Vasoactivity of DVR

Constriction and Dilation of DVR by Vasoactive Agents

DVR are contractile microvessels (Figure 3). A large number of mediators have been shown to constrict or dilate DVR, and a number of receptors have been identified in medullary vascular bundles by ligand binding, autoradiog-raphy, RT-PCR, or immunochemistry. Table III summarizes the findings. The entries listed in Table III include observations of pharmacological effects on vasomotion of micro-perfused DVR isolated from vascular bundles of the rat as well as receptor studies that have employed a variety of methods. We attribute constriction to the action of pericytes. Vasopressin (compared to angiotensin II and endothelin) is a weak DVR vasoconstrictor. Endothelins constrict primarily via the ETA receptor and are thought to exert a self-limiting vasodilatory effect through ETB receptor stimulation. ET1 and ET2 isoforms are ETA and ETB receptor agonists and have proven to be the most potent vasoconstrictors of DVR thus far observed [11]. Angiotensin II (AngII) also reliably constricts isolated DVR. AT2 receptor antagonists enhance AngII constriction and AT2 receptor expression has been verified in DVR by RT-PCR. Effects of adenosine effects are concentration dependent. At low concentration, adenosine A1 receptor stimulation induces DVR constriction. At high concentration, A2 effects predominate

Table II Solute Permeability of Vasa Recta.

Permeability

OMDVR3

IMDVRb

IMAVRb

Species

x10-5 cm/sec

PNa

28

51

Hamster

PNa

76

75

115

Rat

PNa

67

116

Rat

PUrea

47

Rat

PUrea

360

76

121

Rat

PUrea

343 ^ 191c

Rat

Pd

476d

Rat

Praffinose

40

Rat

Permeability

OMDVR3

IMDVRb

IMAVRb

Species

ratio

PUrea/PNa

1.09

0.98

Rat

PCl/PNa

1.33

Rat

Praffinose/PNa

0.35

Rat

PInulin/PNa

0.22

Rat

Abbreviations: OMDVR, outer medullary descending vasa recta; IMDVR, inner medullary descending vasa recta; IMAVR, inner medullary ascending vasa recta.

3 Values obtained with in vitro microperfusion are highly dependent upon perfusion rate.

b Values obtained with in vivo microperfusion in the exposed papilla are probably underestimated due to boundary layer effects.

c Values are before and after inhibition with 50 mM thiourea. d Diffusional water permeability measured with 3H2O efflux. References to original data in Ref. [6].

Table III Mediators That Constrict and Dilate DVR.

Constriction3

Dilation

Receptor studiesb

Angiotensin

+++

+

AT1, AT2

ATl

at2

Endothelins

++++

-

eta, etb

eta > etb

Vasopressin

++

+

V1

V1

V2

Adenosine

++

+

A1, A2a, A2b

Al

A2a, A2b

Prostaglandin E2

-

+

-

Nitric oxide

-

+

-

Kinins

+

+

-

Bl

B2

Acetylcholine

++

+

-

Norepinephrine

++

-

a2B

3 Table entries refer to observations of constriction or dilation in response to receptor specific or nonspecific agonists. The intensity of constriction is graded from (-) to (++++).

b Table entries show the receptor subtypes expressed in DVR. Information is derived from studies that employed various methods, including RT-PCR, radioligand binding, and immunochemistry.

Figure 3 Vasoconstriction of outer medullary DVR. (A) DVR isolated and microperfused in vitro is exposed to AngII (10 nM) by abluminal application from the bath. Panels a and b show the vessel prior to and after constriction. Two cell types can be seen. Pericyte cell bodies project from the abluminal surface and endothelia line the lumen. (B) The graph shows quantification of DVR constriction through measurement of luminal diameter. Results are expressed as percent constriction = 100 x (Do - D)/Do, where Do is basal diameter and D is after constriction. The mean luminal diameter of perfused DVR is ~14 mm. Constriction has been induced by abluminal exposure to endothelin 1 (0.1 nM, n = 6) or AngII (10 nM, n = 15), or by raising extracellular K+ concentration from 5 to 100 mM by isosmotic substitution for NaCl (n = 6). Reproduced with permission from Ref. [2]. Renal Medullary Microcirculation in Encyclopedia of the Microcirculation, edited by David Shepro, Elsevier Inc.

Figure 3 Vasoconstriction of outer medullary DVR. (A) DVR isolated and microperfused in vitro is exposed to AngII (10 nM) by abluminal application from the bath. Panels a and b show the vessel prior to and after constriction. Two cell types can be seen. Pericyte cell bodies project from the abluminal surface and endothelia line the lumen. (B) The graph shows quantification of DVR constriction through measurement of luminal diameter. Results are expressed as percent constriction = 100 x (Do - D)/Do, where Do is basal diameter and D is after constriction. The mean luminal diameter of perfused DVR is ~14 mm. Constriction has been induced by abluminal exposure to endothelin 1 (0.1 nM, n = 6) or AngII (10 nM, n = 15), or by raising extracellular K+ concentration from 5 to 100 mM by isosmotic substitution for NaCl (n = 6). Reproduced with permission from Ref. [2]. Renal Medullary Microcirculation in Encyclopedia of the Microcirculation, edited by David Shepro, Elsevier Inc.

and adenosine dilates preconstricted DVR. Kinins increase DVR endothelial intracellular calcium concentration and promote nitric oxide generation through the bradykinin, B2 receptor. The cataloging of agents in Table III does not provide an integrated hypothesis of renal medullary function; however, the large number of agents to which DVR respond is clear. We assume that, in vivo, DVR vasomotor tone is governed by the integrated response to many hormonal and paracrine influences.

