Acta med. scand. Vol. 195, pp. 345-350, 1974

EFFECT OF BICARBONATE AND PHOSPHATE ON THE ADRENERGIC RECEPTOR RESPONSE IN HUMAN ADIPOSE TISSUE IN VITRO

Urban Rosenqvist

From the Department of Endocrinology and Metabolism, Karolinska Hospital. Stockholm, Sweden

Abstract. The adrenergic receptor response of human adipose tissue has been tested in vitro in the presence and absence of bicarbonate and phosphate. It could be shown that omission of these ions from the medium resulted in an enhanced a-adrenergic response of the tissue. The effect of HCO, was inhibited by Diamox". It is concluded that the adrenergic response is sensitive to the presence of these ions and it is suggested that it could be due to a disturbance of the calcium uptake into the mitochondria.

Human adipose tissue contains two kinds of adrenergic receptors with opposite actions on lipolysis (4). Thus, stimulation of the P-adren- ergic receptor increases the rate of lipolysis while a-adrenergic agonists inhibit both basal and stimulated lipolysis. The mode of action of the a- receptor in this tissue has not been clarified but several observations indicate that stimulation of the receptor inhibits the formation of cyclic AMP (6, 9). The a-receptor responsiveness of the tis- sue can vary and is markedly enhanced in adipose tissue from hypothyroid subjects (10, 11, 15). A similar enhancement of the a-adrenergic re- sponse was recently observed in normal adipose tissue specimens incubated in a sodium-deficient medium which permitted calcium to accumulate intracellularly (13).

The object of the present series of experiments was to study whether lack of ions that are of importance for the mitochondria1 uptake of Ca++ (1, 7) would change the adrenergic receptor re- sponse.

any medication. Anaesthesia was induced by a short- acting barbiturate (Nembutal") and was maintained with Halothane", nitrous oxide and oxygen. The tissue was transported in 0.9% NaCl at 37°C from the operating theatre. The adipose tissue was then cut into 50-100 mg pieces and preincubated for one hour at pH 7.4 at 37°C in the same buffer as used in the incubation except that it contained only 1 mglml albumin and no glucose. Separate specimens were then transferred to 1.5 ml medium containing 1 mglml glucose and 3 mglml albumin. The incubation was maintained for two hours at 37°C using air as the gas phase. The composition of the media used is shown in Table 1. The release of glycerol was used as an index of lipolysis and assayed according to Chernic (3).

The following agents were used: I-noradrenaline bi- tartrate (Astra), phentolamine, Regitine" (Ciba), Ne- 2'O-dibutyryl-3',5'-adenosine monophosphate (dibu- tyryl CAMP) (BoehringerlMannheim), acetazolamide (Diamox") (Lederle)

RESULTS

The effect of the regular KHB media and the NACI-Tris medium lacking HCO; and PO:- on the response to different lipolytic agents is shown in Table 11. The basal release of glycerol was significantly lower @<0.001) in the Tris medium. The lipolytic response to 2x M noradrenaline was also significantly lower in the NaCI-Tris buffer both when the absolute values @<0.005) and when the net responses @<0.025) were com- pared. The addition of 5 pglml phentolamine abolished the difference in noradrenaline response between the two kinds of buffers. Phentolamine alone did not significantly modify the basal release

subjects in the KHB group was not significantly different from that of the NaCI-Tris group (44.8+

METHODS Subcutaneous adipose tissue was obtained during Of glycerol in either group. The mean age Of the laparotomy. The patients did not have any malignant or metabolic disorders or jaundice, nor did they take

Acta med. scond. 195

346 U . Rosenqvist

Tahe I. lonic composition of the buffers used ( m M )

Na+ K + Mg+ Ca2+ CI- HC0:- PO:- SO:- Tris

Krets-Henseleit-bicarbonate (KHB) 164 6.1 1.4 2.5 129 25 1.4 1.4 - NaCI-Tris 160 4.6 I 1.5 169.6 - - - 2 NaCI-H COF-Tri s 160 4.6 I 1.5 140 25 2 NaCI-PO:--Tris 160 4.6 I 1.5 164.6 - 5 - 2 NaCI-H CO;-PO:--Tri s 160 4.6 I 1.5 135 25 5 - 2

- -

2.8 and 37.4f3.5 years, mean fS.E.M.), nor was there any difference in percent of ideal body weight (16) between the KHB and NaCI-Tris group (102.4k5.0and 101.0f3.8%, mean 2S.E.M.).

