Endothelin-1, but not angiotensin II, induces afferent arteriolar myosin diphosphorylation as a potential contributor to prolonged vasoconstriction.

Bolus administration of endothelin-1 elicits long-lasting renal afferent arteriolar vasoconstriction, in contrast to transient constriction induced by angiotensin II. Vasoconstriction is generally evoked by myosin regulatory light chain (LC20) phosphorylation at Ser19 by myosin light chain kinase (MLCK), which is enhanced by Rho-associated kinase (ROCK)-mediated inhibition of myosin light chain phosphatase (MLCP). LC20 can be diphosphorylated at Ser19 and Thr18, resulting in reduced rates of dephosphorylation and relaxation. Here we tested whether LC20 diphosphorylation contributes to sustained endothelin-1 but not transient angiotensin II-induced vasoconstriction. Endothelin-1 treatment of isolated arterioles elicited a concentration- and time-dependent increase in LC20 diphosphorylation at Thr18 and Ser19. Inhibition of MLCK or ROCK reduced endothelin-1-evoked LC20 mono- and diphosphorylation. Pretreatment with an ETB but not an ETA receptor antagonist abolished LC20 diphosphorylation, and an ETB receptor agonist induced LC20 diphosphorylation. In contrast, angiotensin II caused phosphorylation exclusively at Ser19. Thus, endothelin-1 and angiotensin II induce afferent arteriolar constriction via LC20 phosphorylation at Ser19 due to calcium activation of MLCK and ROCK-mediated inhibition of MLCP. Endothelin-1, but not angiotensin II, induces phosphorylation of LC20 at Thr18. This could contribute to the prolonged vasoconstrictor response to endothelin-1.

Involvement of myosin regulatory light chain diphosphorylation in sustained vasoconstriction under pathophysiological conditions.

Smooth muscle contraction is activated primarily by phosphorylation at Ser19 of the regulatory light chain subunits (LC20) of myosin II, catalysed by Ca(2+)/calmodulin-dependent myosin light chain kinase. Ca(2+)-independent contraction can be induced by inhibition of myosin light chain phosphatase, which correlates with diphosphorylation of LC20 at Ser19 and Thr18, catalysed by integrin-linked kinase (ILK) and zipper-interacting protein kinase (ZIPK). LC20 diphosphorylation at Ser19 and Thr18 has been detected in mammalian vascular smooth muscle tissues in response to specific contractile stimuli (e.g. endothelin-1 stimulation of rat renal afferent arterioles) and in pathophysiological situations associated with hypercontractility (e.g. cerebral vasospasm following subarachnoid hemorrhage). Comparison of the effects of LC 20 monophosphorylation at Ser19 and diphosphorylation at Ser19 and Thr18 on contraction and relaxation of Triton-skinned rat caudal arterial smooth muscle revealed that phosphorylation at Thr18 has no effect on steady-state force induced by Ser19 phosphorylation. On the other hand, the rates of dephosphorylation and relaxation are significantly slower following diphosphorylation at Thr18 and Ser19 compared to monophosphorylation at Ser19. We propose that this diphosphorylation mechanism underlies the prolonged contractile response of particular vascular smooth muscle tissues to specific stimuli, e.g. endothelin-1 stimulation of renal afferent arterioles, and the vasospastic behavior observed in pathological conditions such as cerebral vasospasm following subarachnoid hemorrhage and coronary arterial vasospasm. ILK and ZIPK may, therefore, be useful therapeutic targets for the treatment of such conditions.

Voltage-activated Ca(2+) channels in rat renal afferent and efferent myocytes: no evidence for the T-type Ca(2+) current.

