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Catecholamines and vasopressin in forebrain nuclei of hypertension prone and resistant rats
Brain Research
Bulletin, Vol. 7, pp. 671-676,
1981. Printed in the
U.S.A.
Catecholamines and Vasopressin in Forebrain Nuclei of Hypertension Prone and Resistant Rats GIORA
FEUERSTEIN,’ ROBERT L. ZERBE, DRORI BEN-ISHAY,* IRWIN J. KOPIN AND DAVID M. JACOBOWITZ
Laboratory of Clinical Science, NZMH, Bethesda, MD 20205 and *Department of Medicine, Hypertension Unit, Hadassah Hospital, Jerusalem, Received FEUERSTEIN,
Israel
9 July 1981
I. J. KOPIN AND D. M. JACOBOWITZ. Catecholamines and and resistant rats. BRAIN RES. BULL. 7(6) ~671-676, 1981.Catecholamine and vasopressin content were studied in discrete brain nuclei of the Sabra strain of hypertension prone (SBH) and resistant (SBN) rats. Higher concentrations of dopamine, norepinephrine and epinephrine were observed in the median eminence of SBN compared to SBH or controls (SB) rats. Dopamine and epinephrine levels were higher in the lateral septal nucleus of SBH rats as compared to SBN or SB. Vasopressin content in discrete regions along the hypothalamo-pituitary axis was elevated in both SBH and SBN as compared to SB, but were especially elevated in the SBH group. The catecholamine and vasopressin changes found in SBH are different than those described in other genetically hypertensive rats indicating a difference in either the pathogenesis or central response to hypertension of this strain. vasopressin
G., R. L. ZERBE, D. BEN-ISHAY,
in forebrain
Brain nuclei
nuclei
Epinephrine
of hypertension
Norepinephrine
prone
Dopamine
of hypertension prone (SBH) and hyper(SBN) rats are genetically inbred rats that are sensitive and resistant, respectively, to blood pressureelevating procedures such as DOCA-salt treatment or appli-
THE
Sabra
strain
tension resistant
cation of a renal arterial clip [1,2]. Several studies have focused attention on the possible pathogenesis of hypertension by central catecholamine alterations in spontaneously hypertensive (SHR) rats [17, 21, 261. The present experiment was designed to examine the relationship between catecholamine levels in specific forebrain nuclei of hypertension prone and/or resistant (genetic) Sabra strains. Changes in monoamine levels in discrete areas could reveal functional abnormalities which underlie the sensitivity or resistance phenomenon to hypertensive stimuli. In addition, the content of vasopressin in major vasopressin containing nuclei along the hypothalamo-pituitary axis was studied since vasopressin has been implicated in the pathogenesis of hypertension in a number of experimental models of hypertension[3, 4, 15, 181. Furthermore, pituitary vasopressin is elevated in the spontaneous (genetic) hypertensive rats [3,16]. METHOD
Male SBH, SBN and normotensive (SB) rats 14-16 weeks old (matched by date of birth) derived from the parent strain from which the other strains were derived, were maintained in Jerusalem, Israel under the same conditions regarding IAddress for reprint requests: Dr. G. Feuerstein,
Copyright
@ 1981 ANKHO
Vasopressin
Genetic hypertension
light-dark cycle (12 hr:12 hr), temperature (21°C) and humidity. Normal rat Purina chow and tap water were supplied ad lib. The systolic blood pressure was measured by tailplethysmography in unanesthetized rats, 3 days prior to killing the animals. The rats were killed by decapitation between l&l1 a.m. The brain was quickly removed and immediately frozen on dry ice, transported by air freight to the U.S. and stored in the deep freeze (-70°C). The brains were mounted on specimen plates and 300 pm coronal sections were cut on a cryostat at -8°C. Specific areas from the sections were then microdissected by the technique of Palkovits [19,20]. The size and number of punches of the microdissected regions are presented in Tables 2-4. Assay of Norepinephrine, Epinephrine and Dopamine Brain punches were sonicated in 100 ~1 of ice cold 0.1 N perchloric acid, and 5 ~1 of the homogenate were removed for protein assay [13]. After centrifugation, aliquots of the supematant fluid were removed and norepinephrine, dopamine and epinephrine were assayed simultaneously by a single isotope radioenzymatic, thin layer chromatographic method described previously [6,251. In brief, 50 ~1 of the protein-free solutions were incubated with catechol-Omethyltransferase and tritiated S-adenosylmethionine (13.1 Ci/mmol, New England Nuclear). After incubation, the reaction was stopped by addition of borate buffer (pH 8.0)
Laboratory of Clinical Science, NIMH, Bldg. 10, Rm. 2D46, Bethesda, MD
International
Inc.-0361-9230/81/120671-06$01.10/O
20205.
FEUERSTEIN
671
containing metanephrine, normetanephrine and 3-methoxytyramine. The amines were extracted into toluene: isoamyl alcohol (3:2) and then into 0.1 M acetic acid. The radioactive products were separated by thin-layer chromatography and the appropriate areas separately scraped into counting vials. After periodate oxidation of the 0-methylated compounds to vanillin (for norepinephrine and epinephrine only), phosphor containing toluene was added and tritium assayed by liquid scintillation spectrometry. Vusopressin
Assay
Vasopressin was measured by radioimmunoassay. The assay utilizes a rabbit anti-vasopressin antibody, kindly provided by Dr. Jacques Diirr (University of Chicago, IL) in a final dilution of 1:2,500,000. Iodinated vasopressin (New England Nuclear, Boston, MA; 40 IU/mg) and synthetic arginine vasopressin (Calbiochem, San Diego, CA) served as tracer (1500 cpm/tube, SA=1330 $Zi/pg) and standard, respectively. The total assay volume of 0.5 ml was incubated for 7 days at 4°C and bound vasopressin was separated from the free peptide using polyethylene glycol [7]. The assay reliably detects 0.4 to 40 pg of standard vasopressin per tube with an intra-assay coefficient of variation of 3.9% at 1 pg/tube and 6.2% at 10 pg/tube. Cross reactivity with oxytotin is less than 1%. Punches were sonicated in 0.1 N HCl and diluted in assay buffer (0.1 M phosphate, 0.3% NaCl, 0.1% bovine serum albumin, pH 7.6) so that measurements would fall in the midrange of the standard curve. All samples from specific brain regions were diluted identically and assayed at the same time. Statistical analysis for differences among groups was done by the Duncan’s Multiple Range Tests. Data are reported as mean (SEM) values for the number of animals indicated in the tables. Concentrations of the catecholamines and vasopressin are expressed as pglpg protein.
