| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 First Department of
Physiology, Physiological
roles of ATP-sensitive K+ channels
for spontaneous activity in isolated rat mesenteric lymph microvessels
(maximum diameter ~80-150 µm) were investigated. The lymph
microvessels were cannulated with glass micropipettes and pressurized
at a perfusion pressure of 6 cmH2O. Changes in the diameter and
frequency of spontaneous contractions in the lymphatics were measured
with videomicroscopy. Pinacidil
(K+-channel opener) inhibited the
spontaneous activity. In the presence of glibenclamide (selective
ATP-sensitive K+-channel blocker;
10
pinacidil; glibenclamide; iberiotoxin; tetraethylammonium
THE LYMPHATIC SYSTEM plays an
important role in regulating the transport of extracellular fluids and
macromolecular substances in tissues. Thus lymphatic vessels act to
return fluid and protein that escape from the capillary blood vessels
to the circulation. In the process of transport, the escaped fluid and
protein enter into the initial microlymphatics by a transient pressure
gradient between the interstitial space and the initial lymphatics (1, 14). To accomplish these tasks, larger collecting lymph vessels work as
a series of lymphatic pumps that propel the lymph fluid centripetally
by rhythmic constriction and dilation.
It is well known that humoral and neural factors affect the spontaneous
activity of lymphatic smooth muscles in the collecting lymphatics (9).
The spontaneous activity in rat (6) and bovine (2) collecting
lymphatics, as well as blood vessels (5, 16), depends on extracellular
Ca2+ and a voltage-dependent
Ca2+ channel. The electrogenic
sodium pump has also been known to modulate spontaneous activity in
isolated bovine mesenteric lymphatics (10). Recently, some
investigators reported that K+
channels regulate membrane potential of lymphatic smooth muscles in
sheep and guinea pig mesenteric lymphatics (4, 17).
ATP-sensitive K+ channels, which
were first found in cardiac muscle (8), are located in the plasma
membrane of cells including vascular and nonvascular smooth muscle
cells (13) and participate in the regulation of the membrane potential.
The intracellular concentration of ATP is a determinant to activate and
deactivate ATP-sensitive K+
channels. Thus ATP produced by respiratory activity and metabolic demand in the cells may contribute to feedback mechanisms that control
cell functions through an activation of ATP-sensitive K+ channels. In addition, it is
well known that lymphatic smooth muscles containing numerous
mitochondria and glycogen granules and blood capillaries within the
lymphatic walls (12) may play an essential role in maintaining rigorous
spontaneous contractions in bovine mesenteric lymph vessels. The
presence of numerous mitochondria and glycogen granules also seems to
produce a lot of ATP in the cells in collecting lymphatics with
spontaneous activity and seems to reflect a high metabolic activity of
the lymphatic smooth muscles. We hypothesized that lymphatic
spontaneous activity may be related to the ATP-sensitive
K+ channels of lymphatic smooth
muscles. The present study was undertaken to investigate the
involvement of ATP-sensitive K+
channels in the regulation of spontaneous activity in isolated rat
mesenteric lymph microvessels.
Six-week-old male Wistar rats (~150 g,
n = 15) were used for the present
studies. The rats were housed in an environmentally controlled vivarium
and were fed a standard pellet diet and water ad libitum. All
experimental protocols were approved by the Animal Ethics Committee,
Shinshu University School of Medicine, in accordance with the guiding
principles of the American Physiological Society.
Isolation and cannulation of lymph microvessels.
The rats were anesthetized with pentobarbital sodium (50 mg/kg ip). The
mesenteries were exposed by an incision of the abdomen, excised, and
placed on a petri dish containing cold (4°C) Krebs solution. The
Krebs solution contained (in mM) 120.0 NaCl, 5.9 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 NaH2PO4,
5.5 glucose, and 25.0 NaHCO3. With
microsurgical instruments and an operating microscope, we isolated
lymph microvessels (n = 15; ~80- to
150-µm maximum diameter, 3-mm length) and transferred them to a
vessel chamber (10 ml) containing two glass micropipettes and Krebs
solution at room temperature.
