Download In vivo evaluation of 2-adrenoceptors in cats with

In vivo evaluation of α2-adrenoceptors
in cats with idiopathic cystitis
Jodi L. Westropp, DVM, PhD; Philip H. Kass, DVM, PhD; C. A. Tony Buffington, DVM, PhD
Objective—To evaluate the in vivo response of α2-adrenoceptors to medetomidine administration in cats with feline idiopathic cystitis (FIC) during periods of stress and after environmental enrichment.
Animals—13 cats with FIC and 12 healthy cats.
Procedures—Cats were subjected to an acute-onset moderate stressor for 8 days. After
stress, 20 µg of medetomidine/kg was administered IM on days 1, 3, and 8. Heart rate,
blood pressure, pupil diameter, respiratory rate, and level of sedation were evaluated before
and after administration of the drug. After day 8, cats were moved to an enriched environment, and tests were repeated on day 35.
Results—Heart rate decreased and pupil diameter increased significantly after medetomidine administration in healthy cats, compared with cats with FIC. Cats with FIC had significantly lower respiratory rates. No significant differences in blood pressure or sedation level
were found.
Conclusions and Clinical Relevance—Increased plasma catecholamine concentrations
during the enrichment phase, which have been reported elsewhere, may have contributed
to the differences in α2-adrenoceptor responses detected in cats with FIC. (Am J Vet Res
2007;68:203–207)
D
iseases affecting the lower portion of the urinary tract
of cats are characterized by variable combinations
of stranguria, hematuria, urinations in inappropriate locations (periuria), and pollakiuria. There are several differential diagnoses for these clinical signs, including urolithiasis, urinary tract infections, and primary behavioral
abnormalities. When no underlying cause for these clinical signs can be found, a diagnosis of FIC is often made.
The etiology of this disease is unknown, and it appears to
affect male and female cats equally.
The clinical signs of FIC wax and wane and
are often reported by owners to be exacerbated by
stressful circumstances. Chronic stress from internal
and external sources can increase tyrosine hydroxylase activity, the rate-limiting step in catecholamine
synthesis, in the LC1 with accompanying increases
in autonomic outflow. Increased tyrosine hydroxylase immunoreactivity in the LC2 of cats with FIC
has been reported. Increased plasma catecholamine
concentrations, including dihydroxyphenylalanine,
norepinephrine, and dihyroxyphenylglycol, in cats
with FIC, compared with healthy cats, also have been
reported.3
Received April 14, 2006.
Accepted August 11, 2006.
From the Departments of Medicine and Epidemiology (Westropp)
and Population Health and Reproduction (Kass), School of
Veterinary Medicine, University of California, Davis, CA 95616;
and the Department of Veterinary Clinical Sciences, College of
Veterinary Medicine, The Ohio State University, Columbus, OH
43210 (Buffington).
Supported in part by NIH grants F32 DK09958 (JLW) and P50
DK64539 (CATB).
Address correspondence to Dr. Westropp.
FIC
LC
α2-AR
MEMO
Abbreviations
Feline idiopathic cystitis
Locus coeruleus
α2-Adrenoceptor
Multimodal environmental modification
The LC plays an integral part in modulating the
body’s response to stressful circumstances,4 mediated
largely through norepinephrine-containing neurons of the
sympathetic nervous system. Release of norepinephrine
from these neurons is controlled in part by a rich supply
of presynaptic autoinhibitory α2-ARs.5 Postsynaptic α2ARs also are present on some norepinephrine-containing
neurons, such as those modulating cardiovascular function6 and iris diameter.7 Neurons in the LC are relatively
inactive during periods of quiescence. During periods of
arousal, however, the firing rate of these neurons increases
and greater amounts of norepinephrine are released. The
α2-ARs are important in modulating those responses, and
in the spinal cord, they can help reduce nociceptive input
to the brain. The antinociceptive effects of the α2-ARs are
mediated via inhibition of adenylcyclase activity, activation of K+ currents, inhibition of Ca++ channels, and increased mitogen-activated protein kinase phosphorylation
through both pre- and postsynaptic neuronal membrane
receptors.8
Because high plasma catecholamine concentrations have been detected in cats with FIC, we hypothesized that α2-ARs could become downregulated because of increased exposure to their agonist. Results of
preliminary studies suggest abnormalities in the α2-ARs
of cats with FIC9,a and women with interstitial cystitis,10
an analogous disease in humans.
