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Norepinephrine-epinephrine and serotonin content of nasal mucosa in relation to hemorrhage, reserpine and excitement
Life Scieacas Vol . 18, pp . 211-214 Printed in the U .S .A .
Pergamoa Press
AASAL MITWSA JF RSIATICLQ TO ~RABAag, R AFD ~CC ITP~PP Sharon A . Arihood and Richard T . Jackson Division of Otolaryngology Rmory University School oß Medicine, Atlanta, Georgia
30322
(Received in final form December 29, 1975)
The norepinephrine-sp in~ *+" and serotonin contanta oß rabbit and. dog nasal mucosa xere determined by a fluorescent method . Norepinephrine-epinephrine levels (4 ug~gm) in the dog xere reduced by hemorrhage . In rabbits, the normal level xas loner (1 .3 ug/~) and xaa reduced by reaerpine and 5-20 seconds oß violent struggle . The serotonin level xas reduced by treatment xith reaerpine . The dopamine content of na~al mucosa xas be].or the detection limit oß the method (0 .2 ug~gm oß xet tissue) . The amrnmt oß blood Blaring thmugh the nasal mucosa can quickly regulate the potency of the nasal aiz~rey . Norma].lf, the nasal blood vessels are partialljt constricted due to a sympathetic tone (1) . Ar4r interruption of this tone results in nasal vasodilation and a deoreased nasal aixvay . A common esemple is the nasal congestion seen in hypertensive patients treate8 xith reaerpine . Dahlstram and lwae (2) studied the histological localization and distribution oß norepinephriae in the nasal muoosa oß rats ; guinea pigs, rabbits sad cats using the fluorescent histologiel method . They found an extensive, rich netxork of sympathetic fibers . One goal oß this xork xas to quantitatively determine the norepinephrineepinephrine and serotonin content oß animal nasal mucosa . Another goal xas to attempt to determine the ]ability oß these compounds by employing procedures li]cely to induce changes in their concentration . Methode üe used the procedure oß Anaell and season (3) to determine the norepinephriae-epinephrine (103-E), serotonin and dopamine co~ate~ of the nasal muooaa in the adult rabbit and dog . The method does not separate the norepi++~±r+nR from the epi~~,.~rp content . The dogs and rabbits xere killed by a .~ overdose oß eodi~ pentobaz+bital . The nasal maoosa xas removed Pram the nasal septum an8 lateral xa11 oß the nasal foam . Tut'binate tiaeue xas not used. Care xas taken to exclude cartil age and bone fragments . The xet xeight of the tissue varied from 0 .1 to 1 .9 gnome, the rabbit having much less mucosa than the dog . After removal, the tisane xas rinsed briefly in distilled xater and quickly Brown .
212
Norepinaphrine in Nasal Hucoea
Vol . 18, No . 2
The frozen tissue and a little dry ice rare placed in a mortar and pulverized xith a pestle . After most of the dry ice had sublimed, the tissue xas treasferred to a-glaes homogenizer and homogenized in about 10 ml of acidbutanol per gram of tissue . The volume of acid-butanol xas adjusted to produce meaningful readings rhea the final solutions rare read on the fluorometer . A Pew modifications of the procedure rare used . The pH 6 .5, O .1M EDTA solution had 3 .72 grams dissolved in 95 ml of 1M sodium acetate instead of 950 ml as reported . The 2M, pH 7 .6 sodium acetate, 0 .5M, pH 6 .5 Phosphate buffer, stock catechalamine solutions sad sodium sulfite solution rare pre pared the day of the analysis . The borate buffer xas made every reek . The refrigerated phosphate buffer, pH 7 .0, xas stable for several months . The alternate method given by Ansell and Besson xas used for the last part of the analysis . 5 ml of the supernatant from above the alumina tubes xas used to analyze for serotonin . The remainder of the supernatant over the alumina tube xas discarded, and 0 .8 ml of 0 .5M, pH 6 .5 phosphate buffer xas added to elute the amines . Then 0 .5 ml of the shaken sad centrifuged supernatant xas tr~ted according to the rest of the procedure . The percent recoveries rare as follows : serotonin 42 .6 + 3 .6 (12), NE-E 74 .2 + 3 .4 (12) and dopamine 32 .6 + 3 .7 (11) . The amberôf determinations is in peaeatheses . The analyses rare performed on non-pregnant (male and female) dogs and those pregnant and near-term . The comparison of pregnant versus non-pregnant xas made because roman often experience a nasal congestion during pregnancy eapecially during the last trimester . We xished to determine if the nasal