RESULTS
Table 1 shows the systolic blood pressure and body weights of the rats of the three strains used. It is evident that SBH rats are heavier and have slightly higher blood pressure than the other strains of rats. The SBN rats are somewhat lighter and have a slightly lower blood pressure than the normal SB rats of the same age. Catecholamine
Concentration
in Different Brain Nuclei
Dopamine concentration in the lateral septal nucleus of SBH rats was 2.5-3 fold higher than dopamine concentration in this same nucleus of SB and SBN rats (Table 2). The concentration of dopamine in the median eminence of the SBN rats was significantly higher (about 30%) than the levels found in either the SB or SBH rats (Table 2). Norepinephrine levels in the various nuclei were not significantly different, except for the median eminence in which SBN rats had higher levels than did SBH or SB rats (Table 3). The three strains of rats also differed in the concentration of epinephrine in several brain nuclei. In the lateral septal nucleus of the SBH, epinephrine concentration was more than 2 fold greater than in SB or SBN rats (Table 4). However, both SBH and SBN rats had higher levels of epinephrine in the caudate-putamen than the parent SB strain (Table 4). The SBN rats had a higher concentration of epinephrine
TABLE
E7‘ A I<.
1
BLOOD PRESSURE AND BODY WEIGHT OF 14-16 WEEKS SBH, SBN AND SB RATS
Strain of Rats SBH (n=9)
SB (n=8)
Body weight (grams)
239 i- 4*
229 + 5
213 -z 6;-‘-
Systolic blood pressure (mm Hg)
144 t 3t
130 ir 31
112 + 3:
SBN (n=9)
*=p
in the median eminence ble 4). Vasopressin
than either the SB or SBH rats (Ta-
Concentrations
in Brain Nuclei
Vasopressin concentration was found to increase along the hypothalamo-median eminence-pituitary axis in both the SB and SBN rats (Table 5). The vasopressin concentration of both the SBH and SBN were higher than in SB rats in all four areas. Extremely high vasopressin levels were found in the supraoptic and paraventricular nuclei of the SBH rats, which were 7-8 fold greater than the vasopressin levels found in these areas in SB rats. DISCUSSION
By selective inbreeding of the Hebrew University Sabra rats (SB), two substrains were obtained: SBH-hypertension prone and SBN-hypertension resistant. SBN rats are remarkably resistant to DOCA-salt or renal clip hypertension, whereas SBH rats are excessively sensitive to these procedures [ 1,2]. Previous biochemical studies conducted with the SBH, SBN and SB rats have demonstrated that the hypertension prone rats have lower 18,271 brain stem norepinephrine content but higher tyrosine hydroxylase activity than the hypertension resistant strain. Moreover, hypothalamic norepinephrine-dependent adenylate cyclase of SBH rats was found to be more responsive to norepinephrine than SBN rats [l 11.In these studies, large brain areas were examined. The present study extends these observations and further demonstrates that the two substrains obtained from normal SB rats differ in catecholamine concentration in a few specific brain nuclei. The most significant difference was found in the median eminence, in which levels of all the catecholainines (dopamine, epinephrine and norepinephrine) were higher in the SBN than in the other substrains. This difference between the SBN and SBH strains is in contrast to the absence of differences in the amount of norepinephrine in the median eminence found in SHR and WKY rats at comparable ages [21,24]. On the other hand, several hypothalamic nuclei in which no differences in norepinephrine concentrations were found among the various strains examined in the present study were shown to contain higher norepinephrine levels in normotensive WKY rats as compared to aged-matched SHR rats [ll]. The levels of norepinephrine
CATECHOLAMINES
673
AND HYPERTENSION TABLE 2
DOPAMINE
CONCENTRATION
(pg&
PROTEIN ‘- S.E.M.) IN BRAIN NUCLEI OF SB, SBH AND SBN RATS Strain
Region accumbens tractus diagonalis lateralis septi interstitialis striae terminalis N. caudatus-putamen Cingulate cortex N. preopticus medialis N. anterior ventralis thalami N. periventricularis hypothalami N. hypothalamic anterior N. supraopticus N. paraventricularis Median eminence N. arcuatus N. ventromedialis N. dorsomedialis N. hypothalamic posterior Hippocampus Pituitary gland
No. of punches per rat
SBH
(mm) 0.5
N. N. N. N.
of Rats
Size of cannula SBN
SB
0.5 0.5 0.5
83.3 8.9 24.9 17.1
+ 2 k ?