AJVR, Vol 68, No. 2, February 2007
203
The purpose of the study reported here was to
evaluate the in vivo response of α2-AR function by assessing sedation, heart rate, blood pressure, and pupil
diameter in healthy cats and cats with FIC after administration of the selective α2-AR agonist medetomidine.
Because clinical signs of FIC can increase during stress,
cats were evaluated at various times during and after a
moderate stress protocol, as previously described.3
Materials and Methods
Thirteen cats (3 neutered males, 1 sexually intact female, and 9 spayed females) obtained as donations from
clients because of a history of severe, recurrent stranguria;
hematuria; pollakiuria; and periuria or a combination of
these signs were evaluated at The Ohio State University
Veterinary Teaching Hospital. Twelve clinically normal
cats (3 sexually intact males, 1 neutered male, 7 sexually
intact females, and 1 spayed female) of similar age were
used as controls. All cats were initially housed in stainless steel cages in the animal colony and allowed to acclimate to their environment for at least 3 months. Cats
received a diagnosis of FIC or were deemed healthy on
the basis of evaluations that have been reported.3 The Animal Care and Use Committee of The Ohio State University
approved all experimental procedures. Other results from
these cats have been reported.3
A moderate stress protocol was administered for
8 days and additional procedures, including administration of medetomidine, were performed on each cat
on days 1, 3, and 8, as reported.3 Prior to medetomidine administration, a lux meterb was placed next to
the right eye and each cat was allowed to acclimate
to the light for 1 to 2 minutes. A midlateral pupil
measurement was obtained to the nearest millimeter. All values were approximately 430 lux for each
cat. Resting heart and respiratory rates and indirect
systolic blood pressure measured with an ultrasonic
Doppler flow detectorc with a No. 3 cuff on the left
pelvic limb were also evaluated (cats were placed in
right lateral recumbency for this measurement). As
part of the stress protocol, after these measurements,
a venous blood sample was obtained from the jugular
vein for analyses of catecholamine and sodium fluorescein concentrations, as reported.3
After venipuncture, 20 µg of medetomidine/kg
was administered IM into the epaxial muscles of each
cat and the time was recorded. Exactly 10 minutes
later, heart rate, blood pressure, pupil measurement,
respiratory rate, and level of sedation were recorded.
Level of sedation was assessed from 0 to 20 by evaluation of a number of variables including the posture
of the cat, resistance to being placed in lateral recumbency, and degree of jaw relaxation. A higher
value was interpreted to indicate a higher total sedation score.11 Atipamezole (300 µg/kg) was then administered to counteract any delayed effects of the
medetomidine.
Cats were tested in groups of 4, after which they
were placed in clean metabolism cages to continue the
stress protocol. Each cat received 100 g/d of a standard
feline commercial diet, and intake was recorded daily.
At the end of the moderate stress period (day 9), the
cats were moved to an enriched environment. Cages
204
were larger, and each cage contained a covered bed, 2
types of approved toys, and a larger litter pan. The cats
were fed the commercial dry food as before and also
were offered a commercial canned cat food. All food
was weighed daily, and the intake of each type was recorded. All cats had interaction with humans (in addition to the animal caretakers) ≥ 15 min/d. Music also
was played in the room during the day. On day 35, food
was withheld the night before testing and all the previous testing procedures were repeated.
Statistical analysis—The effects of experimental group (ie, cats with FIC vs healthy controls), time
(1, 3, 8, and 35 days), and treatment (before vs after
treatment) and their interactions were simultaneously
analyzed by use of 3-way repeated-measures ANOVA.