congestion might be related to the level of these compounds . Reversible hemorrhage had been shorn to reduce NE levels in the heart, liver and spleen of rabbits sad several tissues in the dog (4) . We therefore applied the ana~rsis to nasal tissue from four dogs that had been hemorrhaged to e systolic blood pressure of 50 mm Ag " The rabbits rare divided into three groups . The Pirst rare normal rabbits that had been killed xith en overdose of i~ravenous sodium pento barbital in such a ray that no signs of straggle rare evident . The second group xea killed with the same anesthetic but had struggled viole~ly for 5 to 20 seconds before death . The struggle usually occurred because xe had not ca].mly handled the animal or did not ezercise meticulous care in the insertion of the needle or the delivery of the anesthetic . The third group of rabbits had been treated xith 0 .75 mg/kg of reaerpine 24 hours before being killed quietly by an overdose of sodium pentobarbital . Samples of rat corpus striat~ rare analyzed xith the nasal mucosa samples to verifq our dopamine analysis . Results Dopamine - With the dilutions that rare used, the estimated dopamine concentration xas be].cm our detection limits, approzimately 0 .2 ug/gm of tissue . For verification, our value of 4 .43 ug/gm of dopamine in the rat corpus striatum is samexhat lower than the values (5 .12 to 8 .58 ug/gm) reported using other methods (5-8) . Because of these uncertainties, xe feel it is best to say that the concentration of dopamine in nasal mucosa is be].ow the threshold of this technique as xe used it . 3erotonin and NE-E - The content of these compounds in our dogs is shown in Table l . The data in the tables is sot corrected for percent recovery . There xas no diPfereace betxeen the values obtained in non-pregnant and preg nant dogs . In hemorrhaged dogs, the NE-E content dropped significantly . The aerotonin and NE-E levels in our rabbit groups are shown in Table 11 . The NE-E levels in the normal, unstressed rabbits ere less than in the dog sad more variable . The rabbits that struggled showed a drastic drop in the NE-S co~eat and a slight drop in serotonin . The reserpinized rabbits had serotonin levels similar to the dog and a NE-E coateat below the detection limit of the procedure .
Vol . 18, No . 2
Norepinephriae in Nasal Mucosa
213
TABLE 1 Serotonin and HE-E Content of Dog Halal Mucosa u8/gm Wet Weight Source
8erotonia
HE-E
Hon pregnant dogs
0 .206 + 0 .03 ~8
4 .]1 + 0 .33
Pregnant dogs
0 .201 + 0 .02 n~+
3 .99 ± 0.84
Hemorrhaged dogs
0 .194 + 0 .05 n~+
O .ô2 _+ 0 .13
TART .F ]1 Serotonin and HE-E Content of Rabbit Halal Mucosa ug/gm Wet Weight
Source
Serotonin
FE-E
Untreated rabbits (pentobarbitalno struggle)
1 .53 + 0 .48 n=9
1 .36 _+ 0 .42
Untreated rabbits (pentobarbitalstruggle)
1 .10 + 0 .33 na+
~ 0 .25
Reserpiaieed rabbits, pentobarbital, ao struggle
0 .24 + 0 .13 nt9
~ 0 .25
Discussion Ho accurate estimate can be made of the dopamine content of ra,sal mucosa Prom these experiments . It appears that the coaceatxstion must be leas than 0 .2 ug/geo of xet tissue . The 1PS-E content of dog nasal mucoea is higher than brain (9) xhich varies Pram 0 .01 ug/gm in the posterior roots to 1 .04 u8/gm in the arm postre®n . Levels o! HE in the rabbit brain vary from 0 .05 ug/gm in the medulla oblongata to 0 .85 in the nucleus caudatus, an area also very high in dopamine (10) . Estimates of HE-E in a more vascular tissue, egleen, vary from 1 .65 (ll to 4 .75 ue/8~+ (~) " It is not surprising that hemorrhage reduces the HE-E content of nasal mucosa . In rabbits, heart, liver and spleen lose approximately 50~ of their H$ comment xhen subjected to reversible hemorrhagic shock (ll) . Ho eign ific~at changes xere found in the epinephrine level in the latter experiments . The N8 concentration in individual tissues is the dog vary in their response to hemorrhage . Ia dogs, the lung and liver retain their HE content (12) . It is xell known that reeerpine depletes HE and serotoaia (l9) . Thus the drop in the HS-E and serotoain content of rabbit nasal muoosa xae eapecteb It semis reasonable that a struggle should reduce the HE-E level because of the accompanying mesa sympathetic discharge and the very large store of these campotimds in nasal mucoea . It xould appear that the PE-E and eerotoain content of animal nasal mueosa is relative],y high, dynamic and responsive to manipulation .
