F. Markos

Aldosterone Rapidly Activates Na+/H+ Exchange in M-1 Cortical Collecting Duct Cells via a PKC-MAPK Pathway

Background: In this study, the mechanism of the rapid non-genomic effect of aldosterone on Na+/H+ exchanger (NHE)-mediated intracellular pH (pHi) recovery from an acid load in murine M-1 cortical collecting duct cells was assessed. Methods: Spectrofluorescence microscopy and Western blot analysis was carried out and NH4Cl was used to induce the acid load. Results: Aldosterone (10 nM) induced a rapid (<5 min) concentration-dependent increase in pHi recovery in M-1 cells, an effect mimicked by its precursor deoxycorticosterone (1 nM). This response was unaffected by the mineralocorticoid receptor (MR) antagonist spironolactone (10 µM) but was significantly reduced by the NHE antagonists 5′-(N-ethyl- N-isopropyl)amiloride (EIPA) (20 µM) and cariporide (1 µM). The PKC inhibitor chelerythrine chloride (1 µM) significantly attenuated the aldosterone-induced increase in NHE1 activity. HBDDE (80 µM), a PKCα inhibitor, inhibited the rapid aldosterone effect whereas rottlerin (15 µM), a PKCδ antagonist, did not. The glucocorticoid receptor agonists hydrocortisone (1 µM) and dexamethasone (100 nM) decreased NHE activity, whereas the synthetic mineralocorticoid fludrocortisone (1 nM) had no significant effect. MAPK inhibition using PD98059 (25 µM) significantly attenuated the rapid aldosterone effect; Western blot analysis showed that aldosterone activation of ERK 1/2 was unaffected by pretreatment with spironolactone but was inhibited following chelerythrine chloride. Conclusion: Aldosterone causes a rapid non-genomic increase in NHE1 activity in M-1 cells via a PKCα /MAPK pathway independent of the classical MR.

Characteristics of arterial wall shear stress which cause endothelium-dependent vasodilatation in the anaesthetized dog

1 The effects of changes in the mean and amplitude of arterial wall shear stress on endothelium‐dependent arterial dilatation of the iliac artery of the anaesthetized dog were examined. 2 Changes in the mean and amplitude of blood flow and wall shear stress were brought about by varying local peripheral resistance and stroke volume using a distal infusion of acetylcholine and the stimulation of the left ansa subclavia. Changes in the diameter of a segment of the iliac artery with the endothelium intact, relative to a segment with no endothelium, were used as an index of the release of nitric oxide. 3 The increase in mean blood flow was from 84 ± 12 to 527 ± 53 ml min−1 and in amplitude was from 365 ± 18 to 695 ± 38 ml min−1 (means ±s.e.m.). The increase in mean wall shear stress was from 1.78 ± 0.30 to 7.66 ± 1.01 N m−2 and in amplitude was from 7.37 ± 0.46 to 13.9 ± 2.00 N m−2 (means ±s.e.m.). 4 Increases in mean shear stress caused an increase in the diameter only of the section of artery with endothelium; the slope of the relationship was 0.064 ± 0.006 mm N−1 m2 (mean ±s.e.m., P < 0.001); changes in the amplitude of shear stress did not cause an increase in diameter. Changes in both the mean and amplitude of shear stress had no significant effect on the diameter of the section of artery with no endothelium. 5 These findings coupled with the known anti‐atheroma effects of nitric oxide and the effect of shear stress on cell adhesion and platelet aggregation offer a possible explanation for the disposition of atheroma in those parts of the arterial system which have low mean and high amplitude of wall shear stress.

Nitric oxide facilitates vagal control of heart rate via actions in the cardiac parasympathetic ganglia of the anaesthetised dog

The effects of neuronal inhibition of nitric oxide (NO) production on the bradycardia resulting from stimulation of preganglionic and postganglionic parasympathetic fibres were investigated in an anaesthetised dog preparation following transection of the cervical vagi and in the presence of a β‐adrenoreceptor antagonist. Injection of 1‐(2‐trifluoromethylphenyl) imidazole (TRIM), an inhibitor of neuronally released NO, into the sinus node artery reduced the bradycardia evoked by right cervical vagal stimulation. In contrast, when the response to preganglionic stimulation had been abolished by hexamethonium (10 mg kg−1), the bradycardia following stimulation of postganglionic parasympathetic fibres on the atrial epicardium was unaffected by TRIM. First, these results confirm the facilitatory actions of neuronally released NO on vagal heart responses in the dog. Second, they indicate that this modulatory and facilitatory role of NO is likely to be exerted at vagal preganglionic‐postganglionic synaptic mechanisms in the cardiac parasympathetic ganglia and not at the postganglionic‐sinoatrial node synapse.