For dependent measures with ordinal scores, the MannWhitney test was used to compare groups at each time
point. Values of P < 0.05 were considered significant.
Results
No differences in food intake were detected
between the 2 groups at any time during the study.
Abnormalities pertaining to the lower portion of the
urinary tract were found in 1 healthy cat (a sexually
intact male) shortly after the study began. The cat was
removed from the study, and data obtained from the cat
were not included in the statistical analysis. In all 13
cats with FIC and 11 healthy cats, all variables were
evaluated on all testing days except for days 8 and 35;
variables could not be determined in 1 healthy cat on
each of these days because of injection errors.
Heart rates before administration of medetomidine were
not different between groups (Figure 1). After administration
of medetomidine, the decrease in heart rate was significantly
greater in healthy cats, compared with cats with FIC, except
on day 35 (Table 1). Cats with FIC had a significantly (P =
0.03) lower respiratory rate, compared with that of healthy
cats, throughout the study. Both groups had a significant (P
< 0.001) decrease in respiratory rate after medetomidine was
administered (Figure 2). No significant difference was detected across days (P = 0.43).
No significant difference in resting blood pressure
was identified between groups (P = 0.42). Mean blood
Figure 1—Heart rate (HR; mean ± SEM) on various days before
(white symbols) and after (black symbols) IM administration of
medetomidine (20 µg/kg) in 12 healthy cats (triangles) and 13
cats with FIC (squares); on days 1 through 8, cats were stressed,
whereas on days 9 through 35, cats were housed in a nonstress
enriched environment.
AJVR, Vol 68, No. 2, February 2007
Table 1—Mean ± SD percentage changes in heart rate 10
minutes after medetomidine (20 µg/kg, IM) was administered to
12 stressed healthy cats and 13 stressed cats with FIC; on days
1 through 8 cats were stressed, whereas on days 9 through 35
cats were housed in a nonstress enriched environment.
Day
1
3
8
35
FIC
Healthy
23  15
15  14
24  9
20  10
34  10
37  12
32  13
26  16
Figure 4—Sedation scores in the same cats as in Figure 1,
obtained 10 minutes after administration of medetomidine.
Table 2—Mean ± SD percentage changes in pupil diameter in the
same cats as in Table 1.
Day
Figure 2—Respiratory rate (RR; mean ± SEM) in the same cats
as in Figure 1.
Figure 3—Blood pressure (BP; mean ± SEM) in the same cats
as in Figure 1.
pressure was significantly (P < 0.001) lower 10 minutes
after medetomidine was administered (Figure 3) in
both groups. No differences were detected in percentage change in blood pressure between the 2 groups, although the change was slightly greater in healthy cats.
No significant difference in sedation score was identified between the 2 groups (P = 0.15; Figure 4). The
increase in pupil diameter was significantly (P = 0.004)
greater in healthy cats, compared with cats with FIC,
after medetomidine administration (Table 2). Furthermore, the percentage change in pupil diameter was significantly (P = 0.003) greater on days 3, 8, and 35 in
healthy cats, compared with that recorded on day 1.
Discussion
Significant differences in pupil and heart rate responses to medetomidine were detected between cats
with FIC and healthy cats. Medetomidine, a bridgemethylated derivative of detomidine used often in vet-
1
3
8
35
FIC Healthy
15  21
39  18
32  19
31  25
35  29
53  13
41  26
45  25
erinary medicine for sedation and restraint,12 is a potent
and selective pre- and postsynaptic α2-AR agonist and
appears to have more selectivity for the α2a-ARs than
for other ARs. The dose (20 µg/kg, IM) was chosen on
the basis of results of previous administration of this
drug to healthy, middle-aged cats, which resulted in
moderate sedation in most cats, as well as from doses
reported in the literature.13,14 Although medetomidine
has a much stronger α2-to-α1 AR selectivity ratio than
other α2-AR agonists do, higher doses were not used to
attempt to reduce the likelihood of stimulating α1-ARs,
which would counteract the sedation.12
The α2-ARs have a wide variety of functions, particularly in the cardiovascular and nociceptive systems.