Pericyte Ca2+ Signaling and Channel Architecture

Recently, the mechanisms by which AngII induces vasoconstriction have been evaluated using fluorescent probes of intracellular calcium concentration and membrane potential and by electrophysiological recording. As expected for signaling via the AngII AT1 receptor, a classical peak and plateau [Ca2+]i response is elicited in globally fura2-loaded pericytes. Both electrophysiological recording and measurements with a potential-sensitive fluorescent probe showed that AngII depolarizes the pericyte, mediated primarily through activation of a Ca2+-sensitive Cl- conductance that shifts membrane potential away from the equilibrium potential of K+ ion toward that of Cl-. An 11 pS Ca2+-sensitive Cl- channel has been identified in DVR pericytes. Membrane potential of Angll-treated pericytes often oscillates and voltage-clamped cells held at -70 mV exhibit classical spontaneous transient inward currents (STICs) typical of various smooth muscle preparations [12, 13].

The role of membrane depolarization to gate Ca2+ entry had been well established in the afferent arteriole. Until recently, however, the existence of voltage-gated calcium entry pathways in the efferent circulation and DVR pericyte was uncertain. RT-PCR, immunochemistry, and examination of vasoreactivity in isolated arterioles verified expression of T-type and L-type calcium channel a subunits in efferent arterioles of juxtamedullary (but not superficial) glomeruli and in DVR [14]. Indeed, the L-type channel blocker diltiazem vasodilates Angll constricted DVR and reduces [Ca2+]i of AngII-treated pericytes. Both high external K+ concentration and the L-type agonist BAYK8644 are weak DVR vasoconstrictors. Finally, agents that repolarize pericytes, bradykinin and the KATP channel opener pinacidil, are effective vasodilators (Figure 4) [15]. The many down-

Figure 4 Repolarization of Angll depolarized pericytes by vasodilators. (A) Recording of membrane potential from a DVR pericyte successively exposed to Angll (10 nM) and then bradykinin (100 nM). A biphasic repolarization occurs after exposure to bradykinin. (B) Similar recording from a DVR pericyte exposed to AngII (10 nM) and then the KATP channel opener, pinacidil (10 mM). Both bradykinin and pinacidil repolarize pericytes and vasodilate preconstricted DVR. Reproduced with permission from Ref. [2]. Renal Medullary Microcirculation in Encyclopedia of the Microcirculation, edited by David Shepro, Elsevier Inc.

Figure 4 Repolarization of Angll depolarized pericytes by vasodilators. (A) Recording of membrane potential from a DVR pericyte successively exposed to Angll (10 nM) and then bradykinin (100 nM). A biphasic repolarization occurs after exposure to bradykinin. (B) Similar recording from a DVR pericyte exposed to AngII (10 nM) and then the KATP channel opener, pinacidil (10 mM). Both bradykinin and pinacidil repolarize pericytes and vasodilate preconstricted DVR. Reproduced with permission from Ref. [2]. Renal Medullary Microcirculation in Encyclopedia of the Microcirculation, edited by David Shepro, Elsevier Inc.

stream effects of pericyte Angll and endothelin receptor activation remain unknown, however actions independent of [Ca2+]i elevation must occur. Principally, depolarization in the absence of agonist induces far less intense constriction than does Angll or endothelins. Phosphorylation events that sensitize the intracellular contractile machinery to the effects of Ca2+ are likely to be implicated.

Endothelial Ca2+ Signaling and NO Production

The calcium-sensitive fluorophore fura2 loads avidly into DVR endothelia (to the near exclusion of pericytes) and so it has been possible to examine global intracellular Ca2+ transients generated by endothelium dependent vasodilators. Bradykinin (BK) generates a peak and plateau calcium response, enhances NO generation, and induces vasodilation. An unexpected finding is that the vasoconstrictor Angll suppresses basal calcium and inhibits BK, acetylcholine, thapsigargin, and cyclopiazonic acid induced calcium responses in DVR endothelia [16]. This is surprising because the effect is inhibited by high concentrations of the ATI blocker losartan and modulated by AnglI AT2 receptors [17]. AnglI and ATI receptors mediate the vast majority of effects by signaling through IP3 generation and calcium mobilization. Second, infusion of AngII has been observed to lead to secondary enhancement of NO levels within the medulla and in isolated cortical microvessels. Given that eNOS/NOS3 is a calcium-dependent isoform of nitric oxide synthase (NOS), suppression of calcium would be expected to block rather than enhance endothelial NO generation. Possibly, adjacent nephrons that also express NOS isoforms might be responding to generate NO on the vascular bundle periphery, providing a feedback loop through which those structures regulate their own perfusion. It has been hypothesized that AngII might suppress DVR endothelial Ca2+ signaling as a means of turning regulation of DVR vasoactivity away from the endothelium to the medullary thick ascending limb (mTAL).

The physiological roles of NO cannot be thoroughly evaluated without considering interactions with oxygen free radicals. Oxygen radicals result from reductions of O2 to generate superoxide (O2-), hydrogen peroxide (H2O2), hypochlorous acid, and hydroxyl radical (OH), the "reactive oxygen species" (ROS). O2- reacts with NO to form perox-ynitrite (ONOO-), a product that is a weak vasodilator. ROS are generated by "leak" of electrons from the mitochondrial electron transport chain and a variety of enzymatic processes. Intrinsic mechanisms limit cellular levels of ROS. Several isoforms of superoxide dismutase (SOD) convert O 2- to O2 and H2O2. The SOD mimetic tempol has been shown to enhance medullary perfusion. NO production by, and AngII constriction of, isolated DVR are both enhanced by tempol. Given the importance of NO in the maintenance of medullary blood flow, it is inviting to speculate that the level of "oxidative stress" in the renal medulla has physiological and pathophysiological regulatory roles.

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