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
7 and
106 M) and
tetraethylammonium (TEA; nonselective
K+-channel blocker;
104 and
103 M), the
pinacidil-induced inhibition of the spontaneous contractions in lymph
microvessels was significantly reversed. Glibenclamide and TEA
themselves, however, did not affect the frequency of spontaneous activity in the lymph microvessels. These results suggest that ATP-sensitive K+ channels are
involved in the regulation of spontaneous activity in the smooth
muscles of isolated lymph microvessels of rat mesenteries.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Measurement of diameter in lymph microvessels. The lymph microvessel image was obtained through an objective lens (×4), a photo eyepiece lens (×5), and a monochrome charge-coupled device camera (KP-M1, Hitachi). Changes in the diameter of lymph microvessels in response to vasoactive agents were manually and automatically measured with a domestically made diameter detection device, calibrated with a stage micrometer (Nikon), and recorded on a videocassette recorder (Sony) and a direct-writing recorder (Sanei-Sokki, Recti 8K). The perfusion pressure in the microvessels was increased to 6 cmH2O by elevating a 50-ml syringe connected to the inflow tubing while the outflow tubing was closed with a stopcock throughout the experiment. Because oscillation in the diameter of lymph microvessels causes a small change in the volume of the vessels (6, 7), we used a 50-ml syringe as a reservoir to minimize the changes in pressure. The height of the pressure column was constant throughout the experiment. This perfusion pressure is somewhat at the high end of the normal physiological pressure range for these vessels but is known to be optimal for producing stable spontaneous activity (6, 7). Thus the activity may be overstretch-induced spontaneous contractions of the isolated microlymph vessels.
Experimental protocols.
A single concentration
(107, 3 × 107,
106, 3 × 106, or
105 M) of pinacidil
(K+-channel opener) was perfused
into the vessel chamber over 3 min to construct a single dose-response
curve for pinacidil in each lymph microvessel. Dose-dependent responses
of the lymph microvessels for pinacidil were also obtained in the
absence and presence of glibenclamide (selective ATP-sensitive
K+-channel blocker;
107 and
106 M), tetraethylammonium
(TEA; nonselective K+-channel
blocker, 104 and
103 M), and iberiotoxin
(IbTX; selective Ca2+-activated
K+-channel blocker,
109 and
108 M), respectively. The
vessels were incubated with the various blockers for 30 min before
responses to the vasoactive agents were measured.
Drugs. Salts were obtained from Wako, pinacidil, glibenclamide, and IbTX from Research Biochemicals International, and TEA from Sigma. Pinacidil and glibenclamide were diluted with DMSO. The DMSO concentration did not exceed 0.025% in the vessel chamber. DMSO in the concentration used did not affect the spontaneous activity of lymph microvessels. Concentrations of drugs were expressed as a final concentration in the vessel chamber. All salts and drugs were prepared on the day of the experiment.
Statistical analyses. Pinacidil-induced inhibitory response of spontaneous activity was expressed as a percentage of total numbers of spontaneous contractions developed during 3-min superfusion of pinacidil against those elicited during 3 min before the administration of the agent. The data are presented as means ± SE, and n indicates the number of vessels. Significant differences (P < 0.05) were determined by one-way ANOVA, followed by Duncan's post hoc test.
| |
RESULTS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Isolated lymph microvessels of rat mesenteries exhibited
overstretch-induced spontaneous constriction and dilation at an
intraluminal pressure of 6 cmH2O.
The maximum and minimum diameters of lymph microvessels were 119.5 ± 4.4 and 47.7 ± 3.3 µm, respectively (n = 15). The frequency of
spontaneous activity of the lymph microvessels was 21.6 ± 0.7 min1
(n = 15).
Effects of pinacidil on lymphatic spontaneous activity.
Figure 1 shows representative tracings of
the effects of pinacidil (3 × 107-105
M) on spontaneous activity in an isolated lymph microvessel. Pinacidil
at 3 × 107 M produced
a slight increase in the periods between spontaneous contractions just
before the agent was washed out. Similar increases in the
periods between spontaneous contractions were observed at 2-3 min
after the superfusion of
106 M pinacidil. Pinacidil
caused a dose-dependent inhibition of spontaneous lymphatic activity
and then resulted in an increase in the periods of cessation of
spontaneous contractions in lymph microvessels. The higher the
concentrations of pinacidil used, the longer the cessation periods of
spontaneous contraction seemed to become during the superfusion of
pinacidil and after the agent was washed out. Glibenclamide, TEA, and
IbTX themselves did not affect the frequency of the spontaneous
contractions in the lymph microvessels (Table
1).
|
|
Effects of
K+-channel
blockers on pinacidil-induced response in lymphatic spontaneous
activity.