In addition to modulating vasoconstriction and iris diameter, α2-ARs are involved in platelet aggregation (via
the α2a-ARs), neurotransmitter release, and antinociception.15 In the urinary bladder, α2-ARs are found primarily in the urethral submucosa and bladder mucosa
(not the muscle), suggesting a role in the regulation of
blood flow and urethral lubrication, but not on smooth
muscle.16 However, α2-ARs function as autoreceptors to
decrease norepinephrine outflow and could alter contractility through this mechanism. Alternatively, the
mucosa could influence contractility via its sensor and
transducer functions.17
Decreased response to medetomidine administration was found in cats with FIC, compared with healthy
cats, for heart rate and pupil dilation, but the reason
for these differences was unclear. We hypothesized that
high norepinephrine concentrations would adversely
affect the α2-ARs in cats with FIC because of chronic
agonist stimulation, although the exact mechanism for
the differences could not be elucidated from this study.
Heart rate is controlled, in part, by central postsynaptic
α2-ARs in the nucleus of the solitary tract.18 Pupil dilation occurs via stimulation of centrally located, post-
AJVR, Vol 68, No. 2, February 2007
205
synaptic α2-ARs located in the Edinger-Westfal nucleus.
Stimulation of these receptors results in pupil dilation
caused by CNS inhibition of the parasympathetic tone
to the iris.19,20 After medetomidine administration to
both groups of cats, both of these variables were significantly different between groups. Throughout the stress
protocol, catecholamine concentrations increased in
cats with FIC to a far greater extent than in healthy
cats. This difference was diminished after environmental enrichment, a time during which norepinephrine
(and other catecholamine) concentrations substantially
decreased.3
Results of an in vitro study9 of electrical field stimulation of bladder strips from cats with FIC indicated
that atipamezole, an α2-AR antagonist, does not alter
the relaxing effect of norepinephrine, providing further evidence for abnormalities in these receptors. In
other species, chronic stress is also reported to cause
desensitization of the α2-ARs. For example, results of
an in vitro study21 evaluating various brain sections
from tree shrews subjected to chronic stress indicate
that the number of binding sites for the α2 antagonist
3[H]rauwolscine is decreased, suggesting downregulation of the α2-ARs because of high noradrenergic activity. An in vivo study by Gomez et al22 also provides
evidence that chronic stress may lead to desensitization
of the postsynaptic α2-ARs in rats via evaluation of the
clonidine-induced jaw-opening reflex. Other authors23
have detected functional desensitization of central
postsynpatic α2-ARs in rats after repeated tail pinching
as a form of chronic variable stress. In that study, the
postsynaptic α2-ARs appeared to become functionally
desensitized first. Desensitization is usually rapid and
reversible, suggesting that receptors are internalized
but not lost.
In the present study, no differences were detected between the 2 groups in regards to sedation and blood pressure after medetomidine administration. Sedative effects
of α2-AR agonists are mediated from stimulation of the
presynaptic α2-AR located primarily on the LC in the pons
and lower portion of the brainstem,24 whereas heart rate,18
blood pressure,12 and pupil dilation20 are primarily postsynaptic. A variable response in sedation in both groups
was also evident. This could be attributable to variable
drug absorption when medetomidine is administered IM,
as has been reported in dogs.25 Sex or individual animal
differences in α2-AR susceptibility to norepinephrine-mediated desensitization also may have been present. Moreover, if abnormalities exist in the α2-ARs, this could affect
peripheral perfusion and absorption of the drug. In future
studies, the drug dose should be calculated and the drug
administered IV to avoid this potential problem.
