Recent studies have improved our understanding of the beneficial actions of calcium antagonists on myocardial microcirculation and metabolism. The effect of calcium antagonists on the microcirculation of the left ventricular rat myocardium was studied using in vivo microscopic techniques. Intravenous verapamil 0.3 mg/kg and nifedipine 75 µg/kg produced a 15 to 18% increase in the diameter of larger A1 and A2 coronary arterioles (range 31 to 300µm); diameters of terminal (A4) arterioles and capillaries did not change significantly.

This study investigated whether intrarenal endothelin-1(ET-1) contributes to sodium excretion in aged rats. Metabolic function studies were performed in male Wistar rats (3 and 24 months) treated with placebo or the orally active ET(A) receptor antagonist darusentan (20 mg/kg/d) for 4 weeks. Mean arterial pressure was measured using an intra-arterial catheter. Electrolytes, aldosterone levels, renin activity, and angiotensin converting enzyme activity were determined in plasma, and mRNA expression of epithelial sodium channel (ENaC) and Na(+), K(+)-ATPase subunits was measured in the renal cortex and medulla. Aging was associated with a marked decrease in urinary excretion of sodium, chloride, and potassium (all P < 0.001) as well as renin activity (P < 0.05), but had no significant effect on gene expression of ENaC or Na(+), K(+)-ATPase subunits. In aged rats, darusentan treatment increased ion excretion (P < 0.05), reduced cortical gene expression of alphaENaC and alpha(1)-Na(+), K(+)-ATPase (both P < 0.05), and increased plasma aldosterone levels (P < 0.01). These data demonstrate a decrease of sodium and potassium excretion in aged rats, changes that are partly sensitive to ETA receptor blockade. Treatment with darusentan also reduced cortical expression of alphaENaC and alpha(1)-Na(+), K(+)-ATPase and increased plasma aldosterone levels independently of blood pressure, electrolytes, renin activity, or angiotensin converting enzyme activity. These findings may provide new pathogenetic links between aging and sodium sensitivity.

The cause of focal-segmental glomerulosclerosis as a consequence of physiological aging, which is believed to be inexorable, is unknown. This study investigated whether inhibition of endothelin-1, a growth-promoting peptide contributing to renal injury in hypertension and diabetes, affects established glomerulosclerosis and proteinuria in the aged kidney. We also determined the role of endothelin receptors for podocyte injury in vivo and in vitro. Aged Wistar rats, a model of spontaneous age-dependent glomerulosclerosis, were treated with the orally active endothelin subtype A ( ETA) receptor antagonist darusentan, and evaluation of renal histology, renal function studies, and expression analyses were performed. In vitro experiments using puromycin aminonucleoside to induce podocyte injury investigated the role of ETA receptor signaling for apoptosis, cytoskeletal injury, and DNA synthesis. In aged Wistar rats, established glomerulosclerosis and proteinuria were reduced by > 50% after 4 weeks of darusentan treatment, whereas blood pressure, glomerular filtration rate, or tubulo-interstitial renal injury remained unaffected. Improvement of structural injury in glomeruli and podocytes was accompanied by a reduction of the expression of matrix metalloproteinase-9 and p21(Cip1/WAF1). In vitro experiments blocking ETA receptors using specific antagonists or RNA interference prevented apoptosis and structural damage to podocytes induced by puromycin aminonucleoside. In conclusion, these results support the hypothesis that endogenous endothelin contributes to glomerulosclerosis and proteinuria in the aging kidney. The results further suggest that age-dependent glomerulosclerosis is not merely a "degenerative" but a reversible process locally confined to the glomerulus involving recovery of podocytes from previous injury.

