‘ ,g'g" 3 .-. no n-:%" ”0.. r.M.Q“‘.OhD-.q- ~43un C-.~.Qv—w--I‘-~‘a-Q*Oq - o - -v— ‘3-- , , - 5‘... -1. ~ - o u . _' _ ' O ‘ . - l .5 ‘ CHEMICAL” EVOKED HYPOTHERMiA. ' {N THE MOUSE - Thesis for the Degree of M, S. WCHiGAN STATE UN VERSITY -~ ARDREW STUART JANOFF - . I 1976 ” - . . . , _ J (. 5 . v I -‘ - . v ' - . o f ‘ . . . . -'--' o . ‘ ' l ' .' . ,g' ‘ u g . n I - , a . '.' n , . . , ‘ . ., ’ . . 4 .« . - _ 1 r. - . ' . , . . -' ' o . 'I.‘ . ‘- - .. I . a ‘ I LIBRARY _ Michigan Stase *' "ii. University ABSTRACT CHEMICALLY EVOKED HYPOTHERMIA IN THE MOUSE By Andrew Stuart Janoff The hypothermic response after parenteral administration of triflupromazine, promazine, chlorpromazine, L-dopa, reserpine, parachlorophenylalanine, and procaine was investigated in the mouse. Mice injected at 24 hour intervals with triflupromazine, promazine, or chlorpromazine exhibited tolerance (a diminished response) with repeated administration. The degree of induced hypothermia and resistance to tolerance after chlorpromazine was greater in male mice than in female mice. Repeated injections of triflupromazine produced less tolerance in 5 week old mice then in adult mice, but this compound failed to induce hypothermia in 5 week old mice after the first injection. Prior administration of triflupromazine dramatically potentiated the hypothermic effect of promazine. L-dopa was unable to potentiate chlorpromazine hypothermia but did evoke a nonadapting hypothermic response when administered alone. Reserpine also evoked a nonadapting hypothermia. Further, animals treated with small doses of reserpine exhibited an increasing response with repeated injections. Parachlorophenylalanine induced hypothermy that became intermittent with successive administration. Procaine Andrew Stuart Janoff did not affect core body temperature at all. The results presented here indicate that of the compounds studied only reserpine and L-dOpa would be of value as hypothermic agents in any long term investigation. CHEMICALLY EVOKED HYPOTHERMIA IN THE MOUSE BY Andrew Stuart Janoff A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Biophysics 1976 This is for my Mother and my Father ii ACKNOWLEDGEMENTS The author wishes to express sincere and grateful appreciation to Dr. Barnett Rosenberg for his continual encouragement and advice over the course of this work. The author is further indebted to Dr. Gabor Kemeny for many stimulating discussions, to Loretta VanCamp for her valuable suggestions, and to Paula Faber and Steve Wallace for their excellent technical assistance. Deep gratitude is expressed to Julie, whose support and understanding have, in a large way, made this thesis possible. 111 TABLE OF CONTENTS Page INTRODUCTION.................................................. 1 Thermodynamics of Ageing and Death....................... 1 Hypothalamic Mechanisms of Thermoregulation.............. 3 Disruption of Thermoregulation by the Systematic Administration Of DrugSOOO0.0000000000000000000.0.0.... 6 Phenothiazines...................................... 6 Procaine............................................ 8 Parachlorophenylalanine............................. 8 Reserpine........................................... 9 METHODS....................................................... 10 General Procedures....................................... 10 Phenothiazines........ .......... . ..... ................... 11 Promazine........................................... ll Triflupromazine..................................... 11 Chlorpromazine and L-Dopa........................... 11 Determination of cross tolerance between triflupromazine, promazine, and chlorpromazine.... 12 ProcaineOOOOOOOOOOOOOOI.0.0.0.0.00....OOOOOOOOOOOOOOOOOOO 12 Parachlorophenylalanine.... ..... ......................... 12 ReserpineOOOOOO. ..... O ......... .00... ........ O... ...... O. 13 iv TABLE OF CONTENTS (cont'd-) RESULTS........... ..... ............................ ..... ...... Phenothiazines. ........ ..... ............. . ..... .......... Promazine................ ......... ... ........ ....... Triflupromazine.... ...... . .......... .. .......... .... Chlorpromazine and L-dopa........................... Determination of cross tolerance between triflu- promazine, promazine and chlorpromazine ...... ..... Procaine.............. ...... .................... ....... .. Parachlorophenylalanine ....... -................ ...... ..... Reserpine ....... .. ..... . ........ . ..... . ..... ............. DISCUSSION..... ........ . ..... . ..... ................ ..... ...... APPENDICES.................................................... APPENDIX.A - Pathways of Monoamine Biosynthesis.......... APPENDIX B - Chemical Structures of the Compounds InvestigatedOOOOOOOOOOO......OOIOOOOOOOOOOOI LIST OFREFERWCES ......... ......OOOOOOOOOOOI0.0.0000.......O. 14 14 17 23 26 26 26 30 37 37 38 40 Table 10 LIST OF TABLES Page Mean core body temperature (0C) : S.E. of mature female mice (n-6) injected i.p. with promazine, 25 mg/kg, at 24 hour intervals for 7 consecutive dayBOOOIOOOOOOOOOOO...............OOOOOOOOOOOOOOOOOOOO 15 Mean core body temperature (0C) i S.E. of mature female mice (n86) injected i.p. with triflupromazine, 5 mg/kg, at 24 hr intervals for 6 consecutive days.... 15 Mean core body temperature (OC) 1 S.E. of 5 week old female mice (n86) injected i.p. with triflu- promazine, 5 mg/kg, at 24 hr intervals for 6 consecutive days...................................... 15 Development of tolerance after i.p. administration of promazine (25 mg/kg) at 24 hour intervals for 7 consecutive days in mature female mice................ 16 Development of tolerance after i.p. administration of triflupromazine (5 mg/kg) at 24 hour intervals for 6 consecutive days in mature female mice.......... 16 Development of tolerance after i.p. administration of triflupromazine (5 mg/kg) at 24 hour intervals for 6 consecutive days in 5 week old female mice...... 16 Development of tolerance after i.p. administration of chlorpromazine (5 mg/kg) at 24 hour intervals for 10 consecutive days in mature male mice........... 22 Mean core body temperature (00) i S.E. of mature female (above) and male mice (below) injected i.p. with procaine at dose levels indicated................ 22 Development of increasing sensitivity in mature female mice after successive i.p. administration of reserpine (.5 mg/kg).................................. 22 Mean core body temperature (0C) 1 S.E. of mature female mice injected i.p. with reserpine as indicatedOOOOO.......OOOOOIOOOO......OOOOOOOOOOOO0.... 28 v1 LIST OF FIGURES Figure Page 1 Time vs core body temperature of mature female mice injected with chlorpromazine, chlorpromazine plus L-dopa (above) or L-dopa (below).................. ...... 18 2 Time vs core body temperature of mature male mice injected with chlorpromazine, chlorpromazine plus L—dopa or L-dopa, and mature female mice injected with chlorpromazine..................................... 20 3 Time vs core body temperature of mature female mice injected sequentually with triflupromazine, promazine and chlorpromazine at 24 hour intervals................. 24 4 Time vs core body temperature of mature female mice injected with promazine 24 hours after prior injection of promazine or triflupromazine...... ......... 24 5 Time vs core body temperature of mature male and female mice injected with parachlorophenylalanine methyl ester (425 mg/kg) at 24 hour intervals for 6 consecutive days........................................ 25 6 Time vs core body temperature of mature female mice injected with reserpine as indicated.................... 27 A1 Biosynthesis of Catecholamines..... ..... ................ 37 A2 Biosynthesis of 5-hydroxytryptamine....... ...... ........ 37 Bl Chlorpromazine ........ . ........ . .......... . ............. 38 DZ Promazine. ............. . .................. . ............ . 38 B3 Triflupromazine ..... . ....................... . ........ ... 38 B4 L-dopa.... ............... ..... ................... ....... 38 B5 Reserpine...... ..... ...... ............... .. ....... ...... 39 B6 Procaine................ ......... . ................. ..... 39 B7 Parachlorophenylalanine Methyl Ester .................... 