5.2 1.7 3.l*t 1.9
81.8 10.8 8.84 14.8
” + 2 +
4.1 1.6 1.86 1.9
78.7 5.9 9.44 15.7
t + + ”
2.9 0.6 1.25 1.6
1.0 1.0 0.75 0.5
68.0 2.71 3.78 1.62
+ 2.5 2 0.31 ” 0.54 + 0.16
59.3 1.82 2.62 1.58
k + t ‘-
2.7 0.51 0.43 0.18
62.2 2.61 3.43 1.63
+ 2 2 f
2.5 0.35 0.37 0.19
3
0.5
-
0.75 0.5 0.5 0.75 0.3 0.5 0.5 0.5 1.0 -
6.13 k 0.57 3.23 k 0.77 + 4.33 k 81.0 ? 13.0 + 1.81 k 4.% f 2.98 2 0.59 t 11.11 k
0.59 0.20 0.55 7.3 1.9 0.18 0.52 0.23 0.12 1.36
5.66 k 0.56 2.31 0.33 3.47 75.0 12.5 2.14 4.83 3.12 0.39 7.83
2 0.36 t 0.13 + 0.71 2 4.0 f 2.0 f 0.35 f 0.54 f 0.44 -e 0.07 f 0.50
7.37 + 0.57 2.55 0.42 4.34 105.3 15.8 1.63 5.42 2.64 0.53 7.01
k 0.55 f 0.08 + 0.56 k 5.1*t 2 2.1 f 0.15 r 0.62 k 0.28 +- 0.14 ‘- 1.10
*=p
found in the present study are significantly higher in some nuclei and lower in others, as compared to the levels previously reported in SHR and WKY rats [21,22]. The differences between the brain nuclei norepinephrine levels in the Wistar rat substrains (SHR and WKY) and the Sabra rats (SBH, SB and SBN) may well be the result of strain differences [14], but variation between surgical, anesthetics and assay procedures may play a major role in the differences in brain nuclei catecholamine levels in the present study as compared to previous investigations. In the normal SB rats, levels of epinephrine vary from 0.059 pglpg protein in the hippocampus up to 3.2 pglkg protein in the median eminence; this latter region has a 4 fold greater epinephrine concentration than the amount found in the same region of Sprague Dawley rats [ 121. Though most of the areas studied did not appear to be significantly different among the SBH, SBN and SB rats, a few regions did show such differences. In addition to the moderate increase in epinephrine levels in the median eminence of SBN rats, 2 fold higher levels of epinephrine were found in the lateral septal nucleus of SBH than SB and SBN rats. The SB strain itself had lower epinephrine concentration in the caudateputamen than the SBH and SBN rats. In the recent report by Versteegeg al. [24], it was shown that epinephrine content in the paraventricular nucleus of SHR rats is higher than that found in WKY rats; no differences in epinephrine content
were found in the median eminence or the lateral septal nucleus. Dopamine levels in the median eminence of SBN rats were also higher than the dopamine levels in the SB or SBH rats, while the opposite relationship was found for the lateral septal nucleus where SBH rats had a three fold higher concentration than in SB and SBN. These data differ from a previous report [24] in which dopamine levels in the nucleus terminalis striae medullaris of SHR rats was higher than WKY, but no differences in dopamine levels were found in other telencephalic or diencephalic nuclei. Although the cardiovascular role of various nuclei that show changes in catecholamine levels is not know, it has been suggested that the activity of the norepinephrine terminals may be modulated by events occurring locally in discrete areas [IO]. Therefore, the paucity of changes observed in norepinephrine levels of the Sabra substrains, compared with that of SHR rats, may be a reflection of the marked differences in blood pressure. The lack of significant changes in norepinephrine concentration in the various areas studied (other than the median eminence) may be a reflection of the relatively small elevation of blood pressure of the Sabra substrain SBH compared to that found in SHR rats [21, 24, 261. Although it is possible that various regions in the brain involved in either the production of or the responses to chronic hypertension may activate (or inactivate) norepi-
674
FEUERSTEIN
ET Al,.
TABLE 3 NOREPINEPHRINE CONCENTRATION (p&g PROTEIN 5 S.E.M.) IN BRAIN NUCLEI OF SB, SBH AND SBN RATS Strain of Rats Region N. N. N. N.
accumbens tractus diagonalis lateralis septi interstitialis striae terminalis N. caudatus-putamen Cingulate cortex N. preopticus medialis N. anterior ventralis thalami N. periventricularis hypothalami N . hypothalamic anterior N. supraopticus N . paraventricularis Median eminence N. arcuatus N. ventromedialis N dorsomedialis N. hypothalamic posterior Hippocampus Pituitary gland
No. of punches per rat
cannula (mm)
4 2 2 4
0.5 0.5 0.5 0.5
1.50 10.8 4.18 77.4
2 5 k ?
0.09 1.2 0.35 5.7
1.13 + 11.6 k 3.79 k 60.5 +
2 2 2 2
1.0 1.0 0.75 0.5
0.64 2.90 22.6 22.8
f t lr t
0.04 0.13 1.3 1.5
0.45 2.57 20.2 22.4
3
0.5
43.9 2 3.1
2 2 2 3 6 4 2 2 2 -
0.75 0.5 0.5 0.75 0.3 0.5 0.5 0.5 1.0 -
11.7 16.9 76.1 10.8 26.0 8.35 35.5 14.1 5.46 1.53
SBH
i -t + k + k + r + +
SB
1.2 1.3 3.3 0.7 2.2 0.89 6.6 0.8 0.57 0.25
SBN 0.22 2.8 0.30 8.6
1.83 11.8 3.99 61.0
t + r r
f 0.04 + 0.18 z!z2.6 -t 3.0
0.57 2.48 21.9 22.9
2 0.09 + 0.19 i 1.9 -c 1.2
38.9 2 3.1
44.1
2 2.6
11.1 14.2 71.8 13.3 34.1 13.9 51.2 20.5 3.89 1.05
10.9 17.1 66.2 16.3 28.5 10.1 56.3 15.0 3.59 1.79
f 0.6 t 2.5 2 5.8 k 1.3*1 t 1.2 + 1.0 t 3.1 ‘- 1.8 2 0.20 _t 0.81
t t c + + + + + t +
1.1 0.8 4.8 1.0 4.5 3.1 6.5 2.9 0.31 0.05
0.32 1.3 0.48 3.8
*=p
nephrine neurons in specific regions as reflected in changes in the basal levels of the monoamine, it is also possible that the changes found in the present study are either unrelated to the pathogenesis of hypertension or central regulation of the cardiovascular system. Vasopressin concentrations along the hypothalamopituitary axis in the normal SB rats increased progressively from the hypothalamic nuclei (supraoptic and paraventricular) through the median eminence to the highest concentration in the pituitary gland. This pattern is consistent with previous studies [5, 9, 181. A striking finding was extremely high vasopressin concentrations in SBH rats, especially in the paraventricular and supraoptic nuclei of the hypothalamus. In this regard, the SBH rats differ from the SHR rats, which have lower vasopressin levels than WKY rats [18]. The higher pituitary contents observed in SBH rats are similar to those previously described in SHR [3,16]. Interestingly, the SBN rats, which are hypertension resistant, also have an elevated vasopressin concentration in most of the regions studied. Thus, both SBN and SBH rats have higher vasopressin levels compared to the SB (control) rats. It is also noteworthy that the relative increase (percent) in vasopressin concentration found in the nuclei of SBH is not the same. The hypothalamic nuclei show a marked increase, while the pituitary gland has a mild increase. This phenomenon may be explained by a limitation of hypothalamicneurohypophyseal transport or by unusually rich vasopressinergic innervation to extrahypothalamic regions [23]. Further investigation is needed to clarify these possibilities.
