Clinical and Experimental Hypertension. Part A: Theory and Practice

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Calcium Suppresses Central Angiotensin II Pressor Response Less in Shr Hiroshi Itoh, Kazuo Takeda, Motoo Tanaka, Masahiro Hirata, Shingo Kawasaki, Junko Hayashi, Minako Oguro, Susumu Sasaki & Masao Nakagawa To cite this article: Hiroshi Itoh, Kazuo Takeda, Motoo Tanaka, Masahiro Hirata, Shingo Kawasaki, Junko Hayashi, Minako Oguro, Susumu Sasaki & Masao Nakagawa (1992) Calcium Suppresses Central Angiotensin II Pressor Response Less in Shr, Clinical and Experimental Hypertension. Part A: Theory and Practice, 14:6, 1017-1035, DOI: 10.3109/10641969209038190 To link to this article: http://dx.doi.org/10.3109/10641969209038190

Published online: 03 Jul 2009.

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Date: 16 March 2016, At: 21:53

CLIN. AND EXPER. HYPER.-THEORY AND PRACTICE, A14(6), 1017-1035 (1992)

CALCIUM SUPPRESSES CENTRAL ANGIOTENSIN I1 PRESSOR RESPONSE

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LESS IN SHR

Hiroshi Itoh, Kazuo Takeda, Motoo Tanaka, Masahiro Hirata, Shingo Kawasaki, Junk0 Hayashi, Minako Oguro, Susumu Sasaki and Masao Nakagawa Second Department of Medicine Kyoto Prefectural University of Medicine Kawaramachi Hirokoji, Kamikyo-ku Kyoto, 602 Japan Keywords: angiotensin 11, calcium, central nervous system, blood pressure, heart rate, plasma norepinephrine, intracerebroventricular injection. ABSTRACT

To determine whether calcium alters central cardiovascular regulation, cardiovascular responses to intracerebroventricular (ICV) injection of calcium were recorded in conscious Wistar rats. Calcium injection consistently produced dose-dependent decreases in mean parterial pressure and heart rate. Pretreatment with a calcium channel blocker, diltiazem, attenuated cardiovascular responses to calcium. Decreases in plasma norepinephrine indicated the contribution of sympatho-inhibition to vasodepression by calcium. Preceding calcium injection reduced pressor responses to ICV-injected angiotensin 11. These findings suggest that there is a pharmacological interaction between calcium and angiotensin I1 in the central nervous system. In spontaneously hypertensive rats (SHR), cardiovascular responses to calcium was larger than Wistar Kyoto rats (WKY).By contrast, calcium reduced pressor responses to angiotensin I1 only in WKY but not in SHR. Because the central interaction between calcium and angiotensin I1 has been different in SHR, our results imply that this difference may be related to the maintenance of high blood pressure in SHR.

1017 Copyright 0 1992 by Marcel Dekker, Inc.

1018

ITOH ET AL.

I " Aside from its well known peripheral antihypertensive effects

,)'s'(

calcium

also lowers blood pressure by acting centrally. Thus, calcium lowers blood pressure and heart rate when injected intracerebroventricularly(ICV) (3). However, the mechanisms responsible for the central depressor effect of calcium are still poorly understood. Downloaded by [McMaster University] at 21:53 16 March 2016

Much more is known about the central pressor effects of angiotensin 11. ICV injected angiotensin I1 elevates blood pressure

circumventricular organs release

(*).

u-7)

(4)

probably by acting on

to stimulate sympathetic activity and vasopressin

These pressor mechanisms are stronger in SHR than in WKY

(').

Because angiotensin I1 receptor binding is regulated by cytoplasmic calcium influx (lo),

excess calcium could lower blood pressure by suppressing central angiotensin

I1 pressor mechanisms.