AIMS: Based on indirect methods, it has been suggested that both L- and T-type Ca(2+) channels mediate signalling in the renal afferent arteriole and that T-type Ca(2+) channels are involved in signalling in the efferent arteriole. However, Ca(2+) currents have never been studied in these two vessels. Our study was initiated to directly determine the type of Ca(2+) channels in these vessels for the first time, using patch clamp. METHODS AND RESULTS: Native myocytes were obtained from individually isolated rat renal afferent and efferent arterioles and from rat tail arteries (TA). TA myocytes, which possess both L- and T-type Ca(2+) currents, served as a positive control. Inward Ca(2+) and Ba(2+) currents (I(Ca) and I(Ba)) were measured in 1.5 mmol/L Ca(2+) and 10 mmol/L Ba(2+), respectively, using the whole-cell configuration. By exploiting known differences in activation and inactivation characteristics and differing sensitivities to nifedipine and kurtoxin, the presence of both L- and T-type Ca(2+) channels in TA myocytes was readily demonstrated. Afferent arteriolar myocytes exhibited relatively large I(Ca) densities (-2.0 ± 0.2 pA/pF) in physiological Ca(2+) and the I(Ba) was 3.6-fold greater. These currents were blocked by nifedipine, but not by kurtoxin, and did not exhibit the activation and inactivation characteristics of T-type Ca(2+) channels. Efferent arteriolar myocytes did not exhibit a discernible voltage-activated I(Ca) in physiological Ca(2+). CONCLUSION: Our findings support the physiological role of L-type Ca(2+) channels in the afferent, but not efferent, arteriole, but do not support the premise that functional T-type Ca(2+) channels are present in either vessel.

Segment-specific differences in the inward rectifier K(+) current along the renal interlobular artery.

AIMS: We investigated the role of the inward rectifier K(+) channel (K(IR)) in the renal interlobular artery (ILA). The ILA supplies the afferent arteriole and ranges in diameter from >100 µm near its origin at the arcuate artery to <30 µm at its most distal segment. METHODS AND RESULTS: Vasodilatory responses to elevated extracellular K(+) (15 mmol/L) and vasoconstrictor responses due to K(IR) blockade by Ba(2+) (10-100 µmol/L) were assessed in in vitro perfused hydronephrotic rat kidneys. The distal ILA (26 ± 1 µm) exhibited K(+)-induced dilation and Ba(2+)-induced vasoconstriction, whereas neither response was observed in the proximal ILA (108 ± 3 µm). The intermediate ILA (55 ± 1 µm) exhibited a modest K(+)-induced vasodilatation, but no Ba(2+)-induced vasoconstriction. The K(+)-induced dilations were blocked by Ba(2+), but not by ouabain. Ba(2+)-induced depolarization, measured in ILA segments from normal kidneys, decreased with the increasing diameter. Patch-clamp studies demonstrated that the K(IR) current (I(KIR)) density also was inversely correlated with ILA segment diameter. Myocytes from afferent arterioles and distal ILAs exhibited similarly large I(KIR), whereas this current was absent in proximal ILA myocytes. Finally, we found that Ba(2+) attenuated myogenic vasoconstriction, suggesting an involvement of I(KIR). The previously shown pattern of myogenic reactivity of the ILA (distal > intermediate > proximal) mirrors the distribution of I(KIR) reported in the present study, further supporting a role for I(KIR). CONCLUSION: Our findings indicate differences in the magnitude of I(KIR) along the ILA and suggest that the influence of K(IR) on reactivity increases as vessel diameter decreases from proximal to distal regions.

Effects of amiloride, benzamil, and alterations in extracellular Na+ on the rat afferent arteriole and its myogenic response.

Recent studies have implicated epithelial Na+ channels (ENaC) in myogenic signaling. The present study was undertaken to determine if ENaC and/or Na+ entry are involved in the myogenic response of the rat afferent arteriole. Myogenic responses were assessed in the in vitro hydronephrotic kidney model. ENaC expression and membrane potential responses were evaluated with afferent arterioles isolated from normal rat kidneys. Our findings do not support a role of ENaC, in that ENaC channel blockers did not reduce myogenic responses and ENaC expression could not be demonstrated in this vessel. Reducing extracellular Na+ concentration ([Na+]o; 100 mmol/l) did not attenuate myogenic responses, and amiloride had no effect on membrane potential. Benzamil, an inhibitor of ENaC that also blocks Na+/Ca2+ exchange (NCX), potentiated myogenic vasoconstriction. Benzamil and low [Na+]o elicited vasoconstriction; however, these responses were attenuated by diltiazem and were associated with significant membrane depolarization, suggesting a contribution of mechanisms other than a reduction in NCX. Na+ repletion induced a vasodilation in pressurized afferent arterioles preequilibrated in low [Na+]o, a hallmark of NCX, and this response was reduced by 10 micromol/l benzamil. The dilation was eliminated, however, by a combination of benzamil plus ouabain, suggesting an involvement of the electrogenic Na+-K+-ATPase. In concert, these findings refute the premise that ENaC plays a significant role in the rat afferent arteriole and instead suggest that reducing [Na+](o) and/or Na+ entry is coupled to membrane depolarization. The mechanisms underlying these unexpected and paradoxical effects of Na+ are not resolved at the present time.