When the net lipolytic response to 2x M noradrenaline was calculated as percent of that obtained in the presence of 5 pg/ml phentol- amine it was 49.9f7.7% in the KHB group and 19.823.9% (mean 2S.E.M.) in the NaC1-Tris group. The difference between the two groups was highly significant (p<0.005).

In order to test whether the observed differences between the two media could be accounted for by the lack of HCO; or POI- the effect of these ions was further investigated (Table H I ) . The supplementation with 25 mM HCO; slightly re- duced the basal release of glycerol. However, neither HCO; or PO:- had any significant ef- fect on basal lipolysis when tested by analysis of variance (8). In the presence of HCO; or PO:- noradrenaline caused a significant (p<0.05) stimulation of lipolysis and this was further in- creased when both anions were added together (p<O.Ol ) . The responses to noradrenaline plus phentolamine were of the same magnitude in the presence or absence of PO:- or HCO; + PO:-.

Table 11. Effect o f K H B and NaCI-Tris on the

The response to dibutyryl cyclic AMP was slightly but not significantly increased in these two latter buffer combinations. In all buffer combinations the responses to either noradrenaline in the pres- ence of phentolamine or dibutyryl cyclic AMP were of the same magnitude and did not differ significantly when tested by paired means.

When the net lipolytic response to ~ X I O - ~ M noradrenaline was calculated as percent of the net response obtained with 2x M noradrenaline in the presence of 5 pg/ml phentolamine the re- sults indicate that the response was significantly increased by HCO; (pc0.05) alone or in com- bination with PO:- @<0.02), while PO:- alone did not have any effect (Fig. I ) .

Damox@, an inhibitor of the carbonic an- hydrase enzyme, was added to the medium in order to further investigate the effect of the HCO; on the adrenergic response. In this series of experiments the response to noradrenalhe+ phentolamine was significantly reduced @<0.05) by HCO;, while the response to noradrenaline alone was unaffected (Table IV). The addition of 0.45 rnM Diarnoxa abolished the difference in re- sponse to noradrenaline+phentolamine (Table IV). This was also apparent when the lipolytic effect of

lipolytic response to 2 X I O - 5 M I-norudrenuline . . ""

in presence and absence of 5 pglml phentolamine (mean 2 S . E . M . )

KHB (N = 16P NaCl-Tris (N=16)" Differencesb

Basal 0.885+0.116 0.448 f 0.074 0.437k0.138 p<0.005 I-noradrenaline 2 x M 1.843f0.207 1.021f0.131 0.882 k 0.245 p < 0.005 I-noradrenaline-basal 0.958f0.129 p<O.001 0.573k0.096 p<O.001 0.384k0.161 ~ ~ 0 . 0 2 5 I-noradrenatine + phentol- amine 5 pg/ml 2.956f 0.39 1 3.494f0.58 1 0.538k0.700 N.S.

(I-noradrenatine + phentol- amine 5 pg/ml)-phentolamine 2.161f0.310 p<O.001 3.lOOf0.562 p<O.001 0.939f0.642 N.S.

Phentolamine 5 pg/ml 0.7WkO. 126 0.394f0.07 1 0.400f0.144 p<O.O2 Phentolamine 5 pg/ml-basal 0.091 f0.066 N.S. -0.054 f 0.050 N . S . 0.037+0.083 N.S.

a The significance was calculated from the paired differences (8). The significance between the means was calculated by Student's r-test (8).

Acto med. scand. 195

Adrenergic receptor response to bicarbonate and phosphate 341

Table 111. Effect of 25 m M HCO; and 5 m M PO:- on the lipolytic, response to 2x10-5 M I-nor- adrenaline in presence and absence 1 , f 5 pglml phentolamine and M dibuturyl cvclic AMP ( N = 5 )

NaCI-HCOF- NaC1-Tris NaCI-HC0,;-Tris NaCI-POf--Tris P04*--Tris

Basal I-noradrenaline I-noradrenaline-basal Phen tolamine Phentolamine-basal 1-noradrenahne+phentolamine (I-norad renab ne + phentolamine) -