Figure 2 demonstrates representative
recordings of the effects of glibenclamide
(107 and
106 M) on pinacidil (3 × 106 M)-induced
inhibition of the overstretch-induced spontaneous activity in the lymph
microvessel. The pinacidil-induced responses were significantly
reversed by pretreatment with
106 M glibenclamide (Fig.
2). Glibenclamide (107
and 106 M) significantly
reduced the pinacidil (3 × 106 M)-induced inhibition
(control; 54.3 ± 3.9%) to 81.1 ± 6.1 and 98.2 ± 1.8%,
respectively. These experimental findings are summarized in
Fig. 3.
|
|
4
and 103 M), the pinacidil
(3 × 106 M)-induced
inhibition of spontaneous activity in the lymph microvessel was also
significantly reversed (Fig. 4). Such
responses to TEA are summarized in Fig. 5.
The percent change in the inhibition of spontaneous activity produced
by 3 × 106 M
pinacidil in the absence of TEA and with
104 and
103 M TEA were 48.6 ± 9.3, 70.4 ± 7.8, and 92.9 ± 3.1%
(P < 0.05 vs. control),
respectively. IbTX (109 and
108 M) did not have an
effect (data not shown) on
105 M pinacidil-induced
inhibition of spontaneous activity in isolated rat mesenteric lymph
microvessels (control, 28.4 ± 6.0%;
109 M IbTX, 24.7 ± 6.3%; 108 M IbTX, 22.0 ± 9.1%).
|
|
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
The salient findings of the present study are that overstretched (6 cmH2O) rat mesenteric lymph microvessels exhibit spontaneous mechanical activities in vitro and that ATP-sensitive K+ channels on lymphatic smooth muscles may regulate spontaneous activity in the lymph microvessels through modification of pacemaker activity, contractile activity, or myo-myogenic conduction.
Spontaneous activity in isolated rat mesenteric lymph microvessels.
In the present study, afferent lymph microvessels isolated from rat
mesenteric lymph nodes demonstrated stable spontaneous mechanical
activity at a perfusion pressure of 6 cmH2O. The maximum and minimum
diameters in the spontaneous activity were 119.5 ± 4.4 and 47.7 ± 3.3 µm, respectively (n = 15).
Frequency of the spontaneous activity was 21.6 ± 0.7 min1. The mechanical
parameters of spontaneous activity in isolated rat mesenteric lymphatic
microvessels are quite compatible with those obtained with afferent
lymph microvessels isolated from rat iliac lymph nodes (6). In in vivo
studies of rat mesenteries, the lymph vessels also showed spontaneous
pumping activity. The frequency of the beatings was ~5
min1 (3, 18). The
difference in frequency of spontaneous activity between in vivo and in
vitro experiments may be related to an intraluminal pressure
influencing the wall tension of the lymph vessels (6, 11). Thus the
faster rhythm of spontaneous contractions in the present experiment
may be related to the stimulation of overstretch in the isolated lymph
microvessels. Although in vivo experiments appear to be under
physiological conditions, it is difficult to regulate the intraluminal
pressure and flow rate of lymph, humoral, and neural factors. Thus the
isolated mesenteric lymph microvessels were adapted in the present
study to keep stable phasic spontaneous contractions at a controlled
perfusion pressure.
Effects of K+
channel on spontaneous activity in lymph microvessels.
