Kathy Loutzenhiser

Angiotensin II–Induced Ca2+ Influx in Renal Afferent and Efferent Arterioles

Angiotensin II (Ang II)-induced Ca(2+) signaling was studied in isolated rat renal arterioles using fura-2. Ang II (10 nmol/L) caused a sustained elevation in [Ca(2+)](i), which was dependent on [Ca(2+)](o) in both vessel types. This response was blocked by nifedipine in only the afferent arteriole. Using the Mn(2+) quench technique, we found that Ang II stimulates Ca(2+) influx in both vessels. Nifedipine blocked the Ang II-induced Ca(2+) influx in afferent arterioles but not in efferent arterioles. In contrast to Ang II, KCl-induced depolarization stimulated Ca(2+) influx in only the afferent arteriole. Cyclopiazonic acid (CPA, 30 micromol/L) was used to examine the presence of store-operated Ca(2+) entry in myocytes isolated from each arteriole. In efferent myocytes, CPA induced a sustained Ca(2+) increase that was dependent on [Ca(2+)](o) and insensitive to nifedipine. This mechanism was absent in afferent myocytes. SKF 96365 inhibited Ang II-induced Ca(2+) entry in efferent arterioles and CPA-induced Ca(2+) entry in efferent myocytes over identical concentrations. Our findings thus indicate that Ang II activates differing Ca(2+) influx mechanisms in pre- and postglomerular arterioles. In the afferent arteriole, Ang II activates dihydropyridine-sensitive L-type Ca(2+) channels, presumably by membrane depolarization. In the efferent arteriole, Ang II appears to stimulate Ca(2+) entry via store-operated Ca(2+) influx.

Biphasic actions of prostaglandin E(2) on the renal afferent arteriole : role of EP(3) and EP(4) receptors.

Prostaglandin (PG) E(2) is an important modulator of the actions of angiotensin (Ang) II. In the present study, we investigated the renal microvascular actions of PGE(2) and the EP receptor subtypes involved. Ibuprofen potentiated Ang II-induced vasoconstriction in in vitro perfused normal rat kidneys and augmented afferent arteriolar, but not efferent arteriolar, responses in the hydronephrotic rat kidney model. This preglomerular effect of endogenous prostanoids was mimicked by exogenous PGE(2), which reversed Ang II-induced afferent arteriolar vasoconstriction at concentrations of 0.1 to 10 nmol/L without affecting the efferent arteriole. The PGE(2)-induced vasodilation was potentiated by the phosphodiesterase inhibitor Ro 20-1724 and was mimicked by 11-deoxy-PGE(1) (0.01 to 1 nmol/L). Butaprost, which acts preferentially at EP(2) receptors, was relatively ineffective. Whereas 0.1 to 10 nmol/L PGE(2) elicited vasodilation, higher concentrations (1 to 10 micromol/L) restored Ang II-induced afferent arteriolar vasoconstriction. This response was blocked by pertussis toxin (200 microg/mL) and was mimicked by the EP(1)/EP(3) agonist sulprostone (1 to 300 nmol/L). Reverse transcription-polymerase chain reaction of individually isolated afferent arterioles revealed the presence of message for EP(4) and all 3 EP(3) splice variants (alpha, beta, and gamma) but not EP(1) or EP(2). Our findings thus indicate that PGE(2) elicits both vasodilatory and vasoconstrictor actions on the afferent arteriole. The vasodilation is mediated by EP(4) receptors coupled to cAMP, presumably via G(alphas). The vasoconstriction is mediated by an EP(3) receptor coupled to G(alphai) and appears to reflect a functional antagonism of the EP(4)-induced vasodilation.

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.

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.

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.

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 ~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.

Voltage-Activated Ca2+ Channels in Rat Renal Afferent and Efferent Myocytes: No Evidence for the T-Type Ca2+ 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.

The 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 Ca2+/calmodulin-dependent myosin light chain kinase. Ca2+-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 LC20 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.

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.