214
Norapinephrine in Nasal Mucoea
Vol . 18, No . 2
References 1.
D.id . Rooker, R.T . Jacksoa, Ann. Otol . Rrinol . Iary~~gol. 78 402-414
12 .
H. HiPt, M. i~7.peria, 8. Hegedur, W . Thaaas, Proc . 8od . ~cp. Biol . Med. ~ 883-887 (1965) . B. F[ovacic, R. Robinson, J. Pharmadol. E~cp . Ther . 152 37-41 (1966) .
(1969) " 2. A . Dahlstrdm, K. P~xe, Acta 0bolaryng. ~ 65-72 (1965) . 3 . G.B . Anrell, M.F . Beeson, Ana1 . Biocrem. ~ 196-206 (1968) . 4. A .B . Horn, A .C . Cuello, R.J . Miller, J . Neurochem . 22 265-270 (1974) 5" J.G. Wood, R.E . Budkley, Sxperieatia 2~ 834-837 (1973 6. W .J . Vaatstra, L. Tigeman, F~rop . J . Pharm . 25 185-190 (lgT4) . 7" M.K. 8hellenberger, J.H . Cordon, Anal . Biodran. 356-372 (1971) . 8. N.J . Uretslgr, J . Pharmacol. akp . Ther . 18~ 359(1974) . 9. J. Crorsland, The Clinicetl Che®istry of Monamines, P.J . Yarley, A .H . Goaealodk, Eds . Eleevier Publ . Co . Amsterdam (1962) . l0 . M. Matsuoka, H. Yosida, R. Imai zumi, Biodhim . Biopàprs . Adta 82 439-441 (1964) . 13 .
Pergamoa Press
AASAL MITWSA JF RSIATICLQ TO ~RABAag, R AFD ~CC ITP~PP Sharon A . Arihood and Richard T . Jackson Division of Otolaryngology Rmory University School oß Medicine, Atlanta, Georgia
30322
(Received in final form December 29, 1975)
The norepinephrine-sp in~ *+" and serotonin contanta oß rabbit and. dog nasal mucosa xere determined by a fluorescent method . Norepinephrine-epinephrine levels (4 ug~gm) in the dog xere reduced by hemorrhage . In rabbits, the normal level xas loner (1 .3 ug/~) and xaa reduced by reaerpine and 5-20 seconds oß violent struggle . The serotonin level xas reduced by treatment xith reaerpine . The dopamine content of na~al mucosa xas be].or the detection limit oß the method (0 .2 ug~gm oß xet tissue) . The amrnmt oß blood Blaring thmugh the nasal mucosa can quickly regulate the potency of the nasal aiz~rey . Norma].lf, the nasal blood vessels are partialljt constricted due to a sympathetic tone (1) . Ar4r interruption of this tone results in nasal vasodilation and a deoreased nasal aixvay . A common esemple is the nasal congestion seen in hypertensive patients treate8 xith reaerpine . Dahlstram and lwae (2) studied the histological localization and distribution oß norepinephriae in the nasal muoosa oß rats ; guinea pigs, rabbits sad cats using the fluorescent histologiel method . They found an extensive, rich netxork of sympathetic fibers . One goal oß this xork xas to quantitatively determine the norepinephrineepinephrine and serotonin content oß animal nasal mucosa . Another goal xas to attempt to determine the ]ability oß these compounds by employing procedures li]cely to induce changes in their concentration . Methode üe used the procedure oß Anaell and season (3) to determine the norepinephriae-epinephrine (103-E), serotonin and dopamine co~ate~ of the nasal muooaa in the adult rabbit and dog . The method does not separate the norepi++~±r+nR from the epi~~,.~rp content . The dogs and rabbits xere killed by a .~ overdose oß eodi~ pentobaz+bital . The nasal maoosa xas removed Pram the nasal septum an8 lateral xa11 oß the nasal foam . Tut'binate tiaeue xas not used. Care xas taken to exclude cartil age and bone fragments . The xet xeight of the tissue varied from 0 .1 to 1 .9 gnome, the rabbit having much less mucosa than the dog . After removal, the tisane xas rinsed briefly in distilled xater and quickly Brown .