The following conclusions can be drawn from these studies: (a) Both the SBH and SBN, which were inbred from the SB strain to have opposite sensitivities to pressor procedures, have high levels of vasopressin along the hypothalamo-pituitary axis; this phenomenon therefore may be separate from the inverse susceptibility of the SBH and SBN to hypertensive stimuli. Whether the increase in vasopressin levels along the hypothalamo-pituitary axis is due to increased synthesis or decreased release has not been determined. (b) The SBH, SB and SBN rats differ from each other in respect to the catecholamine concentrations in only a very few forebrain nuclei. The most consistent change was observed in the hypertension resistant prone (SBN) rats which had higher levels of all the catecholamines in the median eminence. The catecholamines in the median eminence are in opposition with the vascular system which transports the release or inhibitory factors to the pituitary. The higher catecholamine levels in the median eminence of the hypertension-resistant strain (SBN) suggests the possibility that a neuroendocrine imbalance may play a role in the development or compensation responses to blood pressure elevation. The possibility that the median eminence has a role in cardiovascular regulation is intriguing, but at present appears to be a matter of speculation. Thus, whether the changes found in the catecholamine and vasopressin systems are causative or adaptive phenomena in the pathogenesis of hypertension susceptibility or -resistance awaits further investigation.
CATECHOLAMINES
615
AND HYPERTENSION TABLE 4
EPINEPHRINE
CONCENTRATION
(pg/pg PROTEIN
k S.E.M.) IN BRAIN
NUCLEI
OF SB, SBH AND SBN RATS
Strain of Rats No. of punches per rat
Region
cannula (mm)
SBN
SB
SBH
N . accumbens
0.5
N . tractus diagonalis N. lateralis septi N. interstitialis striae terminalis N. caudatus-putamen Cingulate cortex N . preopticus medialis N. anterior ventralis thalami N. periventricularis hypothalami N. hypothalamic anterior N. supraopticus N. paraventricularis Median eminence N . arcuatus N. ventromedialis N. dorsomedialis N. hypothalamic posterior Hippocampus Pituitary gland
0.5 0.5 0.5
3.27 0.66 1.07 1.81
% 0.17 f 0.10 2 0.11*t ? 0.16
2.83 0.69 0.45 1.37
+ 2 + +
0.19 0.13 0.08 0.22
3.19 0.72 0.49 1.86
? ? 2 *
0.26 0.09 0.05 0.15
1.0 1.0 0.75 0.5 0.5
2.61 0.142 1.09 0.352 2.49
t + rt 2 2
0.11 0.014 0.05 0.028 0.21
1.98 0.112 0.92 0.285 1.99
+ * 2 -t r
O.l6*t 0.014 0.11 0.042 0.21
2.63 0.134 1.31 0.365 2.60
-t ? + 2 2
0.16 0.017 0.18 0.040 0.21
0.75 0.5 0.5 0.75 0.3 0.5 0.5 0.5 1.0 -
0.680 0.321 3.07 3.95 2.17 0.54 2.29 0.704 0.082 0.586
? + + 2 ? * t 2 2 ”
0.082 0.042 0.17 0.33 0.16 0.06 0.44 0.051 0.014 0.053
0.479 0.291 2.98 3.20 2.17 0.72 2.48 0.835 0.059 0.493
+ 2 2 + + 2 + ” ” *
0.051 0.060 0.21 0.17 0.29 0.15 0.30 0.160 0.006 0.063
0.740 0.257 2.90 5.78 2.58 0.62 2.86 0.754 0.080 0.468
? 2 f 2 ? ? 2 ? 2 2
0.150 0.044 0.34
-
0.32*t
0.15 0.06 0.33 0.086 0.009 0.073
*=p
TABLE 5 VASOPRESSIN CONCENTRATION (p&g PROTEIN ? S.E.M.) IN BRAIN NUCLEI SB, SBH AND SBN RATS
OF
Strain of Rats Region*
n
N . paraventricularis
8 8 8 15
N. supraopticus Median eminence pituitary gland
SBH 1268 ? !%t 1184k99.t 1071 * 727” 1781 ? 63$
n
SB
n
7 7 7 7
162 ? 461‘ 159-c27$ 723 2 47 982 5 48$
9 8 9 15
SBN 435 2 664 2 811 ? 1407 -c
69t 104$ 79$b 86$
n=Number of rats in each group. *No. of punches and cannula size are given in Tables 2-4. *All three groups are significantly different from each other at 0.05 level. SAll three groups are significantly different from each other at 0.01 level. §pP
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Bulletin, Vol. 7, pp. 671-676,
1981. Printed in the
U.S.A.