What is the mechanism of flow-mediated arterial dilatation

The present review attempts to explain the controversies concerning the mechanism of shear stress‐mediated arterial dilatation, commonly called flow‐mediated arterial dilatation (FMD). Flow‐mediated dilatation occurs in an artery when the blood flow to the organ supplied by the artery is increased. There are two hypotheses regarding the stimulus for FMD: (i) a wave of endothelial and smooth muscle hyperpolarization, conducted in a retrograde fashion from the vasodilated peripheral vascular bed towards the relevant conduit artery; and (ii) an increase in shear stress sensed by the endothelial cells. The latter hypothesis is associated with two further postulates concerning the method of mechanotransduction of the shear stress stimulus: (i) direct transmission from endothelial cell cytoskeleton to the vascular smooth muscle to induce dilatation; and (ii) indirect transmission to the endothelial cell cytoskeleton via the glycocalyx. The virtues and inconsistencies of these hypotheses are discussed. The first hypothesis is excluded because a vasodilated peripheral vascular bed does not cause dilation of the upstream conduit artery if an increase in flow within the conduit artery is prevented and because FMD is completely blocked by inhibition of nitric oxide synthase (NOS). It is probable that the stimulus is an increase in shear stress between the blood and the adjacent layer of the arterial wall, the glycocalyx. Ultimately, a change in the endothelial cell cytoskeleton is the likely event that leads to activation of NOS and this activation does not occur without a functioning glycocalyx.

Inhibition of Neuronal Nitric Oxide Reduces Heart Rate Variability in the Anaesthetised Dog

In the vagally intact anaesthetised dog, we have investigated the role of nitric oxide (NO) on a normal sinus arrhythmia using an inhibitor of neuronally released NO, 1‐(2‐trifluoromethylphenyl) imidazole (TRIM). The mean and S.D. of the R‐R interval was used to describe mean heart rate and heart rate variability, respectively. TRIM (0.8 mg I.C.) injected into the sinus node artery increased the mean heart rate slightly but reduced heart rate variability 3‐fold from a control of 790 ± 124 ms (mean ± S.D.; n = 5) to 666 ± 36 ms (P < 0.01 Students paired t test, n = 5). These results suggest that neuronally released NO may have a vagal facilitatory role in the maintenance of sinus arrhythmia in the normal heart.

The effect of tezosentan, a non-selective endothelin receptor antagonist, on shear stress-induced changes in arterial diameter of the anaesthetized dog.

The effects of changes in the mean (Sm) and pulsatile (Sp) components of arterial wall shear stress on arterial dilatation of the iliac artery of the anaesthetized dog were examined in the absence and presence of the endothelin receptor antagonist tezosentan (10 mg kg−1 I.V.; Ro 61‐0612; [5‐isopropyl‐pyridine‐2‐sulphonic acid 6‐(2‐hydroxy‐ethoxy)‐5‐(2‐methoxy‐phenoxy)‐2‐(2‐1H‐tetrazol‐5‐yl‐pyridin‐4‐yl)‐pyrimidin‐4‐ylamide]). Changes in shear stress were brought about by varying local peripheral resistance and stroke volume using a distal infusion of acetylcholine and stimulation of the left ansa subclavia. An increase in Sm from 1.81 ± 0.3 to 7.29 ± 0.7 N m−2 (means ± S.E.M.) before tezosentan caused an endothelium‐dependent arterial dilatation which was unaffected by administration of tezosentan for a similar increase in Sm from 1.34 ± 0.6 to 5.76 ± 1.4 N m−2 (means ± S.E.M.). In contrast, increasing the Sp from 7.1 ± 0.8 to a maximum of 11.5 ± 1.1 N m−2 (means ± S.E.M.) before tezosentan reduced arterial diameter significantly. Importantly, after administration of tezosentan subsequent increases in Sp caused arterial dilatation for the same increase in Sp achieved prior to tezosentan, increasing from a baseline of 4.23 ± 0.4 to a maximum of 9.03 ± 0.9 N m−2 (means ± S.E.M.; P < 0.001). In conclusion, the results of this study provide the first in vivo evidence that pulsatile shear stress is a stimulus for the release of endothelin from the vascular endothelium.

Pharmacokinetics and bio-distribution of novel super paramagnetic iron oxide nanoparticles (SPIONs) in the anaesthetized pig.