39 vii INTRODUCTION Thermodynamics of Ageing and Death Previous work has suggested that thermodynamics is a valid approach to the study of ageing and death in biological systems (Rosenberg, Kemeny, Switzer, Hamilton, 1971). This approach simplifies the study of ageing in that it avoids a description-of complex molecular interactions and poorly understood physiological dysfunctions related to senescence. Procaryotic organisms, virions and yeast, when exposed to lethal temperatures above their optima for growth tend to die according to pseudo first order kinetics such that: dn/dt = -kdn (1) The absolute rate theory equation (Gladstone, Laidler and Eyring, 1941) describes kd as varying exponentially with temperature so: k - KkBT/h exp [AS+/R] X exp [-AH+/RT] (2) d Rosenberg and Kemeny et a1 (1971) have shown that data gathered from a large number of investigations involving the thermal killing of unicellular organisms indicates that: AS+ = aAH+ + b (3) This equation represents a compensation law because changes in 1 2 + AS+ are partially compensated for by changes in AH resulting in a relatively constant value of kd. At the compensation temperature, Tc 3 1/a = 3300K, the compensation is exact, b - -66 cal/moleoK. The fact that in vitro thermal denaturation of a large number of proteins under different experimental conditions can be described by the above equations (1-3) yielding the same values for a and b. in equation 3 (3300K, ~66 cal/moleoK) has lead Rosenberg and Kemeny et al (1971) to advance the general hypothesis that protein denatur- ation is the rate limiting step for thermal death in unicellular organisms. In order to extend a thermodynamic description of ageing and thermal death to multicellular poikilotherms, other methods of analysis are needed due to the nonexponential survivorship curves exhibited by these organisms. Larry Smith (Rosenberg, Kemeny, Smith, Skurnick, Bandurski, 1973) discovered that the square shaped survivorship curves of multicellular organisms are best fit by describing the mortality rate as a power of time such that: _ l dfiit) _ n 110:) — - ———N(t) dt — At (4) in its integrated form: Atn+1 3-551 = e‘ n+1 (5) 0 Therefore by plotting the log log of percent survivors against the log of time, straight lines were obtained with a slope of n + 1 and the extrapolated y intercept yielding the value of A. Survivorship curves of Drosophila melanogaster at different ambient temperatures 3 showed higher rates of mortality at higher temperatures with A being temperature dependent but n being relatively temperature independent. This suggested that: + 'AH R A 2 Ace / T (6) The Arrhenius plot subsequently obtained showed a good straight line. The slope, AHall = 190 kcal/mole yielded the activation enthalpy for the mortality rate of Drosophila. The existence of an activation enthalpy for age related death in Drosophila suggests that simple molecular events are involved. Moreover, the high value determined for AH+suggests that these monomolecular events relate to protein denaturation. Using the value of AH+ - 190 kcal/mole and equations 4-6 it can be shown theoretically that the temperature dependence of ageing and death in multicellular organisms is such that in humans a decrease of core body temperature of only 2°C will extend median survival time from 71.8 years to 100 years (Rosenberg, Kemeny et al, 1973). Any thermodynamic study of ageing in laboratory mammals, however, is obviously dependent on techniques which would allow for the manipulation of the core body temperature of these organisms. Such techniques require an understanding of the neurochemistry of thermoregulation. Hypothalamic Mechanisms of Thermoregulation Feldberg and Myers (1964) have suggested that the balance between the release of norepinephrine and serotonin in the anterior hypothalamus of homeotherms is the mechanism whereby body temperature is regulated. Presumably ascending monoaminergic pathways which terminate in the 4 rostral hypothalamus (Fuxe,1965; Fuxe, Hokfelt and Ungerstedt, 1970) and carry information from temperature receptors in the skin (Bligh, 1966) deep in the body (Hammel, 1968) and the blood (Hayward and Baker, 1968) synapse with monoaminoceptive cholinergic efferents ultimately involved in the control of thermogenesis. Much evidence has accumulated to indicate that the release of 5-HT in the anterior hypothalamus in mammals acts through a cholinergic pathway to signal heat production, while the release of norepinephrine in this region blocks the cholinergic heat producing pathway (Myers, 1969; Myers and Yaksh, 1969; Myers, 1970; Hall, 1972; Hall and Myers, 1972; Knox and Lomax, 1972; Rudy and Wolf, 1972; Myers, 1974; Chawla, Johri, Saxena, Singhal, 1975; Metcalf, Myers, Redgrave, 1975; Myers, 1975). A second cholinergic pathway apparently located in the posterior hypothalamus and modulated by the ratio of Na+ to Ca++ ions in this area functions as a heat loss mechanism.