Catecholamines and Vasopressin in Forebrain Nuclei of Hypertension Prone and Resistant Rats GIORA
FEUERSTEIN,’ ROBERT L. ZERBE, DRORI BEN-ISHAY,* IRWIN J. KOPIN AND DAVID M. JACOBOWITZ
Laboratory of Clinical Science, NZMH, Bethesda, MD 20205 and *Department of Medicine, Hypertension Unit, Hadassah Hospital, Jerusalem, Received FEUERSTEIN,
Israel
9 July 1981
I. J. KOPIN AND D. M. JACOBOWITZ. Catecholamines and and resistant rats. BRAIN RES. BULL. 7(6) ~671-676, 1981.Catecholamine and vasopressin content were studied in discrete brain nuclei of the Sabra strain of hypertension prone (SBH) and resistant (SBN) rats. Higher concentrations of dopamine, norepinephrine and epinephrine were observed in the median eminence of SBN compared to SBH or controls (SB) rats. Dopamine and epinephrine levels were higher in the lateral septal nucleus of SBH rats as compared to SBN or SB. Vasopressin content in discrete regions along the hypothalamo-pituitary axis was elevated in both SBH and SBN as compared to SB, but were especially elevated in the SBH group. The catecholamine and vasopressin changes found in SBH are different than those described in other genetically hypertensive rats indicating a difference in either the pathogenesis or central response to hypertension of this strain. vasopressin
G., R. L. ZERBE, D. BEN-ISHAY,
in forebrain
Brain nuclei
nuclei
Epinephrine
of hypertension
Norepinephrine
prone
Dopamine
of hypertension prone (SBH) and hyper(SBN) rats are genetically inbred rats that are sensitive and resistant, respectively, to blood pressureelevating procedures such as DOCA-salt treatment or appli-
THE
Sabra
strain
tension resistant
cation of a renal arterial clip [1,2]. Several studies have focused attention on the possible pathogenesis of hypertension by central catecholamine alterations in spontaneously hypertensive (SHR) rats [17, 21, 261. The present experiment was designed to examine the relationship between catecholamine levels in specific forebrain nuclei of hypertension prone and/or resistant (genetic) Sabra strains. Changes in monoamine levels in discrete areas could reveal functional abnormalities which underlie the sensitivity or resistance phenomenon to hypertensive stimuli. In addition, the content of vasopressin in major vasopressin containing nuclei along the hypothalamo-pituitary axis was studied since vasopressin has been implicated in the pathogenesis of hypertension in a number of experimental models of hypertension[3, 4, 15, 181. Furthermore, pituitary vasopressin is elevated in the spontaneous (genetic) hypertensive rats [3,16]. METHOD
Male SBH, SBN and normotensive (SB) rats 14-16 weeks old (matched by date of birth) derived from the parent strain from which the other strains were derived, were maintained in Jerusalem, Israel under the same conditions regarding IAddress for reprint requests: Dr. G. Feuerstein,
Copyright
@ 1981 ANKHO
Vasopressin
Genetic hypertension
light-dark cycle (12 hr:12 hr), temperature (21°C) and humidity. Normal rat Purina chow and tap water were supplied ad lib. The systolic blood pressure was measured by tailplethysmography in unanesthetized rats, 3 days prior to killing the animals. The rats were killed by decapitation between l&l1 a.m. The brain was quickly removed and immediately frozen on dry ice, transported by air freight to the U.S. and stored in the deep freeze (-70°C). The brains were mounted on specimen plates and 300 pm coronal sections were cut on a cryostat at -8°C. Specific areas from the sections were then microdissected by the technique of Palkovits [19,20]. The size and number of punches of the microdissected regions are presented in Tables 2-4. Assay of Norepinephrine, Epinephrine and Dopamine Brain punches were sonicated in 100 ~1 of ice cold 0.1 N perchloric acid, and 5 ~1 of the homogenate were removed for protein assay [13]. After centrifugation, aliquots of the supematant fluid were removed and norepinephrine, dopamine and epinephrine were assayed simultaneously by a single isotope radioenzymatic, thin layer chromatographic method described previously [6,251. In brief, 50 ~1 of the protein-free solutions were incubated with catechol-Omethyltransferase and tritiated S-adenosylmethionine (13.1 Ci/mmol, New England Nuclear). After incubation, the reaction was stopped by addition of borate buffer (pH 8.0)
Laboratory of Clinical Science, NIMH, Bldg. 10, Rm. 2D46, Bethesda, MD
International
Inc.-0361-9230/81/120671-06$01.10/O
20205.
FEUERSTEIN
671
containing metanephrine, normetanephrine and 3-methoxytyramine. The amines were extracted into toluene: isoamyl alcohol (3:2) and then into 0.1 M acetic acid. The radioactive products were separated by thin-layer chromatography and the appropriate areas separately scraped into counting vials. After periodate oxidation of the 0-methylated compounds to vanillin (for norepinephrine and epinephrine only), phosphor containing toluene was added and tritium assayed by liquid scintillation spectrometry. Vusopressin
Assay
Vasopressin was measured by radioimmunoassay. The assay utilizes a rabbit anti-vasopressin antibody, kindly provided by Dr. Jacques Diirr (University of Chicago, IL) in a final dilution of 1:2,500,000. Iodinated vasopressin (New England Nuclear, Boston, MA; 40 IU/mg) and synthetic arginine vasopressin (Calbiochem, San Diego, CA) served as tracer (1500 cpm/tube, SA=1330 $Zi/pg) and standard, respectively. The total assay volume of 0.5 ml was incubated for 7 days at 4°C and bound vasopressin was separated from the free peptide using polyethylene glycol [7]. The assay reliably detects 0.4 to 40 pg of standard vasopressin per tube with an intra-assay coefficient of variation of 3.9% at 1 pg/tube and 6.2% at 10 pg/tube. Cross reactivity with oxytotin is less than 1%. Punches were sonicated in 0.1 N HCl and diluted in assay buffer (0.1 M phosphate, 0.3% NaCl, 0.1% bovine serum albumin, pH 7.6) so that measurements would fall in the midrange of the standard curve. All samples from specific brain regions were diluted identically and assayed at the same time. Statistical analysis for differences among groups was done by the Duncan’s Multiple Range Tests. Data are reported as mean (SEM) values for the number of animals indicated in the tables. Concentrations of the catecholamines and vasopressin are expressed as pglpg protein.