In addition to intracellular calcium, which activates myosin light chain (MLC) kinase, MLC phosphorylation and hence contraction is importantly regulated by MLC phosphatase (MLCP). Recent evidence suggests that distinct signaling cascades of vasoactive hormones interact with the Rho/Rho kinase (ROK) pathway, affecting the activity of MLCP. The present study measured the impact of ROK inhibition on vascular F-actin distribution and on vasoconstriction induced by activation/inhibition of distinct signaling pathways in vivo in the microcirculation of the split hydronephrotic rat kidney. Local application of the ROK inhibitors Y-27632 or HA-1077 induced marked dilation of pre- and postglomerular vessels. Activation of phospholipase C with the endothelin ET B agonist IRL 1620, inhibition of soluble guanylyl cyclase with 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), or inhibition of adenylyl cyclase with the adenosine A1 agonist N6-cyclopentyladenosine (CPA) reduced glomerular blood flow (GBF) by about 50% through vasoconstriction at different vascular levels. ROK inhibition with Y-27632 or HA-1077, but not protein kinase C inhibition with Ro 31-8220, blunted ET B-induced vasoconstriction. Furthermore, the reduction of GBF and of vascular diameters in response to ODQ or CPA were abolished by pretreatment with Y-27632. ROK inhibitors prevented constriction of preglomerular vessels and of efferent arterioles with equal effectiveness. Confocal microscopy demonstrated that Y-27632 did not change F-actin content and distribution in renal vessels. The results suggest that ROK inhibition might be considered as a potent treatment of renal vasoconstriction, because it interferes with constriction induced by distinct signaling pathways in renal vessels without affecting F-actin structure.

A novel peptide, PNP (Pseudocerastes persicus natriuretic peptide), was isolated from the venom of the Iranian viper P. persicus. Amino acid sequencing revealed that the 37-residue peptide belongs to the family of natriuretic peptides. The physiological effects of intra-venously PNP infused into anesthetized rats on urine flow, sodium excretion and blood pressure were comparable to those of atrial natriuretic peptide (ANP). In PC12 cells that were treated with either PNP, ANP, or C-type natriuretic peptide, PNP induced a similar cGMP response as ANP. Since PC12 cells only express the natriuretic peptide receptor (NPR)-A receptor we conclude that PNP binds to the NPR-A receptor. The solution conformation of PNP was characterized using 1H nuclear magnetic resonance spectroscopy and indicates a high degree of conformational flexibility.

BACKGROUND:Major cell signaling pathways involved in agonist-induced vasoconstriction are recognized to be Ca2+ mobilization via inositol-1,4,5 triphosphate (IP3), Ca2+ influx through l-type channels, activation of protein kinase C (PKC), and of Rho-associated kinase (ROK). However, their contribution for renal vasoconstriction induced by different agonists is not well characterized.METHODS:Increasing doses of angiotensin II (Ang II), norepinephrine, and arginine vasopressin (AVP) were infused into the left renal artery of anesthetized rats to reduce renal blood flow from a threshold value to about 50%. Rightward shift of the dose-response curves due to coinfusion of inhibitors served to assess contribution of different pathways: trimethoxybenzoate (TMB-8) against Ca2+ mobilization, nifedipine against Ca2+ influx, staurosporine and Ro-318220 against PKC, and Y-27632 and HA-1077 against ROK. Effects of inhibitors were also determined for renal response to a single dose of U-46619, a thromboxane A2 agonist. Composite response to U-46619 consisting of a fast and slow component did not permit determination of dose-response curves.RESULTS:Inhibition of ROK by Y-27632 or HA-1077 had the largest effect on renal responses to agonists. They shifted dose-response curves of Ang II, norepinephrine, and AVP to sevenfold and higher values. Staurosporine, nifedipine, and TMB-8 had variable effect on agonist responses. They attenuated effects of Ang II and norepinephrine in an additive manner, and each of them increased effective dose values about fourfold. TMB-8 did not attenuate response to AVP and U-46619. Staurosporine and nifedipine diminished effects of AVP in a nonadditive manner, and attenuated additively the fast component of U-46619 response.CONCLUSION:In contrast to other cell signaling pathways, ROK plays a common role for all vasoconstrictor agonistsis in renal circulation.