In blood vessels, glibenclamide is a selective inhibitor of
ATP-sensitive K+ current that
produces relaxation of arterial smooth muscles (15). However, the
physiological roles of the ATP-sensitive
K+ current in lymphatic smooth
muscles remain unclear. The present study is the first demonstration of
the direct effects of a K+-channel
opener, pinacidil, on the spontaneous activity of the isolated lymph
microvessels. Low concentrations (3 × 107 M) of
pinacidil caused a slight increase in the periods between spontaneous
contractions in the lymph microvessels. Higher concentrations of
pinacidil caused a significant increase in the periods of cessation of
spontaneous activity in a dose-dependent manner. Glibenclamide significantly reduced the pinacidil-induced inhibitory responses on the
spontaneous activity of the lymph microvessels. The experimental findings suggest that activation of ATP-sensitive
K+ channels on the lymphatic
smooth muscles has produced a cessation of the spontaneous contractions
through an inhibition of the pacemaker activity, contractile activity,
and/or myo-myogenic conduction of the contractions. In contrast,
glibenclamide itself produces no effect on ACh-induced nitric
oxide-mediated hyperpolarization in lymphatic smooth muscles (17). In
the present study, glibenclamide itself caused no significant effect on
the spontaneous activity of the lymphatic smooth muscles. This
conclusion may be strongly supported by the present experimental
findings obtained with IbTX and TEA. Thus IbTX, a selective
Ca2+-activated
K+-channel blocker, did not affect
the pinacidil-induced inhibitory responses on the spontaneous activity
in isolated lymph microvessels. However, TEA, a nonselective
K+-channel blocker, significantly
reversed the pinacidil-induced inhibitory responses in isolated lymph microvessels.
| |
ACKNOWLEDGEMENTS |
|---|
This study was supported by Grants-in-Aid for Scientific Research (08457009 and 0987708) from the Japanese Ministry of Education, Science, Sports and Culture.
| |
FOOTNOTES |
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: T. Ohhashi, 1st Dept. of Physiology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, Japan (E-mail: ohhashi{at}sch.md.shinshu-u.ac.jp).
Received 21 December 1998; accepted in final form 7 June 1999.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
1.
Aukland, K.,
and
R. K. Reed.
Interstitial-lymphatic mechanisms in the control of extracellular fluid volume.
Physiol. Rev.
73:
1-78,
1993
2.
Azuma, T.,
T. Ohhashi,
and
I. C. Roddie.
Bradykinin-induced contractions of bovine mesenteric lymphatics.
J. Physiol. (Lond.)
342:
217-227,
1983
3.
Benoit, J. N.,
D. C. Zawieja,
A. H. Goodman,
and
H. J. Granger.
Characterization of intact mesenteric lymphatic pump and its responsiveness to acute edemagenic stress.
Am. J. Physiol.
257 (Heart Circ. Physiol. 26):
H2059-H2069,
1989
4.
Cotton, K. D.,
M. A. Hollywood,
N. G. McHale,
and
K. D. Thornbury.
Outward currents in smooth muscle cells isolated from sheep mesenteric lymphatics.
J. Physiol. (Lond.)
503:
1-11,
1997
5.
Dörnyei, G.,
E. Monos,
G. Kaley,
and
A. Koller.
Myogenic responses of isolated rat skeletal muscle venules: modulation by norepinephrine and endothelium.
Am. J. Physiol.
271 (Heart Circ. Physiol. 40):
H267-H272,
1996
6.
Mizuno, R.,
G. Dörnyei,
A. Koller,
and
G. Kaley.
Myogenic responses of isolated lymphatics: modulation by endothelium.
Microcirculation
4:
413-420,
1997[Medline].
7.
Mizuno, R.,
A. Koller,
and
G. Kaley.
Regulation of the vasomotor activity of lymph microvessels by nitric oxide and prostaglandins.
Am. J. Physiol.
274 (Regulatory Integrative Comp. Physiol. 43):
R790-R796,
1998
8.
Noma, A.
ATP-regulated K+ channels in cardiac muscle.
Nature
305:
147-148,
1983[Medline].
9.
Ohhashi, T.
Mechanisms for regulating tone in lymphatic vessels.
Biochem. Pharmacol.
45:
1941-1946,
1993[Medline].
10.
Ohhashi, T.,
and
T. Azuma.
Effect of potassium on membrane potential and tension development in bovine mesenteric lymphatics.
Microvasc. Res.
23:
93-98,
1982[Medline].
11.