(Myers, 1974). Thus the set-point core body temperature for homeotherms is represented physiologically as the Na+ to Ca++ ratio in the posterior hypothalamus. Increases in the Ca++/Na+ ratio in this area depress while decreases elevate the set-point temperature (Myers and Veale, 1970; Veale and Yaksh, 1971; Myers and Veale, 1971; Myers and Yaksh, 1971; Myers and Brophy, 1972; Myers and Buckman, 1972; Myers and Tytell, 1972). Homeotherms, therefore, are able to regulate their core body temperature around an ionically determined set-point by regulating the levels of monoamines released in the anterior hypothalamusT Heat production is generally thought to be associated with cellular oxidation, increased +Appendix A details pathways of monoamine biosynthesis. 5 oxygen consumption and shivering (Prusiner and Poe, 1968) while heat loss is related to peripheral blood flow, body posture and panting (Cremer and Bligh, 1969; Feldberg, 1975). The importance of this model is that it exhibits distinct species continuity. When injected directly into the anterior hypothalamus 5-HT elevates and norepinephrine lowers the body temperature of the unanesthetized cat (Feldberg and Myers, 1965), monkey (Myers and Yaksh, 1969), rat (Avery, 1971; Crawshaw, 1972), ground squirrel (Beckman and Satinoff, 1972), and dog (Myers, 1975). Systemic administration of SHIP which is rapidly decarboxylated to form 5-HT in brain tissue results in a hyperthermic response in the rabbit that can be blocked by the serotonin antagonist 2-brom LSD (Horita and Gogerty, 1958), while the blockage of central 5-HT receptors in the rat and mouse results in hypothermia (Grabowska, Michaluk,Antkiewicz, 1973). Further, hyperthermia induced in the rat by systemic administration of drugs is correlated with an increased turnover of 5-HT in the brain (Reid, 1970; Bruinvels and Kemper, 1971) and hypothermia is associated with increased levels of norepinephrine in the hypothalamus (Bruinvels and Kemper, 1971). Finally, peripheral cooling of the monkey elevates the release of 5-HT from the anterior hypothalamus (Myers and Beleslin, 1971) and peripheral warming of the cat evokes the release of norepinephrine from this region (Myers and Chinn, 1973). While inconsistent effects of biogenic amines on temperature have been reported, these effects are generally obtained in studies not involving the central administration of amines, so that direct peripheral effects cannot be discounted, or in studies involving the injection into the hypothalamus of a high dose of the amine in a large volume. Myers (1968, 1970) and Avery (1971) have shown that when increasingly high doses or large volumes of a biogenic amine are administered centrally a reversal of the initial effect occurs. Disruption of Thermoregulation by the Systemic Administration of Drugs Although it is possible to alter core body temperature in homeotherms by the central administration of biogenic amines, this technique does not seem to be feasible for the kinds of long term studies that would be necessary in the investigation of the temperature dependence of ageing in mammals. Simpler techniques that could nevertheless render homeotherms hypothermic for a part or all of the duration of their lives would be more desirable. The experiments reported here investigate the potential of various drugs to evoke hypothermia in the mouse after systemic administration. The purpose of these investigations was to determine for each of the compounds or combination of compounds tested the existence or nonexistence of a hypothermic response and the development or nondevelopment of a diminished response (tolerance) with repeated trials. The compounds chosen were generally either those previously