RESULTS
Table 1 shows the systolic blood pressure and body weights of the rats of the three strains used. It is evident that SBH rats are heavier and have slightly higher blood pressure than the other strains of rats. The SBN rats are somewhat lighter and have a slightly lower blood pressure than the normal SB rats of the same age. Catecholamine
Concentration
in Different Brain Nuclei
Dopamine concentration in the lateral septal nucleus of SBH rats was 2.5-3 fold higher than dopamine concentration in this same nucleus of SB and SBN rats (Table 2). The concentration of dopamine in the median eminence of the SBN rats was significantly higher (about 30%) than the levels found in either the SB or SBH rats (Table 2). Norepinephrine levels in the various nuclei were not significantly different, except for the median eminence in which SBN rats had higher levels than did SBH or SB rats (Table 3). The three strains of rats also differed in the concentration of epinephrine in several brain nuclei. In the lateral septal nucleus of the SBH, epinephrine concentration was more than 2 fold greater than in SB or SBN rats (Table 4). However, both SBH and SBN rats had higher levels of epinephrine in the caudate-putamen than the parent SB strain (Table 4). The SBN rats had a higher concentration of epinephrine
TABLE
E7‘ A I<.
1
BLOOD PRESSURE AND BODY WEIGHT OF 14-16 WEEKS SBH, SBN AND SB RATS
Strain of Rats SBH (n=9)
SB (n=8)
Body weight (grams)
239 i- 4*
229 + 5
213 -z 6;-‘-
Systolic blood pressure (mm Hg)
144 t 3t
130 ir 31
112 + 3:
SBN (n=9)
*=p
in the median eminence ble 4). Vasopressin
than either the SB or SBH rats (Ta-
Concentrations
in Brain Nuclei
Vasopressin concentration was found to increase along the hypothalamo-median eminence-pituitary axis in both the SB and SBN rats (Table 5). The vasopressin concentration of both the SBH and SBN were higher than in SB rats in all four areas. Extremely high vasopressin levels were found in the supraoptic and paraventricular nuclei of the SBH rats, which were 7-8 fold greater than the vasopressin levels found in these areas in SB rats. DISCUSSION
By selective inbreeding of the Hebrew University Sabra rats (SB), two substrains were obtained: SBH-hypertension prone and SBN-hypertension resistant. SBN rats are remarkably resistant to DOCA-salt or renal clip hypertension, whereas SBH rats are excessively sensitive to these procedures [ 1,2]. Previous biochemical studies conducted with the SBH, SBN and SB rats have demonstrated that the hypertension prone rats have lower 18,271 brain stem norepinephrine content but higher tyrosine hydroxylase activity than the hypertension resistant strain. Moreover, hypothalamic norepinephrine-dependent adenylate cyclase of SBH rats was found to be more responsive to norepinephrine than SBN rats [l 11.In these studies, large brain areas were examined. The present study extends these observations and further demonstrates that the two substrains obtained from normal SB rats differ in catecholamine concentration in a few specific brain nuclei. The most significant difference was found in the median eminence, in which levels of all the catecholainines (dopamine, epinephrine and norepinephrine) were higher in the SBN than in the other substrains. This difference between the SBN and SBH strains is in contrast to the absence of differences in the amount of norepinephrine in the median eminence found in SHR and WKY rats at comparable ages [21,24]. On the other hand, several hypothalamic nuclei in which no differences in norepinephrine concentrations were found among the various strains examined in the present study were shown to contain higher norepinephrine levels in normotensive WKY rats as compared to aged-matched SHR rats [ll]. The levels of norepinephrine
CATECHOLAMINES
673
AND HYPERTENSION TABLE 2
DOPAMINE
CONCENTRATION
(pg&
PROTEIN ‘- S.E.M.) IN BRAIN NUCLEI OF SB, SBH AND SBN RATS Strain
Region accumbens tractus diagonalis lateralis septi interstitialis striae terminalis N. caudatus-putamen Cingulate cortex N. preopticus medialis N. anterior ventralis thalami N. periventricularis hypothalami N. hypothalamic anterior N. supraopticus N. paraventricularis Median eminence N. arcuatus N. ventromedialis N. dorsomedialis N. hypothalamic posterior Hippocampus Pituitary gland
No. of punches per rat
SBH
(mm) 0.5
N. N. N. N.
of Rats
Size of cannula SBN
SB
0.5 0.5 0.5
83.3 8.9 24.9 17.1
+ 2 k ?