Ohhashi, T.,
T. Azuma,
and
M. Sakaguchi.
Active and passive mechanical characteristics of bovine mesenteric lymphatics.
Am. J. Physiol.
239 (Heart Circ. Physiol. 8):
H88-H95,
1980.
12.
Ohhashi, T.,
S. Fukushima,
and
T. Azuma.
Vasa vasorum within the media of bovine mesenteric lymphatics.
Proc. Soc. Exp. Biol. Med.
154:
582-586,
1977[Medline].
13.
Quayle, J. M.,
M. T. Nelson,
and
N. B. Standen.
ATP-sensitive and inwardly rectifying potassium channels in smooth muscle.
Physiol. Rev.
77:
1165-1232,
1997
14.
Schmid-Schönbein, G. W.
Microlymphatics and lymph flow.
Physiol. Rev.
70:
987-1028,
1990
15.
Standen, N. B.,
J. M. Quayle,
N. W. Davies,
J. E. Brayden,
Y. Huang,
and
M. T. Nelson.
Hyperpolarizing vasodilators activate ATP-sensitive K+ channels in arterial smooth muscle.
Science
245:
177-180,
1989
16.
Sun, D.,
G. Kaley,
and
A. Koller.
Characteristics and origin of myogenic response in isolated gracilis muscle arterioles.
Am. J. Physiol.
266 (Heart Circ. Physiol. 35):
H1177-H1183,
1994
17.
Von der Weid, P.-Y.,
and
D. F. Van Helden.
Functional electrical properties of the endothelium in lymphatic vessels of the guinea-pig mesentery.
J. Physiol. (Lond.)
504:
439-451,
1997
18.
Zhang, J.-L.,
S. Yokoyama,
and
T. Ohhashi.
Inhibitory effects of fluorescein isothiocyanate photoactivation on lymphatic pump activity.
Microvasc. Res.
54:
99-107,
1997[Medline].
This article has been cited by other articles:
|
|
 |
|
  W. Wang, Z. Nepiyushchikh, D. C. Zawieja, S. Chakraborty, S. D. Zawieja, A. A. Gashev, M. J. Davis, and M. Muthuchamy Inhibition of myosin light chain phosphorylation decreases rat mesenteric lymphatic contractile activity Am J Physiol Heart Circ Physiol, August 1, 2009; 297(2): H726 - H734. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Kousai, R. Mizuno, F. Ikomi, and T. Ohhashi ATP inhibits pump activity of lymph vessels via adenosine A1 receptor-mediated involvement of NO- and ATP-sensitive K+ channels Am J Physiol Heart Circ Physiol, December 1, 2004; 287(6): H2585 - H2597. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Yashiro and B. R. Duling Participation of intracellular Ca2+ stores in arteriolar conducted responses Am J Physiol Heart Circ Physiol, June 5, 2003; 285(1): H65 - H73. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Hosaka, R. Mizuno, and T. Ohhashi Rho-Rho kinase pathway is involved in the regulation of myogenic tone and pump activity in isolated lymph vessels Am J Physiol Heart Circ Physiol, June 1, 2003; 284(6): H2015 - H2025. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Nakaya, R. Mizuno, and T. Ohhashi B16-BL6 melanoma cells release inhibitory factor(s) of active pump activity in isolated lymph vessels Am J Physiol Cell Physiol, December 1, 2001; 281(6): C1812 - C1818. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Mizuno, N. Ono, and T. Ohhashi Parathyroid hormone-related protein-(1-34) inhibits intrinsic pump activity of isolated murine lymph vessels Am J Physiol Heart Circ Physiol, July 1, 2001; 281(1): H60 - H66. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
, Starting point of superfusion of pinacidil;
, starting point of washing out of pinacidil.
) and
10
) M] on
pinacidil-induced inhibitory responses on spontaneous activity in
isolated rat mesenteric lymph microvessels. Ordinate denotes percent
inhibition of spontaneous contractions, which is defined as
[(total nos. of developed spontaneous contractions during 3-min
superfusion of pinacidil)/(those elicited during 3 min before start of
superfusion)] × 100; abscissa shows concentration of
pinacidil on a logarithmic scale. * Significant difference
(P < 0.05) from control (
).
