypertensive drugs. Taken together, the evidence suggests that structural narrowing of the renal afferent arteriole could be an important link in the pathogenesis of primary hypertension, at least in the SHR.     

Angiotensin II induces a tachyphylactic calcium response in the rabbit afferent arteriole

The influence of repeated administration of angiotensin II (AII) on smooth muscle calcium concentration ([ Ca²⁺]_i) was studied in isolated rabbit renal afferent arterioles loaded with the calcium-sensitive fluorescent probe Fura–2. [Ca²⁺]_i was evaluated in the proximal and distal parts of the afferent arterioles. AII (10^?8m ) increased the [Ca²⁺]_i in both these regions. A second administration of AII, however, did not elicit any response in [Ca²⁺]_i. The response to noradrenaline administration at the end of the experiment was not affected, i.e. there was no fading or cross-desensitization. Since this desensitization was specific for AII, it was of the tachyphylaxis type. Increasing doses of AII (10^?11–10^?8 m ) did not reverse the tachyphylaxis. However, in the proximal part, pretreatment with the voltage-sensitive calcium channel blocker nifedipine (10^?6m ) blunted the tachyphylactic effect of a second administration of AII. When l -arginine (l -Arg) was administered to the bath solution, thus activating the NO system, the development of tachyphylaxis was suppressed in the proximal region. Pretreatment with the protein kinase C (PKC) inhibitor chelerythrine (10^-6m ) did not affect the tachyphylaxis. We conclude that the calcium response to AII in the isolated rabbit afferent arteriole shows tachyphylaxis. This tachyphylaxis cannot be reversed by applying increasing doses of AII (10^?11–10^?8m ). PKC does not seem to be involved in the tachyphylactic phenomenon in this preparation. It was also found that nifedipine and NO reduced the tachyphylaxis.     

Perfusate composition influences nitric oxide homeostasis in rat juxtamedullary afferent arterioles

AIM: Vascular diameters in isolated juxtamedullary nephron preparations perfused with cell-free solutions differ from those perfused with blood. In the present study, the effects of the albumin content of the perfusate on the afferent arteriolar diameter and endothelial nitric oxide were investigated in the isolated juxtamedullary nephron preparation perfused with Krebs-Ringer-bicarbonate buffer containing albumin in different concentrations. METHODS: The endothelium was loaded with DAF-FM DA, a nitric oxide-sensitive fluoroprobe. Perfusion was maintained either with 4% (control group), 10 or 20% albumin in the perfusate or with L-NAME (10-4 m) added to the perfusate. Fluorescent images were obtained and stored for evaluation of DAF-FM fluorescence and vascular diameters (in mid-afferent arterioles) immediately before perfusate change and every 15 min thereafter, for a 2-h period. RESULTS: Increasing the albumin concentration resulted in a decrease in fluorescence. The most rapid decline of fluorescence was obtained following L-NAME administration (relative fluorescence after 2 h: 4% albumin 92.4 +/- 5.3%; 10% albumin 79.5 +/- 4.2%; 20% albumin 66.2 +/- 2.6%; L-NAME 55.4 +/- 3.0%; mean +/- SD, n = 5). A dose-dependent constriction of the afferent arterioles was observed (normalized diameter: 4% albumin 99.8 +/- 3.0%; 10% albumin 80.3 +/- 3.3%; 20% albumin 74.3 +/- 3.2%; L-NAME 70.6 +/- 3.5%). CONCLUSION: We propose that albumin interferes with arteriolar nitric oxide homeostasis, probably by scavenging nitric oxide intra-luminally. In this respect, albumin acts similarly to red blood cells in the circulation. The magnitude of the scavenging determines the effectiveness of autoregulation in the perfused preglomerular vessels. The scavenging properties of the perfusing fluid are important in setting operating levels of endothelial nitric oxide.     

Focal arteriolar insudation. A response of arterioles to chronic nonspecific irritation.

The subcutaneous insertion of sterile, inert plastic pellets over the cremaster muscles of rats induces characteristic focal lesions of the arterioles at a distance from the pellets. These lesions appear with a delay of about 6 hours; by light microscopy they are characterized by a focal dilatation accompanied by endothelial damage and increased permeability. They are more severe if the pellets are loaded with histamine and are inhibited if the pellets are loaded with serotonin. Electron microscopy shows interendothelial gaps; the media is massively infiltrated with blood components and fibrin. The medial smooth muscle cells are stretched and at times necrotic; inflammatory cells are scarce. On the basis of these features the lesion was named focal arteriolar insudation (FAI). Although its pathogenesis is not yet clear, the data at hand suggest that it is caused by endogenous mediators affecting the smooth muscle cells and/or the endothelium. FAI appears to be a specific arteriolar response to chronic nonspecific irritation.     