5.2 1.7 3.l*t 1.9
81.8 10.8 8.84 14.8
” + 2 +
4.1 1.6 1.86 1.9
78.7 5.9 9.44 15.7
t + + ”
2.9 0.6 1.25 1.6
1.0 1.0 0.75 0.5
68.0 2.71 3.78 1.62
+ 2.5 2 0.31 ” 0.54 + 0.16
59.3 1.82 2.62 1.58
k + t ‘-
2.7 0.51 0.43 0.18
62.2 2.61 3.43 1.63
+ 2 2 f
2.5 0.35 0.37 0.19
3
0.5
-
0.75 0.5 0.5 0.75 0.3 0.5 0.5 0.5 1.0 -
6.13 k 0.57 3.23 k 0.77 + 4.33 k 81.0 ? 13.0 + 1.81 k 4.% f 2.98 2 0.59 t 11.11 k
0.59 0.20 0.55 7.3 1.9 0.18 0.52 0.23 0.12 1.36
5.66 k 0.56 2.31 0.33 3.47 75.0 12.5 2.14 4.83 3.12 0.39 7.83
2 0.36 t 0.13 + 0.71 2 4.0 f 2.0 f 0.35 f 0.54 f 0.44 -e 0.07 f 0.50
7.37 + 0.57 2.55 0.42 4.34 105.3 15.8 1.63 5.42 2.64 0.53 7.01
k 0.55 f 0.08 + 0.56 k 5.1*t 2 2.1 f 0.15 r 0.62 k 0.28 +- 0.14 ‘- 1.10
*=p
found in the present study are significantly higher in some nuclei and lower in others, as compared to the levels previously reported in SHR and WKY rats [21,22]. The differences between the brain nuclei norepinephrine levels in the Wistar rat substrains (SHR and WKY) and the Sabra rats (SBH, SB and SBN) may well be the result of strain differences [14], but variation between surgical, anesthetics and assay procedures may play a major role in the differences in brain nuclei catecholamine levels in the present study as compared to previous investigations. In the normal SB rats, levels of epinephrine vary from 0.059 pglpg protein in the hippocampus up to 3.2 pglkg protein in the median eminence; this latter region has a 4 fold greater epinephrine concentration than the amount found in the same region of Sprague Dawley rats [ 121. Though most of the areas studied did not appear to be significantly different among the SBH, SBN and SB rats, a few regions did show such differences. In addition to the moderate increase in epinephrine levels in the median eminence of SBN rats, 2 fold higher levels of epinephrine were found in the lateral septal nucleus of SBH than SB and SBN rats. The SB strain itself had lower epinephrine concentration in the caudateputamen than the SBH and SBN rats. In the recent report by Versteegeg al. [24], it was shown that epinephrine content in the paraventricular nucleus of SHR rats is higher than that found in WKY rats; no differences in epinephrine content
were found in the median eminence or the lateral septal nucleus. Dopamine levels in the median eminence of SBN rats were also higher than the dopamine levels in the SB or SBH rats, while the opposite relationship was found for the lateral septal nucleus where SBH rats had a three fold higher concentration than in SB and SBN. These data differ from a previous report [24] in which dopamine levels in the nucleus terminalis striae medullaris of SHR rats was higher than WKY, but no differences in dopamine levels were found in other telencephalic or diencephalic nuclei. Although the cardiovascular role of various nuclei that show changes in catecholamine levels is not know, it has been suggested that the activity of the norepinephrine terminals may be modulated by events occurring locally in discrete areas [IO]. Therefore, the paucity of changes observed in norepinephrine levels of the Sabra substrains, compared with that of SHR rats, may be a reflection of the marked differences in blood pressure. The lack of significant changes in norepinephrine concentration in the various areas studied (other than the median eminence) may be a reflection of the relatively small elevation of blood pressure of the Sabra substrain SBH compared to that found in SHR rats [21, 24, 261. Although it is possible that various regions in the brain involved in either the production of or the responses to chronic hypertension may activate (or inactivate) norepi-
674
FEUERSTEIN
ET Al,.
TABLE 3 NOREPINEPHRINE CONCENTRATION (p&g PROTEIN 5 S.E.M.) IN BRAIN NUCLEI OF SB, SBH AND SBN RATS Strain of Rats Region N. N. N. N.
accumbens tractus diagonalis lateralis septi interstitialis striae terminalis N. caudatus-putamen Cingulate cortex N. preopticus medialis N. anterior ventralis thalami N. periventricularis hypothalami N . hypothalamic anterior N. supraopticus N . paraventricularis Median eminence N. arcuatus N. ventromedialis N dorsomedialis N. hypothalamic posterior Hippocampus Pituitary gland
No. of punches per rat
cannula (mm)
4 2 2 4
0.5 0.5 0.5 0.5
1.50 10.8 4.18 77.4
2 5 k ?
0.09 1.2 0.35 5.7
1.13 + 11.6 k 3.79 k 60.5 +
2 2 2 2
1.0 1.0 0.75 0.5
0.64 2.90 22.6 22.8
f t lr t
0.04 0.13 1.3 1.5
0.45 2.57 20.2 22.4
3
0.5
43.9 2 3.1
2 2 2 3 6 4 2 2 2 -
0.75 0.5 0.5 0.75 0.3 0.5 0.5 0.5 1.0 -
11.7 16.9 76.1 10.8 26.0 8.35 35.5 14.1 5.46 1.53
SBH
i -t + k + k + r + +
SB
1.2 1.3 3.3 0.7 2.2 0.89 6.6 0.8 0.57 0.25
SBN 0.22 2.8 0.30 8.6
1.83 11.8 3.99 61.0
t + r r
f 0.04 + 0.18 z!z2.6 -t 3.0
0.57 2.48 21.9 22.9
2 0.09 + 0.19 i 1.9 -c 1.2
38.9 2 3.1
44.1
2 2.6
11.1 14.2 71.8 13.3 34.1 13.9 51.2 20.5 3.89 1.05
10.9 17.1 66.2 16.3 28.5 10.1 56.3 15.0 3.59 1.79
f 0.6 t 2.5 2 5.8 k 1.3*1 t 1.2 + 1.0 t 3.1 ‘- 1.8 2 0.20 _t 0.81
t t c + + + + + t +
1.1 0.8 4.8 1.0 4.5 3.1 6.5 2.9 0.31 0.05
0.32 1.3 0.48 3.8
*=p
nephrine neurons in specific regions as reflected in changes in the basal levels of the monoamine, it is also possible that the changes found in the present study are either unrelated to the pathogenesis of hypertension or central regulation of the cardiovascular system. Vasopressin concentrations along the hypothalamopituitary axis in the normal SB rats increased progressively from the hypothalamic nuclei (supraoptic and paraventricular) through the median eminence to the highest concentration in the pituitary gland. This pattern is consistent with previous studies [5, 9, 181. A striking finding was extremely high vasopressin concentrations in SBH rats, especially in the paraventricular and supraoptic nuclei of the hypothalamus. In this regard, the SBH rats differ from the SHR rats, which have lower vasopressin levels than WKY rats [18]. The higher pituitary contents observed in SBH rats are similar to those previously described in SHR [3,16]. Interestingly, the SBN rats, which are hypertension resistant, also have an elevated vasopressin concentration in most of the regions studied. Thus, both SBN and SBH rats have higher vasopressin levels compared to the SB (control) rats. It is also noteworthy that the relative increase (percent) in vasopressin concentration found in the nuclei of SBH is not the same. The hypothalamic nuclei show a marked increase, while the pituitary gland has a mild increase. This phenomenon may be explained by a limitation of hypothalamicneurohypophyseal transport or by unusually rich vasopressinergic innervation to extrahypothalamic regions [23]. Further investigation is needed to clarify these possibilities.