Although both groups had a significant decrease in
blood pressure after administration of medetomidine,
no differences were detected between the groups. Blood
pressure of cats with FIC decreased by only 9% and
8% on days 1 and 3, respectively, whereas it decreased
by 15% and 12% on these days in the healthy cats, although this difference was not significant.
Interestingly, cats with FIC had a significantly
lower respiratory rate than did healthy cats, although
respiratory rates decreased significantly in both groups
after medetomidine was administered. We expected
206
cats with FIC to have higher respiratory rates because
of their greater stress responsiveness.26 Respiratory rate
alone thus may not be a good indicator of stress. Some
cats may respond to stress with inactivity and minimal
movements as part of a defense mechanism to avoid detection by predators. A lower respiratory rate also has
been reported in humans27 after chronic stress.
The α2-ARs are complex receptors; they are coupled
to G proteins (usually inhibitory [Gi proteins]) and use
many second messenger systems to carry out the response to agonist binding. Abnormalities of some of
these downstream effector systems have been detected
in other chronic stressful diseases that commonly occur
in humans with interstitial cystitis. For example, hypofunctional G proteins have been reported in humans
with migraine headaches and fibromyalgia.28,29 Abnormalities in α2-ARs, G protein, or second messengers
could permit increased nociceptive input to the brain
and various other abnormalities that could perpetuate
clinical signs of FIC and interstitial cystitis, especially
during periods of stress. Adrenergic receptor gene polymorphisms also have been reported in humans with interstitial cystitis10 and in cats with FIC.30
Unfortunately, in this study, we were unable to
match the groups regarding sex. Although the male and
female numbers in general were similar (FIC, 3 males
and 10 females; healthy, 3 males and 8 females), the
control population contained more sexually intact cats.
The cats with FIC were obtained as donations, and most
had been neutered, whereas many of the control cats
were purchased and had not been neutered. No data in
the literature were found to evaluate the differences in
α2-ARs in male versus female or sexually intact versus
neutered cats. Results of a study31 in healthy premenopausal women indicate greater α-AR sensitivity than
in men because a lower infusion rate of phenylephrine
(primarily an α1-AR agonist, not an α2-AR agonist) was
required to increase systolic blood pressure. Responses to phenylephrine and clonidine (an α2-AR agonist)
included substantial dose-related vasoconstriction in
healthy men but not in healthy women.32 However, 1
study33 compared α2a-AR binding sites in postmenopausal women with those of healthy women of reproductive age. Although differences were found in imidazoline receptors, no differences were found in platelet
α2a-AR densities and no differences were detected during estrogen replacement therapy.
In the present study, the increases in dihydroxyphenylalanine, norepinephrine, and dihyroxyphenylglycol concentrations3 could have contributed to the
decreased α2-AR responses detected in cats with FIC.
Continual agonist stimulation could cause desensitization of the receptors. By day 35, catecholamine
concentrations were lower and only mild differences
in percentage pupil dilation were detected between
the 2 groups of cats. On the basis of these data, treatment strategies aimed at decreasing sympathetic tone
in hopes of improving α2-AR function have been developed and tested in client-owned cats with FIC.34 In
this study, owners of 46 indoor-housed cats with FIC
were offered recommendations for MEMO based on a
detailed environmental history. Cats had follow-up for
10 months via client contact to determine the effect of
AJVR, Vol 68, No. 2, February 2007
MEMO on lower urinary tract signs and other clinical signs. Significant reductions in lower urinary tract
signs, fearfulness, nervousness, and signs referable to
the respiratory tract were identified. These results suggest that MEMO may be a useful adjunctive approach
to therapy for indoor-housed cats with FIC caused by
decrements in noradrenergic outflow.
a.
b.
c.
Buffington CAT. Functional assessment of α-2 adrenoreceptor
sensitivity in cats with interstitial cystitis (abstr). Soc Neurosci
1998;24:595.
Lutron LX-101 lux meter, Lutron Electronic Enterprise Co Ltd,
Taipei, Taiwan.
Model 811-BL, Parks Medical Electronics Inc, Aloha, Ore.
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