Accordingly, our present studies had two main aims: first, to investigate mechanisms underlying the vasodepressor effects of centrally-administered calcium, and second, to examine calcium involvement in elevating blood pressure in spontaneously hypertensive rats. Preliminary experiments were done on normotensive Wistar rats to determine effective ICV calcium doses and identify mechanisms responsible for the ensuing depressor effects. Next, the possible involvement of calcium in elevating blood pressure of SHR was explored by comparing depressor responses to calcium in SHR and WKY and then determining whether calcium pretreatment would suppress pressor responses to angiotensin I1 differently in the 2 rat groups.

f l Ninety-three 9-week old rats, of which 65 were normotensive Wistar, 14 were Wistar Kyoto (WKY), and 14 were spontaneously hypertensive (SHR) were used. Normotensive Wistar rats were used for preliminary experiments to determine: (1) effective doses of ICV injected calcium, (2) effects of calcium

RESPONSES TO ICV INJECTION OF CALCIUM

1019

channel blockade with diltiazem on calcium-induced vasodepression, and (3) effects of ICV injected calcium on plasma norepinephrine concentration or angiotensin I1 pressor responses. Based on the results thereby obtained, hemodynamic responses to ICV injected calcium were compared in SHR and

WKY. Data from 10 rats were discarded for various technical reasons (i.e.

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incorrect placement of ICV cannulae or plugged vascular catheters). Anesthesia and Suryical C w l a t i on Rats were anesthetized with pentobarbital (3 mg/loO g, intraperitoneally: IP) while indwelling teflon cannulae were inserted separately into a femoral artery

for recording blood pressure and a femoral vein for drug injection and blood sampling. Each cannula was 3.5 cm long with the inner tip placed in the abdominal aorta or inferior vena cava, and the outer end exteriorized at the nape. Both cannulae were filled with heparinized saline (200 units/ml) to keep them

open. To implant a 24 gauge stainless steel guide cannula (2.5 cm long) into the left lateral cerebral ventricle, rats were mounted on the stereotaxic apparatus (David Kopf) using stereotaxic coordinates: anteroposterior, 5.6 mm; lateral, 1.7 mm; and dorsoventral, 3.0 mrn'"). The cannula was then fixed to the skull using dental cement and screws, and rats were allowed to recover for at least 24 hours. Cardiovascular Recor d.m on Conscious Rat After connecting the vascular cannulae with extension vinyl tubing (19 gauge) led out to the pressure transducer or injection syringe, rats were kept in individual cages and left untouched for 30-40 min to allow them adapt to the recording conditions . Pulsatile arterial pressure was measured by connecting the femoral artery cannula to a pressure transducer (MPV 290, NEC-Sanei) placed outside the cage at the same level as rats' heart. Heart rate was counted by a heart rate meter (NEC-Sanei) triggered by the pulsatile arterial pressure signal. Both blood pressure and heart rate were recorded continuously on separate channels of a 5-channel polygraph (141-16, NEC-Sanei).

ITOH ET AL.

1020

. .

merebroventncular In1ecbons For intracerebroventricular (ICV) injections, a piece of 30-gauge stainless steel tubing was used as an inner cannula and inserted into the guide cannula implanted on the skull. ICV injections of calcium chloride, diltiazem, and angiotensin I1 were delivered manually in 10 seconds in a 10 pl volume using a

50 p1 Hamilton microsyringe. To examine the dose-responses to calcium, graded Downloaded by [McMaster University] at 21:53 16 March 2016

doses of calcium chloride (i.e.; vehicle, 0.5, 1.0 and 1.5 pmol/lO pl) were injected ICV in 24 awake Wistar rats (n = 6 for each dose). To determine whether pretreatment with calcium channel blocker would modify the cardiovascular effects of calcium, vehicle or diltiazem (10 mmol/lO pl), was injected ICV 10 min before calcium (1.O pmol/lO pl) injection in 10 other Wistar rats (n

=

5, respectively). To test possible effects on pressor responsiveness to

angiotensin 11, vehicle or calcium chloride was injected ICV 10 min before angiotensin I1 injection. Two different doses (50 and 100 ng) were injected repeatedly on each rat with 5 min of interval. After each experiment, Evans Blue was injected into the ICV cannula to confirm cannula placement in the lateral ventricle. Plasma Norepinephn'ne Assay In six other Wistar rats, 0.6 ml blood samples for measuring plasma norepinephrine were collected from the venous cannula before and after ICV injections of calcium. Immediately after collecting each blood sample, an equivalent volume of donor rats blood was transfused to prevent hypovolemia. Norepinephrine concentrations in plasma samples obtained by centrifugation were measured using radioenzymatic assay (I2).

. .