The following conclusions can be drawn from these studies: (a) Both the SBH and SBN, which were inbred from the SB strain to have opposite sensitivities to pressor procedures, have high levels of vasopressin along the hypothalamo-pituitary axis; this phenomenon therefore may be separate from the inverse susceptibility of the SBH and SBN to hypertensive stimuli. Whether the increase in vasopressin levels along the hypothalamo-pituitary axis is due to increased synthesis or decreased release has not been determined. (b) The SBH, SB and SBN rats differ from each other in respect to the catecholamine concentrations in only a very few forebrain nuclei. The most consistent change was observed in the hypertension resistant prone (SBN) rats which had higher levels of all the catecholamines in the median eminence. The catecholamines in the median eminence are in opposition with the vascular system which transports the release or inhibitory factors to the pituitary. The higher catecholamine levels in the median eminence of the hypertension-resistant strain (SBN) suggests the possibility that a neuroendocrine imbalance may play a role in the development or compensation responses to blood pressure elevation. The possibility that the median eminence has a role in cardiovascular regulation is intriguing, but at present appears to be a matter of speculation. Thus, whether the changes found in the catecholamine and vasopressin systems are causative or adaptive phenomena in the pathogenesis of hypertension susceptibility or -resistance awaits further investigation.
CATECHOLAMINES
615
AND HYPERTENSION TABLE 4
EPINEPHRINE
CONCENTRATION
(pg/pg PROTEIN
k S.E.M.) IN BRAIN
NUCLEI
OF SB, SBH AND SBN RATS
Strain of Rats No. of punches per rat
Region
cannula (mm)
SBN
SB
SBH
N . accumbens
0.5
N . tractus diagonalis N. lateralis septi N. interstitialis striae terminalis N. caudatus-putamen Cingulate cortex N . preopticus medialis N. anterior ventralis thalami N. periventricularis hypothalami N. hypothalamic anterior N. supraopticus N. paraventricularis Median eminence N . arcuatus N. ventromedialis N. dorsomedialis N. hypothalamic posterior Hippocampus Pituitary gland
0.5 0.5 0.5
3.27 0.66 1.07 1.81
% 0.17 f 0.10 2 0.11*t ? 0.16
2.83 0.69 0.45 1.37
+ 2 + +
0.19 0.13 0.08 0.22
3.19 0.72 0.49 1.86
? ? 2 *
0.26 0.09 0.05 0.15
1.0 1.0 0.75 0.5 0.5
2.61 0.142 1.09 0.352 2.49
t + rt 2 2
0.11 0.014 0.05 0.028 0.21
1.98 0.112 0.92 0.285 1.99
+ * 2 -t r
O.l6*t 0.014 0.11 0.042 0.21
2.63 0.134 1.31 0.365 2.60
-t ? + 2 2
0.16 0.017 0.18 0.040 0.21
0.75 0.5 0.5 0.75 0.3 0.5 0.5 0.5 1.0 -
0.680 0.321 3.07 3.95 2.17 0.54 2.29 0.704 0.082 0.586
? + + 2 ? * t 2 2 ”
0.082 0.042 0.17 0.33 0.16 0.06 0.44 0.051 0.014 0.053
0.479 0.291 2.98 3.20 2.17 0.72 2.48 0.835 0.059 0.493
+ 2 2 + + 2 + ” ” *
0.051 0.060 0.21 0.17 0.29 0.15 0.30 0.160 0.006 0.063
0.740 0.257 2.90 5.78 2.58 0.62 2.86 0.754 0.080 0.468
? 2 f 2 ? ? 2 ? 2 2
0.150 0.044 0.34
-
0.32*t
0.15 0.06 0.33 0.086 0.009 0.073
*=p
TABLE 5 VASOPRESSIN CONCENTRATION (p&g PROTEIN ? S.E.M.) IN BRAIN NUCLEI SB, SBH AND SBN RATS
OF
Strain of Rats Region*
n
N . paraventricularis
8 8 8 15
N. supraopticus Median eminence pituitary gland
SBH 1268 ? !%t 1184k99.t 1071 * 727” 1781 ? 63$
n
SB
n
7 7 7 7
162 ? 461‘ 159-c27$ 723 2 47 982 5 48$
9 8 9 15
SBN 435 2 664 2 811 ? 1407 -c
69t 104$ 79$b 86$
n=Number of rats in each group. *No. of punches and cannula size are given in Tables 2-4. *All three groups are significantly different from each other at 0.05 level. SAll three groups are significantly different from each other at 0.01 level. §pP
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