Manufactured nanomaterials have a variety of medical applications, including diagnosis and targeted treatment of cancer. A series of experiments were conducted to determine the pharmacokinetic, biodistribution and biocompatibility of two novel magnetic nanoparticles (MNPs) in the anaesthetized pig. Dimercaptosuccinic acid (DMSA) coated superparamagnetic iron oxide nanoparticles (MF66‐labelled 12 nm, core nominal diameter and OD15 15 nm); at 0.5, or 2.0 mg/kg) were injected intravenously. Particles induced a dose‐dependent decrease in blood pressure following administration which recovered to control levels several minutes after injection. Blood samples were collected for a 5‐h period and stored for determination of particle concentration using particle electron paramagnetic resonance (pEPR). Organs were harvested post‐mortem for magnetic resonance imaging (MRI at 1.5 T field strength) and histology. OD15 (2.0 mg/kg) MNP had a plasma half‐life of approximately 15 min. Both doses of the MF66 (0.5 and 2.0 mg/kg) MNP were below detection limits. MNP accumulation was observed primarily in the liver and spleen with MRI scans which was confirmed by histology. MRI also showed that both MNPs were present in the lungs. The results show that further modifications may be required to improve the biocompatibility of these particles for use as diagnostic and therapeutic agents.

An Evaluation of the Efficacy of Vasoactive Intestinal Polypeptide Antagonists in vivo in the Anaesthetized Dog

The effectiveness of competitive peptide vasoactive intestinal polypeptide (VIP) receptor antagonists was evaluated on heart rate in the anaesthetized dog. Two specific antagonists, VIP (6–28) and [D-p-Cl-Phe6, Leu17]-VIP, and a nonspecific antagonist, pituitary adenylate cyclase activating peptide fragment (6–27) (PACAP), were studied. VIP (6–28) and [D-p-Cl-Phe6, Leu17]-VIP (100 µg i.c.) increased the heart rate, whereas PACAP (100 µg i.c.) reduced the baseline heart rate. All three shifted the VIP dose-response curve to the right by two- to threefold for 30 min. In conclusion, PACAP, VIP (6–28), and [D-p-Cl-Phe6, Leu17]-VIP have a direct effect on the heart rate, are equally effective, and the effects last approximately 30 min in vivo.

Biodistribution and pharmacokinetic studies of SPION using particle electron paramagnetic resonance, MRI and ICP-MS

AIM Superparamagnetic iron oxide nanoparticles (SPIONs) may play an important role in nanomedicine by serving as drug carriers and imaging agents. In this study, we present the biodistribution and pharmacokinetic properties of SPIONs using a new detection method, particle electron paramagnetic resonance (pEPR). MATERIALS & METHODS The pEPR technique is based on a low-field and low-frequency electron paramagnetic resonance. pEPR was compared with inductively coupled plasma mass spectrometry and MRI, in in vitro and in vivo. RESULTS The pEPR, inductively coupled plasma mass spectrometry and MRI results showed a good correlation between the techniques. CONCLUSION The results indicate that pEPR can be used to detect SPIONs in both preclinical and clinical studies.

Effects of fenoldopam on renal blood flow and its function in a canine model of rhabdomyolysis

Background and objective: Our hypothesis was that fenoldopam, a selective DA1 agonist, would protect against rhabdomyolysis-induced renal injury. Methods: We studied the effects of intravenous fenoldopam (0.1-1.0 μg kg−1 min−1) or saline on renal blood flow and function in 10 anaesthetized Labrador dogs in whom rhabdomyolysis and myoglobinuric acute renal failure had been induced by administration of glycerol 50% (10 mL kg−1) intramuscularly. Haemodynamic measurements including renal blood flow and derived parameters of renal function including creatinine clearance were recorded before and for the 30 min following glycerol injection, and during the 3 h following commencement of each infusion. Serum malondialdehyde concentrations were measured before and 15 min after glycerol intramuscularly, and 30 and 150 min after commencement of the infusion. Results: In the fenoldopam group, creatinine clearance was less than placebo at 1 and 2 h after commencing the infusion (12.7 ± 11.5 versus 31.3 ± 9.9 mL min−1, P = 0.04; 8.5 ± 5.3 versus 20.1 ± 7.4 mL min−1, P = 0.03). A 140-fold increase in serum malondialdehyde concentration occurred in one dog (fenoldopam group). Conclusion: Fenoldopam increased the severity of the renal injury in this canine model of myoglobinuric acute renal failure.