Dibuturyl cyclic AMP Dibuturyl cyclic AMP-basal

phentolamine

0.560_+0.103 1.642f0.391 1.082fO.454 0.43 1 k0.267 0.129fO.214 4.806f0.484

4.376f0.655** 4.555+0.901 3.995 k 0.932*

0.30010.169 1.742kO.367 1.441 f0.3 17* 0.284_+0.113

-0.016f0.068 3.2 19f0.54 1

2.948 f 0.426* * 4 .O 14 f 0.495 3.630 k0.564* *

0.684f 0.178 1.9OOk0.279 1.2 15+0.327* 0.503 k 0.077

5.20 1 k 0.704

4.698+0.664** 6.135+0.%5 5.595+0.985**

-0.18lf0.121

0.605f0.199 2.980k0.598 2.375?0.483** 0.83910.159 0.234k0.088 4.765 k0.807

3.927 k 0.77 I ** 6.056f 1.289 5.334f 1.455*

~ ~ ~~

The p-values were calculated from the paired means; *p<0.05, **p<O.OI.

noradrenaline was expressed in percent of that ob- tained in the presence of phentolamine (Fig. 2).

DISCUSSION

The present investigation has demonstrated that the adrenergic receptor response of normal hu- man adipose tissue in vitro was significantly in- fluenced by the ionic composition of the medium used.

Previous studies have also demonstrated that the a-adrenergic response of human subcuta- neous adipose tissue is markedly changed when incubated in a sodium-deficient medium (13). The a-adrenergic response which inhibits the forma- tion of cyclic AMP, and thus lipolysis, was en- hanced in this type of buffer. The lipolytic effect of noradrenaline was, however, restored by the addition of phentolamine. A more detailed analysis demonstrated that the a-response was enhanced

Table IV. Effect on the lipolytic response to ~ x I O - ~ M I-noradrenaline ( N A ) and 2x10-5 M 1-noradrenaline ( N A ) plus 5 pglml phentolamine (Ph) in presence and absence of 25 m M bicarbonate in the NaCI-Tris medium (N =5)

Diamox 0.45 mM -

Mean Mean diff. Mean Mean diff. Medium Additions kS.E.M. f S. E.M. p" kS.E.M. fS .E.M. Pa

NaC1-Tris NaCI-Tris NaCI-Tri s NaCCTris NaCI-Tris-HCO, NaCI-Tri s-H CO, NaCI-Tri s-H CO, NaCI-Tri s-HCO,

None NA NA+Ph Ph None NA NA +Ph Ph

0.592k0.052 1.48310.240 6.2%+0.852 0.732k0.228 0.17610.059 1.340f0.295 1.%310.546 0.292k0.230

- 0.891 kO.190 5.564f1.031 0.140k0.232

1.164k0.295 I ,672 k 0.397 0.1 15k0.220

-

- <0.01 <0.01

N.S.

<0.02 <0.02

N.S.

-

-0.273k0.363 N.S.

3.890+ 1.379 <0.05

- N A NaCI-Tn\ NaCI-Trir-HCO,

NA

(NA+Ph) -(NA +Ph) NaCI-Trir NaCI-Tris-HCO,

The p-values were calculated from the paired differences.

0.62510. 179 I ,409f0.323 4.692k0.425 0.94610.243 0.356k0.129 I .276f0.471 3.082 k 0.689 0.3 13k0.146

- 0.784f0.433 3.8 lOk0.478 0.321f0.145

0.920f0.437 2.769k 0.653

-0.043k0.043

-0.136kO.662

-

1.033k0.795

- N.S.

<0.005 N.S.

N.S. C0.02

N.S.

-

N.S.

N.S.

Acta med. wand. 195

348 I/. Rosenqvist

l o o r

50

2 5

- Hco, PO, HC0,Q 0

Fig. I . Effect of 25 mM bicarbonate and 5 mM phos- phate on the lipolytic response to ~ X I O - ~ M l-nor- adrenaline as calculated in percent of that induced by 2 x M I-noradrenaline plus 5 Clglml phentol- amine. The p-values were calculated from paired dif- ferences.

as a result of an increase in the concentration of calcium in the cells induced by the sodium defi- cient medium (13). So far little is known about the mechanism of action of the a-receptor in hu- man adipose tissue. However, since it could be demonstrated that the hypothyroid state induced an enhancement of the a-adrenergic response in human adipose tissue (15) as well as in smooth muscles from rabbit aorta (14). it was hypothesized that the mechanisms in the two kinds of tissue could be of similar nature (12). In smooth muscle more is known about the a-adrenergic receptor re- sponse which induces an increase in ionized cal- cium intracellularly, which in turn causes contrac- tion (2). The recent findings of a Caz+ involve- ment in the a-response in normal adipose tissue would strengthen the above hypothesis (13). The object of the present investigation was to study the effect of anions known to be required for the up- take of Caz+ into mitochondria ( 5 , 7), the idea being that omission of such ions would reduce the uptake of calcium into the mitochondria and thereby potentiate the a-adrenergic response. It was recently shown that the uptake of Ca++ in- to mitochondria requires either PO:- or HCO:- ( 5 , 7). Furthermore, it could be shown that Diamoxm, an inhibitor of the carbanhydrase en-