BACKGROUND:Angiotensin II (Ang II), arginine vasopressin (AVP) and tromboxane A(2) (TxA(2)) are dissimilar vasoconstrictors involved in regulating renal circulation. Whereas Ang II is primarily a physiological modulator, AVP and TxA(2) play important roles under pathological conditions. Previously, we have shown variable importance of intracellular Ca(2+) and protein kinase C for their mode of action (Ang II > AVP >U-46619), but the cell signalling via rho-associated kinase (ROK) is a common pathway. The aim of this study was to determine their sites of action in the renal vascular bed and the corresponding role of ROK at the microvascular level.METHODS:Glomerular blood flow (GBF) and luminal diameter of different vessels (10-70 micro m) were measured in the split hydronephrotic kidney of anaesthetized rats. The tissue bath concentration of Ang II, AVP or the TxA(2) agonist U-46619 was adjusted to reduce GBF by approximately 50%. The measurements were repeated after adding a sub-maximal dose of the ROK inhibitor Y-27632 into the bath.RESULTS:Ang II constricted all vessels significantly, the constriction being least in the proximal segment of the arcuate artery ( approximately 70 micro m). Significant constrictions due to AVP were found only in interlobular and arcuate arteries (20-70 micro m), but not in the afferent and efferent arterioles. U-46619 constricted only the arcuate artery (> or = 50 micro m). Y-27632 (10(-4) M) dilated all vessels significantly and increased GBF by 65%. Thereafter, effects of all agonists were severely attenuated. Control reductions in GBF could be obtained at higher concentrations of AVP (10-fold) and U-46619 (5-fold) and a lesser GBF reduction with Ang II (100-fold) without changes in the respective patterns of vascular constriction.CONCLUSIONS:Our data indicate that the agonists, in the order Ang II, AVP and TxA(2), constrict larger vessels within the renal vascular tree via activation of ROK. Therefore, ROK inhibitors may provide a therapeutic tool to antagonize pathological vasospasm of conduit vessels, which are resistant to other vasodilators.

Studies in vitro have demonstrated that vasoconstrictor agents increase intracellular Ca2+and activate protein kinase C (PKC) to elevate vascular tone. The aim of the present study was to determine the importance of these signaling pathways for angiotensin II (ANG II) and thromboxane A2(TxA2) in regulating mesenteric blood flow (MBF) in vivo. In anesthetized rats increasing doses of ANG II or the TxA2agonist U-46619 were administered into the superior mesenteric artery to reduce MBF. Intra-arterial infusion of inhibitors served to examine the contribution of different pathways: 8-(diethylamino)octyl 3,4,5-trimethoxybenoate hydrochloride (TMB-8) to inhibit intracellular Ca2+release, nifedipine to block transmembrane Ca2+influx through the L-type Ca2+channel, and staurosporine to inhibit PKC. Each of the inhibitors attenuated ANG II-induced reductions in MBF, and all dose-response curves were shifted to the right to an approximately threefold higher ANG II dose. Combinations of the inhibitors revealed that their effects were additive; together they abolished the vasoconstrictor action of ANG II completely. In contrast, the dose-response curve for U-46619 was not affected by any of the inhibitors infused either separately or together. The results demonstrate that a rise in intracellular Ca2+and activation of PKC are major mediators of the vasoconstrictor effect of ANG II in mesenteric circulation, but they play a subordinate role, if any, for the effects of TxA2. Because TxA2plays a major role only under pathological conditions, the uncontrolled vasoconstriction appears to be associated with the recruitment of novel signal transduction pathways.

The aim of the present study was to quantify with a uniform technique the rates of conversion of ANG I to ANG II in the lung and kidney and the degradation of both peptides to biologically inactive products in the pulmonary, renal, and systemic circulation. We infused the peptides intravenously, into the left ventricle, and into the left renal artery of rats and compared their effects on renal blood flow. The measured change in renal blood flow was used as a bioassay parameter to estimate the concentration of circulating ANG II. Mathematical analysis of our data allowed us to calculate conversion and degradation rates. Furthermore, the role of aminopeptidases A (EC 3.4.11.7) and N (EC 3.4.11.2) in the degradation of the peptides in the kidney was investigated by intrarenal infusion of the inhibitor amastatin. Our results show that the conversion rate of ANG I is 75% in the pulmonary and 21% in the renal circulation. Both peptides are degraded by 5% in the pulmonary, by 67% in the systemic, and by 93% in the renal circulation. Amastatin prevented 60% of the renal degradation of the peptides to inactive products, and this effect could be attributed to inhibition of aminopeptidase N. The results indicate that the converting capacity of the kidney is of minor importance for endocrine generation of ANG II but could be useful for the paracrine production.

Cyclosporine A (CsA), a widely used immunosuppressive agent, causes renal vasoconstriction and systemic hypertension. Recent data suggest that the renal effect of CsA is possibly mediated by endothelin (ET). We investigated the effects of CsA on renal microvessels and the efficacy of ETA or ETA/ETB receptor antagonists in ameliorating CsA effects in the hydronephrotic rat kidney. Infusion of CsA (30 mg.kg-1) induced a transient increase (20%) in mean arterial pressure (MAP) and a sustained reduction (85%) in glomerular blood flow (GBF) due to preferential constriction of the arcuate artery (39%) and the proximal segment of the interlobular artery (23%). Under basal conditions the ETA receptor antagonist BQ-123 had marginal effects consisting of reduction in MAP, rise in GBF and dilation of preglomerular vessels. The non-selective ETA/ETB receptor antagonist PD 145065 also reduced MAP, but tended to decrease GBF and constrict large preglomerular vessels. The difference in effects of the two antagonists indicated that under basal conditions ETB blockade constricts large preglomerular vessels and reduces GBF. After BQ-123 or PD 145065, the constriction of large preglomerular vessels and reduction in GBF induced by CsA was attenuated by about 50%, but the rise in MAP was not influenced. Our data indicate that a sizable part of renal vasoconstriction due to CsA is mediated via ET production in large preglomerular arteries and can be avoided by the blockade of ETA receptors. Additional blockade of ETB receptors does not attenuate the CsA effects further, possibly because ETB receptors mediate both vasoconstriction and dilation.

1. Vasomotor responses (VMR) induced by local electrical stimulation were studied in the vasculature of the split hydronephrotic rat kidney by in vivo microscopy. 2. Unipolar pulses, which were applied by a micropipette positioned close to the vessel wall, elicited local and propagated VMR. Depolarizing and hyperpolarizing currents caused vasoconstriction and vasodilatation, respectively. 3. The magnitude of VMR could be controlled within seconds by variation of pulse frequency, pulse width and voltage. VMR were abolished by slight retraction of the stimulating micropipette. Repetitive electrical stimulation resulted in reproducibly uniform VMR. 4. Propagated VMR decayed with increasing distance from the stimulation site. They decayed more rapidly in the upstream than in the downstream flow direction in interlobular arteries. The longitudinal decay was well approximated by an exponential function with significantly different length constants of 150 +/- 40 microns (upstream, n = 5) and 420 +/- 90 microns (downstream, n = 8). 5. Our results show that vasomotor responses, which are initiated by changes in membrane potential, are propagated over distances of potential physiological importance in interlobular arteries.

This study investigated effects of renal prostaglandins and their interaction with different vasoactive agents in regulating regional renal blood flow. Using intravenous infusions, we compared effects of different pressor hormones and a nitric oxide (NO) inhibitor under control conditions and after inhibition of cyclooxygenase. Because vasodilator effects of prostanoids are considered to be mediated via opening of ATP-dependent K+ channels, we also studied effects of a prostacyclin analogue (iloprost), a channel opener (lemakalim), and a channel blocker (glibenclamide). Blood flow in renal cortex (CBF) and medulla (MBF) was determined with previously described platinum electrodes inserted into the kidney of anesthetized rats. Angiotensin II and norepinephrine reduced predominantly only CBF (-24 and -19%, respectively). After indomethacin, which selectively reduced MBF (-25%), angiotensin II and norepinephrine also reduced MBF (-45 and -35%, respectively), whereas the corresponding changes in CBF were not affected by indomethacin. Arginine vasopressin and the NO inhibitor NG-nitro-L-arginine methyl ester reduced both CBF and MBF by approximately 30% both under control conditions and after indomethacin. Iloprost and lemakalim increased selectively MBF (15 and 27%, respectively), whereas glibenclamide selectively decreased MBF (-19%). Our data indicate that renal prostaglandins are predominantly involved in regulating medullary circulation. They probably exert their dilatory action on medullary vessels via opening of ATP-dependent K+ channels and are involved in antagonizing medullary effects of pressor hormones in an agonist-specific manner.

This study compared the vasoconstrictor action of angiotensin II (ANG II) and norepinephrine (NE) with different levels of nitric oxide (NO) in the kidney of anesthetized rats. In one series of experiments, the drugs were infused intravenously, and systemic NO content was reduced by a NO synthase inhibitor, nitro-L-arginine methyl ester (L-NAME). L-NAME significantly enhanced the renal blood flow (RBF) reduction produced by ANG II from 26 to 49%, but it had no significant effect on the change in RBF induced by NE. Medullary blood flow was not influenced by either ANG II or NE given alone or given after L-NAME. In the second series of experiments, all drugs were infused into the renal artery to avoid their systemic and, hence, extrarenal effects. In these experiments, renal content of NO was increased by the NO donor sodium nitroprusside (SNP), decreased by L-NAME, or restored by replacing endogenous NO by exogenous NO (L-NAME + SNP). Effects of both ANG II and NE on RBF were similarly and significantly attenuated by SNP (60% of control), enhanced by L-NAME (200% of control), and restored by L-NAME + SNP (90% of control, not significant). Our results indicate that NO attenuates the renal vasoconstriction due to ANG II or NE and that the antagonism between vasoconstrictors and NO is not due to a constrictor-induced production of NO because exogenous and endogenous NO were equally effective.

Parathyroid hormone‐related protein (PTHrP) is expressed in the kidney and acts on vascular PTH/PTHrP receptors to vasodilate the isolated kidney and to stimulate renin release. However, effects of PTHrP on renal blood flow (RBF) and glomerular filtration rate (GFR) in vivo have not been assessed in the absence of its cardiac, peripheral and central effects. We investigated the renal effects of PTH and PTHrP infused into the left renal artery of anaesthetized rats. Intrarenal infusions, adjusted to generate increasing concentrations of human PTHrP(1–34) and rat PTH(1–34) in renal plasma (2 × 10−11 to 6 × 10−9 m) produced a comparable dose‐dependent increase in RBF. The rise was 4% at the lowest and 34% at the highest concentrations of peptides. Up to a concentration of 2 × 10−9 m, mean arterial pressure (MAP) and heart rate were not affected, but at 6 × 10−9 m, intrarenally infused peptides reached the peripheral circulation, and caused a fall in MAP within a few minutes. While MAP returned to basal value after the last peptide infusion, RBF remained more than 10% above control for at least 30 min. Two competitive PTH/PTHrP receptor antagonists, [Nle8,18, Tyr34]‐bPTH(3–34)amide and [Leu11, D‐Trp12]‐hPTHrP(7–34)amide (2 × 10−8 m) were devoid of agonist activity, but markedly antagonized the dose‐dependent increase in RBF elicited by PTHrP. GFR and urine flow were measured in left PTHrP‐infused experimental kidney and right control kidney. Renal PTHrP concentration of 10−10 m elevated left RBF by 10%, and GFR by 20% without significantly increasing filtration fraction, and increased urine flow by 57%. In the right control kidney GFR and diuresis did not change. The results indicate that PTHrP has similar renal haemodynamic effects as PTH and increases RBF, GFR and diuresis in anaesthetized rats.