A highly sensitive technique to measure myosin regulatory light chain phosphorylation: the first quantification in renal arterioles.

Phosphorylation of the 20-kDa myosin regulatory light chains (LC(20)) plays a key role in the regulation of smooth muscle contraction. The level of LC(20) phosphorylation is governed by the relative activities of myosin light chain kinase and phosphatase pathways. The regulation of these two pathways differs in different smooth muscle types and in the actions of different vasoactive stimuli. Little is known concerning the regulation of LC(20) phosphorylation in the renal microcirculation. The available pharmacological probes are often nonspecific, and current techniques to directly measure LC(20) phosphorylation are not sensitive enough for quantification in small arterioles. We describe here a novel approach to address this important issue. Using SDS-PAGE with polyacrylamide-bound Mn(2+)-phosphate-binding tag and enhanced Western blot analysis, we were able to detect LC(20) phosphorylation using as little as 5 pg (250 amol) of isolated LC(20). Phosphorylated and unphosphorylated LC(20) were detected in single isolated afferent arterioles, and LC(20) phosphorylation levels could be accurately quantified in pooled samples of three arterioles (<300 cells). The phosphorylation level of LC(20) in the afferent arteriole was 6.8 +/- 1.7% under basal conditions and increased to 34.7 +/- 5.1% and 44.6 +/- 6.6% in response to 30 mM KCl and 10(-8) M angiotensin II, respectively. The application of this technique will enable investigations of the different determinants of LC(20) phosphorylation in afferent and efferent arterioles and provide insights into the signaling pathways that regulate LC(20) phosphorylation in the renal microvasculature under physiological and pathophysiological conditions.

Inward rectifier K(+) currents and Kir2.1 expression in renal afferent and efferent arterioles.

The afferent and efferent arterioles regulate the inflow and outflow resistance of the glomerulus, acting in concert to control the glomerular capillary pressure and glomerular filtration rate. The myocytes of these two vessels are remarkably different, especially regarding electromechanical coupling. This study investigated the expression and function of inward rectifier K(+) channels in these two vessels using perfused hydronephrotic rat kidneys and arterioles and myocytes isolated from normal rat kidneys. In afferent arterioles pre-constricted with angiotensin II, elevating [K(+)](0) from 5 to 15 mmol/L induced hyperpolarization (-27 +/- 2 to 41 +/- 3 mV) and vasodilation (6.6 +/- 0.9 to 13.1 +/- 0.6 microm). This manipulation also attenuated angiotensin II-induced Ca(2+) signaling, an effect blocked by 100 micromol/LBa(2+). By contrast, elevating [K(+)](o) did not alter angiotensin II-induced Ca2(+) signaling or vasoconstriction in efferent arterioles, even though a significant hyperpolarization was observed (from -30 +/- 1 to 37 +/- 3 mV, P = 0.003). Both vessels expressed mRNA for Kir2.1 and exhibited anti-Kir2.1 antibody labeling.Patch-clamp measurements revealed prominent inwardly rectifying and Ba(2+)-sensitive currents in afferent and efferent arteriolar myocytes. Our findings indicate that both arterioles express an inward rectifier K(+) current, but that modulation of this current alters responsiveness of only the a different arteriole. The expression of Kir in the efferent arteriole, a resistance vessel whose tone is not affected by membrane potential, is intriguing and may suggest a novel function of this channel in the renal microcirculation.

Effects of inhibition of the Na+/K+/2Cl- cotransporter on myogenic and angiotensin II responses of the rat afferent arteriole.