212
Norepinaphrine in Nasal Hucoea
Vol . 18, No . 2
The frozen tissue and a little dry ice rare placed in a mortar and pulverized xith a pestle . After most of the dry ice had sublimed, the tissue xas treasferred to a-glaes homogenizer and homogenized in about 10 ml of acidbutanol per gram of tissue . The volume of acid-butanol xas adjusted to produce meaningful readings rhea the final solutions rare read on the fluorometer . A Pew modifications of the procedure rare used . The pH 6 .5, O .1M EDTA solution had 3 .72 grams dissolved in 95 ml of 1M sodium acetate instead of 950 ml as reported . The 2M, pH 7 .6 sodium acetate, 0 .5M, pH 6 .5 Phosphate buffer, stock catechalamine solutions sad sodium sulfite solution rare pre pared the day of the analysis . The borate buffer xas made every reek . The refrigerated phosphate buffer, pH 7 .0, xas stable for several months . The alternate method given by Ansell and Besson xas used for the last part of the analysis . 5 ml of the supernatant from above the alumina tubes xas used to analyze for serotonin . The remainder of the supernatant over the alumina tube xas discarded, and 0 .8 ml of 0 .5M, pH 6 .5 phosphate buffer xas added to elute the amines . Then 0 .5 ml of the shaken sad centrifuged supernatant xas tr~ted according to the rest of the procedure . The percent recoveries rare as follows : serotonin 42 .6 + 3 .6 (12), NE-E 74 .2 + 3 .4 (12) and dopamine 32 .6 + 3 .7 (11) . The amberôf determinations is in peaeatheses . The analyses rare performed on non-pregnant (male and female) dogs and those pregnant and near-term . The comparison of pregnant versus non-pregnant xas made because roman often experience a nasal congestion during pregnancy eapecially during the last trimester . We xished to determine if the nasal congestion might be related to the level of these compounds . Reversible hemorrhage had been shorn to reduce NE levels in the heart, liver and spleen of rabbits sad several tissues in the dog (4) . We therefore applied the ana~rsis to nasal tissue from four dogs that had been hemorrhaged to e systolic blood pressure of 50 mm Ag " The rabbits rare divided into three groups . The Pirst rare normal rabbits that had been killed xith en overdose of i~ravenous sodium pento barbital in such a ray that no signs of straggle rare evident . The second group xea killed with the same anesthetic but had struggled viole~ly for 5 to 20 seconds before death . The struggle usually occurred because xe had not ca].mly handled the animal or did not ezercise meticulous care in the insertion of the needle or the delivery of the anesthetic . The third group of rabbits had been treated xith 0 .75 mg/kg of reaerpine 24 hours before being killed quietly by an overdose of sodium pentobarbital . Samples of rat corpus striat~ rare analyzed xith the nasal mucosa samples to verifq our dopamine analysis . Results Dopamine - With the dilutions that rare used, the estimated dopamine concentration xas be].cm our detection limits, approzimately 0 .2 ug/gm of tissue . For verification, our value of 4 .43 ug/gm of dopamine in the rat corpus striatum is samexhat lower than the values (5 .12 to 8 .58 ug/gm) reported using other methods (5-8) . Because of these uncertainties, xe feel it is best to say that the concentration of dopamine in nasal mucosa is be].ow the threshold of this technique as xe used it . 3erotonin and NE-E - The content of these compounds in our dogs is shown in Table l . The data in the tables is sot corrected for percent recovery . There xas no diPfereace betxeen the values obtained in non-pregnant and preg nant dogs . In hemorrhaged dogs, the NE-E content dropped significantly . The aerotonin and NE-E levels in our rabbit groups are shown in Table 11 . The NE-E levels in the normal, unstressed rabbits ere less than in the dog sad more variable . The rabbits that struggled showed a drastic drop in the NE-S co~eat and a slight drop in serotonin . The reserpinized rabbits had serotonin levels similar to the dog and a NE-E coateat below the detection limit of the procedure .