reported to have produced hypothermia in mammals or those suspected of the capability to interrupt thermoregulation by altering effective hypothalamic concentrations of monoaminergic transmitters. Appendix B shows the chemical structures of the compounds investigated. Phenothiazines. Chlorpromazine, the prototype phenothiazine, has long been known to produce hypothermia in experimental animals. There is little doubt that its main site of action is on the 7 hypothalamus (Cremer and Bligh, 1969). Centrally the hypothermia evoked by this compound after systemic administration is correlated with decreased 5-HT turnover (Shore, Pletscher, Tomich, Carlsson, Kuntzman, Brodie, 1957; Cox and Potkonyak, 1967) and increased turnover of norepinephrine and dopamine (Moore, 1971). Peripherally, chlorpromazine induced hypothermia is associated with decreased utilization of calorogenic substrates (Mueller and McDonald, 1968), heat loss due to peripheral vasodilation (Decourt, Brunaud, Brunaud, 1953; Baxter, Bolster, McKecnhie, 1954; Dobkin and Gilbert, 1954; Giaja, J. and Markovic-Giaja, 1954), depressed oxidative processes (Courvoisier, Fournel, Ducrot, Kolsky, Roetschet, 1953) and suppression of shivering (Kollias and Bullard, 1964). Tolerance, however, develops rapidly with respect to the hypothermia induced by this compound upon repeated administration in the mouse (Rosenberg, personal communication). In an effort to determine whether other phenothiazine congeners also exhibit tolerance related to their ability to evoke hypothermia, the temporal characteristics of promazine and triflupromazine induced temperature depression were investigated. To determine whether the sequential administration of triflupromazine, promazine and chlor- promazine would provide a more efficacious method of evoking extended hypothermia than the successive administration of either congener alone, experiments were performed to test the existence of cross tolerance with respect to the hypothermic response evoked by these agents. Finally experiments were designed to determine whether an exogenous source of norepinephrine precursor, L-dopa, would offset the tolerance reaction to chlorpromazine. 8 Procaine. Systemic administration of procaine leads to a centrally mediated peripheral vasodilation in laboratory animals (Peterson, 1955; 1955a) and possibly humans (Glasgow and Sinclair, 1962; Gordon, Fudema, Snider, Abrams, Tobin, Kraus, 1965; Livingston and Perrin, 1972). Since peripheral vasodilation is generally associated with heat loss this suggests a possible hypothermic effect of procaine. Aslan (Aslan, Urabiescu, Domilescu, Campeanu, Continiu, Stanescu, (1965) and others (Berger, 1960) have reported that laboratory rodents treated chronically with procaine exhibit an increased survival rate when compared to non-treated controls. Chronic administration of procaine in humans has also been reported to increase longevity (Aslan, 1962). If procaine does in fact have the ability to depress core body temperature this would help to explain these results. Accordingly experiments were performed to test the efficacy of procaine as a hypothermic agent. Parachlorophenylalanine. Parachlorophenylalanine (PCPA) was chosen for investigation because it inhibits tryptophan hydroxylation (Knapp and Mandell, 1972), and thus reduces brain levels of 5-HT (Preziosi, Scapagnini, Nistico, 1968; Aghajanian, Kohor, and Roth, 1973). PCPA may (Roe and Weissman, 1966; Welch and Welch, 1967; Miller and Maickel, 1969) or may not (McDonald and Mueller, 1969; Voicer, 1969) slightly decrease cerebral norepinephrine levels by inhibiting phenylalanine and tyrosine hydroxylase (Roe and Weissman, 1966). Miller and his co~workers (1969a) have reported a 41 and 22 percent reduction respectively of hypothalamic levels of 5-HT and norepinephrine after intraperitoneal injection of PCPA in the rat. 9 Since this compound apparently preferentially reduces 5-HT concentrations in the hypothalamus it should theoretically induce a hypothermic response. Experiments were therefore performed to test this hypothesis. Reserpine. Systemic administration of reserpine in laboratory animals induces a hypothermic response (Shemano and Nickerson, 1958; