J Physiol 561.3 (2004) pp 671–683
Effects of angiotensinII on the pericyte-containing
microvasculature of the rat retina
Hajime Kawamura1, Masato Kobayashi1, Qing Li1, Shigeki Yamanishi1, Kozo Katsumura1,
Masahiro Minami1, David M. Wu1,2and Donald G. Puro1,2,3
1Department of Ophthalmology & Visual Sciences,2Neuroscience Graduate Program and3Department of Molecular and Integrative Physiology,
University of Michigan, Ann Arbor, MI 48105, USA
The aim of this study was to identify the mechanisms by which angiotensinII alters the
physiology of the pericyte-containing microvasculature of the retina. Despite evidence that
this vasoactive signal regulates capillary perfusion by inducing abluminal pericytes to contract
and thereby microvascular lumens to constrict, little is known about the events linking
angiotensin exposure with pericyte contraction. Here, using microvessels freshly isolated from
the adult rat retina, we monitored pericyte currents via perforated-patch pipettes, measured
by time-lapse photography. We found that angiotensin activates nonspecific cation (NSC) and
calcium-activated chloride channels; the opening of these channels induces a depolarization
that is sufficient to activate the voltage-dependent calcium channels (VDCCs) expressed in the
retinal microvasculature. Associated with these changes in ion channel activity, intracellular
calcium levels rise, pericytes contract and microvascular lumens narrow. Our experiments
revealed that an influx of calcium through the NSC channels is an essential step linking the
activation of AT1angiotensin receptors with pericyte contraction. Although not required in
order for angiotensin to induce pericytes to contract, calcium entry via VDCCs serves to
enhance the contractile response of these cells. In addition to activating nonspecific cation,
calcium-activated chloride and voltage-dependent calcium channels, angiotensinII also causes
the functional uncoupling of pericytes from their microvascular neighbours. This inhibition
of gap junction-mediated intercellular communication suggests a previously unappreciated
complexity in the spatiotemporal dynamics of the microvascular response to angiotensinII.
(Received 31 July 2004; accepted after revision 13 October 2004; first published online 14 October 2004)
Corresponding author D. G. Puro: Department of Ophthalmology and Visual Sciences, University of Michigan, 1000
Wall Street, Ann Arbor, MI 48105, USA. Email: firstname.lastname@example.org
The retina contains a rennin–angiotensin system (Kohler
et al. 1997) that may play a role in regulating blood flow
to angiotensinII causes retinal arterioles, capillaries and
more sensitive than the larger vessels (Schonfelder et al.
1998; Kulkarni et al. 1999). Thus, angiotensinII is likely
to serve as a vasoactive signal regulating microvascular
perfusion in the retina.
Candidates for regulating blood flow at the capillary
these cells are thought to control capillary perfusion
(Tilton, 1991; Schonfelder et al. 1998; Kawamura et al.
2003). Suggestive of the particular importance of these
cells in the retinal microvasculature, the density of
pericytes is higher in the retina than in other tissues
(Shepro & Morel, 1993). However, at present, there is
only limited knowledge of the mechanisms by which
vasoactive molecules, such as angiotensinII, regulate
pericyte contractility and thereby lumen diameter and
local blood flow. Consequently, the goal of this study
was to identify events linking exposure of retinal
microvessels to angiotensinII with pericyte contraction
Based on the premise that ion channels are important
in mediating functional responses to vasoactive signals,
we assessed the effects of angiotensinII on the ionic
currents in pericyte-containing microvessels. We now
report that in microvessels freshly isolated from the rat
of ion channels, including ones that provide pathways for
revealed that an influx of calcium via nonspecific cation
C ?The Physiological Society 2004DOI: 10.1113/jphysiol.2004.073098
672H. Kawamura and others
J Physiol 561.3
receptors with pericyte contraction and vasoconstriction.
cell-to-cell communication within retinal microvessels.
of individual pericytes, but also modifies the multicellular
functional organization of the retinal microvasculature.
for Research in Vision and Ophthalmology and the
Animals. As detailed previously (Kawamura et al. 2003),
6- to 8-week Long-Evans rats (Harlan Sprague-Dawley,
Inc., Indianapolis, IN and Charles Rivers, Cambridge,
MA, USA) were killed with a rising concentration of
carbon dioxide, and their retinas were rapidly removed
and incubated in 2.5ml Earle’s balanced salt solution,
which was supplemented with 0.5mm EDTA, 20mm
glucose, 15upapain (Worthington
Freehold, NJ, USA), and 2mm cysteine for 30min
at 30◦C and bubbled with 95% oxygen–5% carbon
dioxide in order to maintain pH and oxygenation.
After transfer to solution A (mm: 140 NaCl, 3 KCl,
1.8 CaCl2, 0.8 MgCl2, 10 Na-Hepes, 15 mannitol,
and 5 glucose at pH7.4 with osmolarity adjusted to
310mosmol1−1), each retina was then gently sandwiched
between two glass coverslips (15mm diameter, Warner
Instrument Corp., Hamden, CT, USA). As reported
previously (Sakagami et al. 1999a; Kawamura et al.
2003), vessels adhered to the coverslip that was in
contact with the vitreal side of the retina. By repeating
this tissue print step, several coverslips containing
microvessels could be obtained from a retina. Figure1
shows a photomicrograph of a segment of a freshly
isolated pericyte-containing microvessel. Other photo-
graphs of retinal microvessels isolated by this method
are in Sakagami et al. (1999), Oku et al. (2001) and Wu
et al. (2001); in addition, a time-lapse video of an isolated
Figure 1. Differential interference contrast photomicrograph of
a portion of a pericyte-containing microvessel freshly isolated
from the rat retina
Arrowheads point to pericytes. Five red blood cells can be seen within
the lumen. Scale bar = 10 µm.
retinal microvessel is in the supplemental material. Our
experiments were performed on microvessels that were
>300µm in length.
As detailed elsewhere (Kawamura et al. 2003; Wu
et al. 2003), a glass coverslip containing isolated
pericyte-containing microvessels was positioned in a
perfusion chamber (volume=200µl) on the stage
of a Nikon Eclipse E800 equipped with differential
interference contrast optics. Microvessels were viewed
at×1000 magnification with the aid of a×100 oil
objective. After a 2.67min control period, microvessels
were exposed for 5.33min to an experimental solution
and then re-exposed to the control perfusate (solution A).
To facilitate detection of pericyte contractions, time-lapse
images were recorded at 8s intervals using a Nikon
DCM1200 digital camera and ImagePro Plus software
(Version 4.5, Media Cybernetics, Silver Spring MD,
contracted and relaxed were not included in our analyses.
As detailed previously (Wu et al. 2003), our calculation
of the probability that responding pericytes were located
near (≤30µm) capillary branch points was based on our
observation that 20% of the length of the monitored
microvessels was within 30µm of a bifurcation.
Measurement of lumen diameters at sites adjacent to
contracting pericytes was facilitated by use of ImagePro
Plus software. Because contracting pericytes could cause
microvascular lumens to move out of the narrow depth
of focus available with differential interference contrast
optics at high magnification, only those lumens that
remained in focus throughout an experiment were
included. During exposure to an experimental perfusate,
lumen diameters were measured when the change in
responsive vessels was maximal.
After freshly isolated microvessels were exposed to
Probes, Eugene, OR, USA) at 37◦C for 30min, the
extracellular fura-2AM was washed out with solution A
for at least 30min. A coverslip containing microvessels
with fura-loaded cells was positioned in a perfusion
chamber (volume=1ml), which was perfused at
∼2mlmin−1. Digital imaging of fluorescence was
performed at room temperature using an intensified
CCD camera with a 12-bit dynamic range (Sensicam,
Cooke Corp., Auburn Hills, MI, USA); the light source
was a high-intensity mercury lamp coupled to a mono-
chromator (Cairn Research Ltd, Faverhsam, UK).
Imaging Workbench 5 (Indec BioSystems, Mountain
View, CA, USA) was used to control the imaging
equipment and collect data. Microvessels were viewed
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682H. Kawamura and others
J Physiol 561.3
of vasa recta pericytes from other microvascular cells, as
it does in the retinal microvessels. Also, it is not known
whether reactive oxygen species mediate angiotensin’s
vasoconstrictive effect in microvessels of the retina, as
they do in the descending vasa recta (Zhang et al. 2004).
Nor do we know whether the pericyte contraction
triggered by the activtion of AT1receptors is modulated
by the concomitant activation of AT2receptors, as occurs
in vasa recta pericytes (Zhang et al. 2004). We speculate
that the mechanisms mediating angiotensin’s effects on a
tissue’s microvasculature have been selected to meet the
specialized functional demands of that tissue. A challenge
for the future is to determine the adaptive advantages of
the various pathways by which angiotensinII can regulate
the physiology of pericyte-containing microvessels.
by which angiotensinII
experiments using freshly isolated retinal microvessels.