Calcium Concentrabon in Cerebrospipal Fluid In twelve Wistar rats anesthetized with urethane (120 mg/100 g IP), ICV cannulae were inserted into the lateral ventricle as described above. The rats were then mounted on a stereotaxic apparatus with the head inclined downward by

RESPONSES TO ICV INJECTION OF CALCIUM

1021

about 45 degrees. After exposing the atlanto-occipital membrane, a 50 p1 sample of cerebrospinal fluid (CSF) was taken from the cisterna magna using a 27 gauge stainless needle cannula connected by polyethylene tubing to a 100 pl microsyringe (Hamilton). CSF was taken manually 10 min after ICV injection of Ca (1.O pmol) or vehicle (n = 6, respectively). Free calcium level was measured

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with fluorescent agent, fura-2 (I3).

. .

Drucs and Statmcs Calcium chloride (CaCl,; 0.5, 1.0 and 1.5 pmol) was dissolved in phosphate buffered solution. Diltiazem (10 mmol) and angiotensin I1 (50, 100 ng) were dissolved in 10 p1 of saline. With data expressed as averages+SEM, blood pressure, heart rate and CSF Ca concentration were compared using an unpaired r-test for comparing 2 groups and one-way ANOVA for comparing 4 groups. Cardiovascular responses to various doses of calcium were compared using a repeated measure of ANOVA (I4).

Whenever calcium injections altered baseline blood pressures, percent changes

from baseline were used for comparing data on pressor responses to angiotensin 11. These percent changes were ranked to verify that they were still distributed

normally and one-way ANOVA was used on both ranked and percent values. If both analysis gave similar results then distribution of percent changes was assumed to be normal. Whenever F-ratio significant at 5 % or less were obtained, the Newman-Keuls’ multiple range test was applied to determine the significance of differences between pairs of means. Plasma catecholamine levels before and after ICV injection of calcium were compared by using a paired t-test.

RESULTS

ICV Iniections of Calcium Decrease Blood Pressure and Heart Rate Graded doses of calcium chloride (i.e; 0.5, 1.0, and 1.5 pmol/lO pl) wereinjected ICV in 18 awake normotensive Wistar rats (n

=

6, for each dose).

Biphasic responses consisting of initial pressor and tachycardiac phases lasting for

ITOH ET AL.

1022 l v m M Ca icv

Phasic Pressure

100

(rnrnHg)

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50

Heart Rate Urnin)

~

0

FIG 1.

10

20

30rnin

The trace of cardiovascular responses to intracerebroventricular injections of CaCl, (1.O pmol) in awake normotensive Wistar rat.

2 min were followed by longer-lasting vasodepressor and bradycardiac responses (Figure 1). Because the initial phases were transient and smaller, the more pronounced second phases were considered representative of the major effects of calcium. These secondary vasodepressor and bradycardiac responses were dosedependent. Average reductions in mean pressure (mm Hg) occurring 10 min after injection were: -11 f l with 0.5 pmol, -24f6 with 1.0 pmol, and -29k-2with 1.5 pmol. Corresponding heart rate (bpm) changes were: -32k13, -84k16, and

-

173+24 respectively. Peak responses for both mean pressure and heart rate were attained within 10 min and lasted for 30 min (Figures 1 and 2). By contrast, control injections of either the vehicle alone ICV (figure 2), or of calcium 1.5 pmol intravenously, had no significant cardiovascular effects (+ 1+2 mm Hg in mean pressure and +2*9 bpm in heart rate 10 min after intravenous injection). Based on these results, a standard calcium dose of 1.0 pmol was used routinely

in subsequent experiments.

1023

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RESPONSES TO ICV INJECTION OF CALCIUM

-40

FIG 2.



I

I

Dose responses in mean pressure and heart rate to intracerebroventricularinjections of CaClz in awake normotensive Wistar rats (n=6 :each dose).

ITOH ET AL.

1024

To determine whether the standard calcium dose would suffice to alter free calcium concentration in the CSF, CSF samples were collected from the cisterna magna following ICV injection of calcium chloride (1.0 pmol) or the vehicle alone in 12 Wistar rats anesthetized with urethane. Free calcium concentration (mg/dl) in CSF samples collected 10 min after calcium injection was significantly higher than that following injection of the vehicle; average concentrations were Downloaded by [McMaster University] at 21:53 16 March 2016

7.2 f l . O and 3.1f0.3 respectively (p

Calcium suppresses central angiotensin II pressor response less in SHR.

To determine whether calcium alters central cardiovascular regulation, cardiovascular responses to intracerebroventricular (ICV) injection of calcium ...
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