Advanced glycosylation end-products and NO-dependent vasodilation in renal afferent arterioles from diabetic rats

Systemic pressor responses to acetylcholine (ACh) are reduced in DM, an effect thought to be related to quenching of nitric oxide (NO) by advanced glycosylation end-products (AGE). We studied the effects of AGE in juxtamedullary (JM) afferent arterioles (AA) from rats with 40-50 days diabetes mellitus (DM) induced via streptozotocin. JM AA were perfused in vitro with solutions containing fresh RBCs suspended in either 6% bovine albumin or 6% AGE-albumin in euglycaemic Krebs-Ringer. Autoregulatory responses were evident in the DM vessels: AA constricted 31 +/- 2% (n=9) when perfusion pressure (PP) was raised from 60 to 140 mmHg. ACh (10 microM) caused a 43 +/- 15% dilation and Ca2+-channel blockade elicited a 95 +/- 14% dilation at 100 mmHg PP, indicating substantial basal vascular tone in DM AA. L-NAME (0.1 mM) constricted DM AA by 21 +/- 2% (n=9) at 100 mmHg PP, indicating significant basal NO production in DM vessels. Segments of renal resistance arteries from DM rats perfused in vitro responded to muscarinic stimulation and elevated glucose levels with significant increments in NO production, as measured with an NO-sensitive electrode. This observation shows that the renal endothelial NO system is intact in DM. While AGE in the perfusate dilated control AA, they had no effect on DM AA at all PP levels, although they blunted ACh-induced dilation. Hence, although AGE do appear to have vasoactive properties in the absence of hyperglycaemia, the results of this study are inconsistent with substantial NO quenching by AGE.     

Effect of volume depletion on the afferent arterioles in the avian kidney

Summary Nine white leghorn chickens were injected i.m. with furosemide (10 to 60 mg/kg body weight) twice daily for 18 days. The birds were then anesthetized with a combination of equithesin and diazepam and the kidneys perfused via the heart. Kidney tissue was sectioned serially and the granular epithelioid cells were counted in the juxtaglomerular apparatuses of the furosemide treated birds and in 3 normal chickens. Hyperplasia and hypergranulation of the epithelioid cells was found to occur in the juxtaglomerular apparatuses of both mammalian and reptilian type nephrons (with and without Henles loop) in the furosemide treated group. This finding was interpreted as an effect of hypovolaemia on the juxtaglomerular apparatuses. Furosemide caused an immediate stop in weight gain, an increase in the erythrocyte volume fraction and a sudden drop in blood pressure. The blood pressure later rose to subnormal levels. The heart rate was not altered. Plasma sodium and chloride fell significantly one day after furosemide administration and remained low throughout the experiment. Potassium fell during the second part of the experimental period. Captopril was injected after 18 days of furosemide treatment and lowered the blood pressure significantly. This was interpreted as indirect evidence for the presence of renin in the granular epithelioid cells and indicates the importance of the renin angiotensin system in maintaining the blood pressure in hypovolaemic conditions.Dedicated to Prof. Dr. Dr. h.c. A. Bohle on his 65th Birthday     

Quantitative ultrastructural study of afferent and efferent arterioles in IgA glomerulonephritis and benign nephrosclerosis

Arteriolosclerosis frequently occurs in IgA nephritis (IgAN), and it is the hallmark of benign nephrosclerosis (BNS). The quantitative ultrastructure of juxtaglomerular arterioles is not known in these disorders. We examined afferent and efferent arterioles in renal biopsies from 25 adult patients with IgAN (hypertension at biopsy: 14 patients) and 9 patients with BNS. Six agematched living renal transplant donors acted as controls. A systematic independent sample of profiles was obtained in thin sections taken at predetermined levels. The thickness of the media (myomedial cells plus the matrix) and the thickness of the medial matrix were estimated stereologically. From these estimates, the matrix/myomedia ratio was calculated. In IgAN with normotension or hypertension, the afferent media and its compartments did not exhibit significant thickening compared with the controls, whereas in BNS the afferent media and its layers were markedly and significantly thickened. The efferent media in IgAN and BNS displayed mild and significant thickening, with significant thickening of the matrix in BNS and IgAN with normotension. The matrix/myomedia ratio was not altered significantly in any group. The results indicate that the afferent arterioles are not the main sites of IgAN-related arteriolosclerosis, that arteriolosclerosis in IgAN and arteriolosclerosis in BNS are different lesions, and that increased efferent arteriolar thickness, demonstrated here for the first time in IgAN and BNS, might be a manifestation of angiotensin II-mediated autoregulatory efferent vasoconstriction exerted to maintain the glomerular filtration pressure.