One benefit of studying microvessels in isolation is that
confounding effects mediated by nonvascular retinal cells
are eliminated. Also, it is possible to precisely control the
concentrations of agonists and inhibitors, as well as the
duration of exposure to these chemicals. Furthermore, in
contrast to cultured pericytes, use of isolated microvessels
time-lapse studies to be performed while these cells are
integral components of the microvasculature. This latter
advantage is of particular importance because pericytes
function as elements of multicellular functional units in
which intercellular communication is mediated via gap
junction pathways, which are regulated by vasoactive
signals (Kawamura et al. 2002; Kawamura et al. 2003)
and can be disrupted by pathophysiological conditions,
such as diabetes (Oku et al. 2001). However, even though
there are many experimental advantages to studying
freshly isolated microvessels, caution must be exercised.
For example, it remains to be demonstrated that the
effects of angiotensinII on ion channels, calcium levels,
pericyte contractility and cell-to-cell coupling occur
in vivo. Hence, an in vivo application of the imaging
and electrophysiological techniques used in this study
would be ideal, although at present this does not appear
to be feasible. Also, because intraluminal pressure is
likely to affect microvascular physiology, development of
methods to internally perfuse isolated microvessels will
be of importance. Yet, despite caveats, freshly isolated
microvessels provide an experimental preparation that
has allowed us to make new observations and propose
new hypotheses concerning the mechanisms by which
angiotensinII regulates the retinal microvasculature.
angiotensinII causes extracellular calcium to enter the
pericytes of retinal microvessels. As a result, these cells
contract, and microvascular lumens narrow. Adding
spatiotemporal complexity to these effects, angiotensinII
also inhibits intercellular communication. Thus, this
vasoactive signal not only regulates pericyte contractility,
but also affects the functional organization of the retinal
Barry PH (1994). JPCalc, a software package for calculating
liquid junction potential corrections in patch-clamp,
intracellular, epithelial and bilayer measurements and for
correcting junction potential measurements. J Neurosci Meth
Fernandes R, Girao H & Pereira P (2004). High glucose down-
regulates intercellular communication in retinal endothelial
cells by enhancing degradation of connexin 43 by a
proteasome-dependent mechanism. J Biol Chem 279,
Grynkiewicz G, Poenie M & Tsien RY (1985). A new generation
of Ca2+indicators with greatly improved fluorescence
properties. J Biol Chem 260, 3440–3450.
Herbert JM, Augereau JM, Gleye J & Maffrand JP (1990).
Chelerythrine is a potent and specific inhibitor of protein
kinase C. Biochem Biophys Res Commun 172,
Kawamura H, Oku H, Li Q, Sakagami K & Puro DG (2002).
Endothelin-induced changes in the physiology of retinal
pericytes. Invest Ophthalmol Vis Sci 43, 882–888.
Kawamura H, Sugiyama T, Wu DM, Kobayashi M, Yamanishi
S, Katsumura K & Puro DG (2003). ATP: a vasoactive signal
in the pericyte-containing microvasculature of the rat retina.
J Physiol 551, 787–799.
Kohler K, Wheeler-Schilling T, Jurklies B, Guenther E &
Zrenner E (1997). Angiotensin II in the rabbit retina. Vis
Neurosci 14, 63–71.
Kulkarni PS, Hamid H, Barati M & Butulija D (1999).
Angiotensin II-induced constrictions are masked by bovine
retinal vessels. Invest Ophthalmol Vis Sci 40, 721–728.
Kuwabara T & Cogan D (1960). Studies of retinal vascular
patterns. 1: normal architecture. Arch Ophthalmol 64,
Lampe PD, TenBroek EM, Burt JM, Kurata WE, Johnson RG &
Lau AF (2000). Phosphorylation of connexin43 on serine368
by protein kinase C regulates gap junctional communication.
J Cell Biol 149, 1503–1512.
Li Q & Puro DG (2001). Adenosine activates ATP-sensitive K+
currents in pericytes of rat retinal microvessels: role of A1
and A2a receptors. Brain Res 907, 93–99.
Li AF, Sato T, Haimovici R, Okamoto T & Roy S (2003). High
glucose alters connexin 43 expression and gap junction
intercellular communication activity in retinal pericytes.
Invest Ophthalmol Vis Sci 44, 5376–5382.