Vol . 18, No . 2
Norepinephriae in Nasal Mucosa
213
TABLE 1 Serotonin and HE-E Content of Dog Halal Mucosa u8/gm Wet Weight Source
8erotonia
HE-E
Hon pregnant dogs
0 .206 + 0 .03 ~8
4 .]1 + 0 .33
Pregnant dogs
0 .201 + 0 .02 n~+
3 .99 ± 0.84
Hemorrhaged dogs
0 .194 + 0 .05 n~+
O .ô2 _+ 0 .13
TART .F ]1 Serotonin and HE-E Content of Rabbit Halal Mucosa ug/gm Wet Weight
Source
Serotonin
FE-E
Untreated rabbits (pentobarbitalno struggle)
1 .53 + 0 .48 n=9
1 .36 _+ 0 .42
Untreated rabbits (pentobarbitalstruggle)
1 .10 + 0 .33 na+
~ 0 .25
Reserpiaieed rabbits, pentobarbital, ao struggle
0 .24 + 0 .13 nt9
~ 0 .25
Discussion Ho accurate estimate can be made of the dopamine content of ra,sal mucosa Prom these experiments . It appears that the coaceatxstion must be leas than 0 .2 ug/geo of xet tissue . The 1PS-E content of dog nasal mucoea is higher than brain (9) xhich varies Pram 0 .01 ug/gm in the posterior roots to 1 .04 u8/gm in the arm postre®n . Levels o! HE in the rabbit brain vary from 0 .05 ug/gm in the medulla oblongata to 0 .85 in the nucleus caudatus, an area also very high in dopamine (10) . Estimates of HE-E in a more vascular tissue, egleen, vary from 1 .65 (ll to 4 .75 ue/8~+ (~) " It is not surprising that hemorrhage reduces the HE-E content of nasal mucosa . In rabbits, heart, liver and spleen lose approximately 50~ of their H$ comment xhen subjected to reversible hemorrhagic shock (ll) . Ho eign ific~at changes xere found in the epinephrine level in the latter experiments . The N8 concentration in individual tissues is the dog vary in their response to hemorrhage . Ia dogs, the lung and liver retain their HE content (12) . It is xell known that reeerpine depletes HE and serotoaia (l9) . Thus the drop in the HS-E and serotoain content of rabbit nasal muoosa xae eapecteb It semis reasonable that a struggle should reduce the HE-E level because of the accompanying mesa sympathetic discharge and the very large store of these campotimds in nasal mucoea . It xould appear that the PE-E and eerotoain content of animal nasal mueosa is relative],y high, dynamic and responsive to manipulation .
214
Norapinephrine in Nasal Mucoea
Vol . 18, No . 2
References 1.
D.id . Rooker, R.T . Jacksoa, Ann. Otol . Rrinol . Iary~~gol. 78 402-414
12 .
H. HiPt, M. i~7.peria, 8. Hegedur, W . Thaaas, Proc . 8od . ~cp. Biol . Med. ~ 883-887 (1965) . B. F[ovacic, R. Robinson, J. Pharmadol. E~cp . Ther . 152 37-41 (1966) .
(1969) " 2. A . Dahlstrdm, K. P~xe, Acta 0bolaryng. ~ 65-72 (1965) . 3 . G.B . Anrell, M.F . Beeson, Ana1 . Biocrem. ~ 196-206 (1968) . 4. A .B . Horn, A .C . Cuello, R.J . Miller, J . Neurochem . 22 265-270 (1974) 5" J.G. Wood, R.E . Budkley, Sxperieatia 2~ 834-837 (1973 6. W .J . Vaatstra, L. Tigeman, F~rop . J . Pharm . 25 185-190 (lgT4) . 7" M.K. 8hellenberger, J.H . Cordon, Anal . Biodran. 356-372 (1971) . 8. N.J . Uretslgr, J . Pharmacol. akp . Ther . 18~ 359(1974) . 9. J. Crorsland, The Clinicetl Che®istry of Monamines, P.J . Yarley, A .H . Goaealodk, Eds . Eleevier Publ . Co . Amsterdam (1962) . l0 . M. Matsuoka, H. Yosida, R. Imai zumi, Biodhim . Biopàprs . Adta 82 439-441 (1964) . 13 .