zyme, would block the effect of HCO;, indi- cating that the important component of the buffer was C 0 2 which was converted to C0:- inside the mitochondrium (5). In addition to these studies Borle (1) has demonstrated that removal of PO:- from the incubation medium causes a rapid release of Ca2+ from cells in tissue culture. Most prob- ably this was a release from the mitochondria in the cells. The present study has shown that re- moval of HCO; and PO:- reduced the lipolytic response of the tissue to noradrenaline, while that to noradrenaline+phentolamine remained, indi- cating that the a-adrenergic response was en- hanced by the omission of bicarbonate and phos- phate. The difference in noradrenaline response could not be accounted for by a difference in age or percent of ideal body weight between the two groups studied.

The KHB and NaCI-Tris differ in the content of three main ions as shown in Table I. The reduc- tion of the Ca++ concentration from 2.5 to 1.5 mM was felt to be of less importance and could prob- ably not account for the enhanced a-response, since the experiments with La+++ would predict the opposite (13). The other difference was the omission of bicarbonate and phosphate in the NaC1-Tris buffer. In order to further study the effect of the lack of these ions, HCO; and PO:-

Diemox 0.45 m M

+<bW I- N.S.4

7 5 I

Fig. 2 . Effect of Diamox' on the response to 25 mM bicarbonate. The response to 2 x M I-noradrenaline was calculated in percent of that of 2X10-s M I-nor- adrenaline plus 5 pg/ml phentolamine. The p-values were calculated from paired differences.

Acra med. scand. 195

Adrenergic receptor response to bicarbonate and phosphate 349

were added back to the NaCI-Tris medium. The results show that the addition of HCO; slightly reduced the response to noradrenaline plus phentolamine, while the addition of PO:- alone or in combination with HCO; significantly stim- ulated lipolysis induced by noradrenaline both in the presence and absence of phentolamine. The response to dibutyryl cyclic AMP was similarly affected as the response to noradrenaline plus phentolamine (Table 111). However, when the nor- adrenaline response was expressed in percent of that obtained in the presence of phentolamine, a significant increase was observed with addition of HC05 alone or HCO; and PO:-. The re- sults show that the adrenergic response is mark- edly influenced by these two anions and that the a-adrenergic response can be suppressed by them.

The study of Elder and Lehninger (5) showed that the mitochondria1 dependence on HCO; for the uptake of CaZ+ was inhibited by Diamoxa or lack of CO, in the medium. This was inter- preted as evidence for the entry of COz into the mitochondria and conversion to HCO;. The ion was then utilized for the trapping of Caz+ inside the mitochondria. Because of these findings it was of interest to test Diamox@ on the effect of bicarbonate on the adrenergic response. In these series of experiments the presence of bi- carbonate reduced the response to noradrenalhe+ phentolamine @<0.05), while the response to noradrenaline alone was not affected. Hence, on a percentage basis the noradrenaline response was significantly increased, as shown in Fig. 2. The addition of 0.45 mM Diamox@ markedly reduced the effect of bicarbonate, while it had little effect on the response obtained in media lacking this ion. Thus it may be anticipated that the effect of bicarbonate can be antagonized by Diamox". This would imply that it might not be the HCO; per se which is active, but the CO, which af- fects the adrenergic response in analogy with the results obtained by Elder and Lehninger (5 ) . How- ever, the above noted effects of bicarbonate and phosphate should be interpreted with care and a final analysis postponed until more is known about the intracellular compartimentalization of Caz+ in this tissue.

In conclusion the present experiments have shown that the a-adrenergic response is poten- tiated in medium lacking bicarbonate and phos-

phate. The mechanism behind this change in adrenergic response could be due to a deficient calcium buffering capacity of the mitochondria in the cells when bicarbonate or phosphate is not present in the incubation medium.

ACKNOWLEDGEMENT This investigation was supported by grants from the Medical Research Committee of the Swedish Life In- surance Companies, Nordic Insulin Foundation, Karl PetrCn Foundation and the Swedish Medical Research Council (B74-19x4223-01).

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