Lindau M & Neher E (1988). Patch-clamp techniques for
time-resolved capacitance measurements in single cells.
Pflugers Arch 411, 137–146.
Mayer ML & Westbrook GL (1987). Permeation and block of
N-methyl-d-aspartic acid receptor channels by divalent
cations in mouse cultured central neurones. J Physiol 394,
C ?The Physiological Society 2004
J Physiol 561.3
Responses of retinal microvessels to angiotensin II 683
Moreno AP, Saez JC, Fishman GI & Spray DC (1994). Human
connexin43 gap junction channels. Regulation of unitary
conductances by phosphorylation. Circ Res 74,
Moriarty P, Dickson AJ, Erichsen JT & Boulton M (2000).
Protein kinase C isoenzyme expression in retinal cells.
Ophthalmic Res 32, 57–60.
Nagahama T, Hayashi K, Ozawa Y, Takenaka T & Saruta T
(2000). Role of protein kinase C in angiotensin II-induced
constriction of renal microvessels. Kidney Int 57, 215–223.
Oku H, Kodama T, Sakagami K & Puro DG (2001).
Diabetes-induced disruption of gap junction pathways
within the retinal microvasculature. Invest Ophthalmol Vis
Sci 42, 1915–.
Pallone TL & Huang JM (2002). Control of descending vasa
recta pericyte membrane potential by angiotensin II.
Am J Physiol Renal Physiol 282, F1064–F1074.
Rhinehart K, Zhang Z & Pallone TL (2002). Ca2+signaling and
membrane potential in descending vasa recta pericytes and
endothelia. Am J Physiol Renal Physiol 283, F852–F860.
Sakagami K, Kawamura H, Wu DM & Puro DG (2001). Nitric
oxide/cGMP-induced inhibition of calcium and chloride
currents in retinal pericytes. Microvasc Res 62,
Sakagami K, Wu DM & Puro DG (1999). Physiology of rat
retinal pericytes: modulation of ion channel activity by
serum-derived molecules. J Physiol 521, 637–650.
Schonfelder U, Hofer A, Paul M & Funk RH (1998). In situ
observation of living pericytes in rat retinal capillaries.
Microvasc Res 56, 22–29.
Shepro D & Morel NM (1993). Pericyte physiology. FASEB J 7,
Suzuma I, Suzuma K, Ueki K, Hata Y, Feener EP, King GL &
Aiello LP (2002). Stretch-induced retinal vascular
endothelial growth factor expression is mediated by
phosphatidylinositol 3-kinase and protein kinase C
(PKC)-zeta but not by stretch-induced ERK1/2, Akt, Ras, or
classical/novel PKC pathways. J Biol Chem 277,
Tilton RG (1991). Capillary pericytes: perspectives and future
trends. J Electron Microsc Tech 19, 327–344.
Wu DM, Kawamura H, Li Q & Puro DG (2001). Dopamine
activates ATP-sensitive K+currents in rat retinal pericytes.
Vis Neurosci 18, 935–940.
Wu DM, Kawamura H, Sakagami K, Kobayashi M & Puro DG
(2003). Cholinergic regulation of pericyte-containing retinal
microvessels. Am J Physiol Heart Circ Physiol 284,
Zhang Z, Rhinehart K, Lee-Kwon W, Weinman E & Pallone T
(2004). AngII signaling in vasa recta pericytes by PKC and
reactive oxygen species. Am J Physiol Heart Circ Physiol 287,
Zhang Z, Rhinehart K & Pallone TL (2002). Membrane
potential controls calcium entry into descending vasa recta
pericytes. Am J Physiol Regul Integr Comp Physiol 283,
The authors thank Bret Hughes for helpful discussions and
use of equipment; Scott Salazay’s technical expertise is greatly
Training Award from the American Diabetes Association
a Harrington Senior Investigator Award (D.G.P.) from Research
the National Institutes of Health.
The online version of this paper can be accessed at:
and contains the following supplementary material.
Time-lapse movie showing a freshly isolated rat retinal
microvessel before, during and after addition of 500 nM
angiotensinII tothe perfusate
Differential interference contrast images were captured at 8 s
indicate when this pericyte-containing microvessel was exposed
to this vasoactive peptide. Two erythrocytes can be seen in
the microvascular lumen. During exposure to angiotensin II,
contraction of the pericyte located on the upper edge of the
microvessel caused the adjacent lumen to narrow.
This material can also be found at:
C ?The Physiological Society 2004