Different action of angiotensin II and noradrenaline on cytosolic calcium concentration in isolated and perfused afferent arterioles

Compensatory, net fluid transfer across the capillaries was studied in the arm of man with plethysmographic technique during experimental hypovolaemia induced by lower body negative pressure (LBNP). Thirty, 60, and 110 cmH₂O LBNP evoked rapid transfer of fluid from tissue to blood at average rates of 0.053, 0.088 and 0.147 ml min^-1 100 ml^-1 soft tissue, i.e. graded responses typical for a true homeostatic regulation. Other experiments demonstrated a net fluid absorption not only from the arm but also from a wide range of skeletal muscle and skin regions in the body during experimental hypovolaemia, i.e. the more or less generalized response required if the absorption process is to contribute importantly to plasma volume regulation. In a third series of experiments it was shown that gradually applied LBNP was a much less efficient stimulus for fluid gain into the circulation than rapidly instituted LBNP, tentatively explaining the fairly slow plasma volume refill in man in previous literature after experimental, true and necessarily slow blood loss. Taken together, the findings described warrant the conclusion that the described process of fluid gain into the circulation may be a very important component in the overall homeostatic circulatory regulation in states of hypovolaemia. The data in fact suggest that the process might be capable of increasing plasma volume by as much as 600 ml within only 10 min, suggesting that such plasma volume control might be much more potent than previously believed.     

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.     

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.     

Endogenous nitric oxide and epoxyeicosatrienoic acids modulate angiotensin II‐induced constriction in the rabbit afferent arteriole

Nitric oxide (NO) and epoxyeicosatrienoic acids (EETs), cytochrome P450 epoxygenase metabolites of arachidonic acid, are released by the vascular endothelium and play important roles in the control of glomerular haemodynamics. We examined whether endogenous NO or EETs modulate angiotensin II‐ (AngII) induced constriction in isolated microperfused afferent arteriole (Af‐Art) of the rabbit kidney.     

Intrarenal administration of angiotensin II does not moderate afferent renal nerve mediated cardiovascular reflexes in the anaesthetized rabbit

Stimulation of the afferent renal nerves in the anaesthetized rabbit by acute reduction in renal perfusion pressure results in a neurally mediated, reflex increase in hindlimb vascular resistance. To determine whether exogenous angiotensin II moderates the reflex, the kidneys of anaesthetized rabbits were vascularly isolated and renal blood flow was occluded acutely, following intrarenal administration of vehicle (0.9% saline) or angiotensin II (0.5 ng), and the hindlimb vascular response was measured. Occlusion of renal blood flow resulted in similar, significant increases in femoral perfusion pressure of 39.7±7.1 mmHg after vehicle and 21.3±8.9 mmHg (P<0.05, n=6) after angiotensin II. The viability of the preparation following repeated episodes of renal blood flow occlusion was tested by a series of three rapid (2–3 min delay) occlusions and three delayed (30 min delay) occlusions. Femoral perfusion pressure rose by 43.1±10.7 mmHg (rapid, P<0.05, n=11) and 64.4±12.3 mmHg (delayed, P<0.05, n = 5) on the first occasion. On the second occasion, the rapid occlusion did not result in a significant increase in femoral perfusion pressure (29.1±8.1 mmHg), but the delayed group did (54.6±22.4 mmHg, P<0.05). On the third occasion, neither group showed a significant change (20.9±16.3 and 30.8±13.5 mmHg). These data suggest that exogenous angiotensin II does not moderate the afferent renal nerve reflex. The decline in hindlimb response following rapid serial occlusion may be attributed to a diminution of an intermediary substance(s) at the nerve receptor site.     

The cytosolic chloride concentration in macula densa and cortical thick ascending limb cells