The Na(+)/K(+)/2Cl(-) cotransporter (NKCC) plays diverse roles in the kidney, contributing sodium reabsorption and tubuloglomerular feedback (TGF). However, NKCC is also expressed in smooth muscle and inhibitors of this transporter affect contractility in both vascular and nonvascular smooth muscle. In the present study, we investigated the effects of NKCC inhibitors on vasoconstrictor responses of the renal afferent arteriole using the in vitro perfused hydronephrotic rat kidney. This preparation has no tubules and no TGF, eliminating this potential complication. Furosemide and bumetanide inhibited myogenic responses in a concentration-dependent manner. Bumetanide was approximately 20-fold more potent (IC(50) 1.0 vs. 20 micromol/l). At 100 and 10 micromol/l, furosemide and bumetanide inhibited myogenic responses by 72 +/- 4 and 68 +/- 5%, respectively. The maximal level of inhibition by bumetanide was not affected by nitric oxide synthase inhibition (100 micromol/l N(G)-nitro-l-arginine methyl ester). However, the time course for the dilation was slowed (from t(1/2) = 4.0 +/- 0.5 to 8.3 +/- 1.7 min, P = 0.04), suggesting either a partial involvement of NO or a permissive effect of NO on relaxation kinetics. Bumetanide also inhibited ANG II-induced afferent arteriolar vasconstriction at similar concentrations. Finally, NKCC1, but not NKCC2, expression was demonstrated in the afferent arteriole by RT-PCR and the presence of NKCC1 in afferent arteriolar myocytes was confirmed by immunohistochemistry. In concert, these results indicate that NKCC modulation is capable of altering myogenic responses by a mechanism that does not involve TGF and suggest a potential role of NKCC1 in the regulation of vasomotor function in the renal microvasculature.

Myosin heavy chain expression in renal afferent and efferent arterioles: relationship to contractile kinetics and function.

The physiological role of smooth muscle myosin heavy chain (MHC) isoform diversity is poorly understood. The expression of MHC-B, which contains an insert at the ATP binding pocket, has been linked to enhanced contractile kinetics. We recently reported that the renal afferent arteriole exhibits an unusually rapid myogenic response and that its kinetic features allow this vessel to modulate tone in response to alterations in systolic blood pressure. In the present study, we examined MHC expression patterns in renal afferent and efferent arterioles. These two vessels regulate glomerular inflow and outflow resistances and control the pressure within the intervening glomerular capillaries (PGC). Whereas the afferent arteriole must respond rapidly to increases in blood pressure, the efferent arteriole plays a distinctly different role, maintaining a tonic elevation in outflow resistance to preserve function when renal perfusion is compromised. Using RT-PCR, Western analysis, and immunofluorescence imaging of intact isolated arterioles, we found that the afferent arteriole predominantly expresses the MHC-B isoform, whereas the efferent arteriole expresses only the slower-cycling MHC-A isoform. We examined the kinetics of angiotensin II- and norepinephrine-induced vasoconstriction and found that the afferent arteriole responds approximately 3-fold faster than the efferent arteriole. Our findings thus point to the renal microcirculation as a unique and important example of smooth muscle adaptation in regard to MHC isoform expression and physiological function.

PAR-2 elicits afferent arteriolar vasodilation by NO-dependent and NO-independent actions.

Proteinase-activated receptors (PARs) are a novel class of G protein-coupled receptors that respond to signals through endogenous proteinases. PAR activation involves enzymatic cleavage of the extracellular NH(2)-terminal domain and unmasking of a new NH(2) terminus, which serves as an anchored ligand to activate the receptor. At least four PAR subtypes have been identified. In the present study, we used the in vitro perfused hydronephrotic rat kidney to examine the effects of activating PAR-2 on the afferent arteriole. The synthetic peptide SLIGRL-NH(2), which corresponds to the exposed ligand sequence and selectively activates PAR-2, did not alter basal afferent arteriolar diameter but caused a concentration-dependent vasodilation (3-30 microM) of arterioles preconstricted by angiotensin II (0.1 nM). A modified peptide sequence (LSIGRL-NH(2), inactive at PAR-2) had no effect. This vasodilation was characterized by an initial transient component followed by a smaller sustained response. A similar pattern of vasodilation was seen when SLIGRL-NH(2) was administered to isolated perfused normal rat kidney. The sustained component of the PAR-2-induced afferent arteriolar vasodilation was eliminated by nitric oxide (NO) synthase inhibition (100 microM nitro-L-arginine methyl ester). In contrast, the transient vasodilation persisted under these conditions. This transient response was not observed when afferent arterioles were preconstricted with elevated KCl, suggesting involvement of an endothelium-derived hyperpolarizing factor. Finally, RT-PCR revealed the presence of PAR-2 mRNA in isolated afferent arterioles. These findings indicate that PAR-2 is expressed in the afferent arteriole and that its activation elicits afferent arteriolar vasodilation by NO-dependent and NO-independent mechanisms.