It is believed that chloride transport through the macula densa (MD) cells is a factor involved in the tubuloglomerular feedback (TGF) mechanism and in MD-mediated renin release. In this study isolated and perfused rabbit kidney cortical thick ascending limb (cTAL) segments containing MD plaques and attached glomeruli were loaded with chloride (CL-sensitive) 6 methoxy-l-fluorophore (sulphanate-propyl) quinolinium (SPQ). MD and cTAL intracellular chloride concentration ([Cl^-]_i) was determined by using image-intensified video microscopy and digital image-processing for measuring the intensity of the emitted SPQfluorescence. With 150 mM NaCl in lumen and bath the [Cl^-], in MD and cTAL cells was 58.8 ± 7.2 mm (n= 20) and 68.7 ± 9.8 mm (n= 14), respectively. When the presumed luminal Na⁺-2Cl^--K⁺ co-transporter was blocked by adding 10^--⁴m furosemide, the [Cl^-]_i was reduced in both, MD and cTAL cells from 55.5 ± 11.9 to 28.6 ± 10.0mm (n= 10) and from 43.8± 2.6 to 13.1± 4.5mm (n= 5), respectively. A reduction in luminal NaCl from 150 to 30 mm also decreased both, MD and cTAL [Cl^-]_i from 69.4± 9.1 to 36.5± 5.1 mm (n= 9) and from 82.9 ±14.5 to 49.4± 8.0 mm (n= 8), respectively. Basolateral addition of the Cl^--channel blocker NPPB increased MD [Cl^-], from 31.1± 2.0 to 100.7± 17.0 mm (n= 5) and cTAL [Cl^-]_i from 44.4 ± 12.9 to 89.7 ± 11.7 mm (n= 5). These results show the existence of a luminal Na⁺-2C1^-K⁺ cotransporter and a basolateral Cl^- conductance, and that chloride transport is directed from the luminal to the basolateral side during symmetrical conditions. They also indicate that sensing of luminal NaCl by the MD cells could be of importance in the TGF mechanism and MD mediated renin release.     

Expression of angiotensin II and its receptors in the normal and hypoxic amoeboid microglial cells and murine BV-2 cells

Involvement of the local angiotensin receptor system in the central nervous system is well documented, yet its cellular localization and role in the glial cells have remained elusive. This study reports expression of angiotensin II and its receptors namely, angiotensin II receptor type 1 (AT₁) and angiotensin II receptor type 2 (AT₂) in the amoeboid microglial cells in the neonatal rat brain. In rats subjected to hypoxia, the amount of angiotensin II released in the corpus callosal tissue was reduced as revealed by enzyme immunoassay. Expression of AT₁ mRNA and protein was down-regulated after hypoxic exposure, but AT₂ was up-regulated. In BV-2 cells exposed to hypoxia for 4 h, expression of AT₁ mRNA was reduced but AT₂ was increased. These changes were further intensified respectively in LPS-stimulated microglia. Edaravone enhanced AT₁ expression but suppressed AT₂ expression significantly in lipopolysaccharide-stimulated cells. Neutralization of AT₂ with its antiserum significantly increased mRNA expression of tumor necrosis factor-α and interleukin-1β but decreased that of transforming growth factor-beta1. In conclusion, the present results suggest that AT₁ may be linked to regulation of vasodilation for increase of blood flow in hypoxic conditions, while up-regulated expression of AT₂ may reduce inflammatory responses through suppression of proinflammatory cytokines and elimination of free radicals.     

Strontium promotes calcium oscillations in mouse meiotic oocytes and early embryos through InsP3 receptors, and requires activation of phospholipase and the synergistic action of InsP3

BACKGROUND: Sr2+ is the most efficient agent for mouse oocyte activation and functions by inducing Ca2+ oscillations. However, its specific mechanism of action remains unknown. Here we investigated the specificity and possible mechanism of Sr2+-induced Ca2+ oscillations in mouse oocytes and early embryos. METHODS: Ca2+ oscillations in oocytes and embryos were measured by ratiometric fluorescence imaging using fura-2AM. The role of phospholipase C (PLC) and inositol trisphosphate (InsP3) receptors in Sr2+-induced Ca2+ oscillations was examined by selective inhibitors. RESULTS: Sr2+ can induce Ca2+ oscillations in both immature and mature oocytes, and in early embryos. A cell cycle stage-dependent phenomenon to Sr2+ stimulation was observed in 1-cell embryos. By using a low molecular weight heparin to antagonize the function of InsP3 receptors, we were able to show that InsP3 receptors are essential for Sr2+-induced Ca2+ oscillations. Treating metaphase II (MII) oocytes with the PLC inhibitor, U73122, abolished Sr2+-induced increases in Ca2+. This inhibitory effect of U73122 could be rescued by microinjection of InsP3, indicating that Sr2+-induced Ca2+ oscillations require the synergistic action of InsP3. CONCLUSIONS: Sr2+-induced calcium oscillations in mouse oocytes and early embryos are mediated through InsP3 receptors, and require PLC activation and the synergistic action of InsP3.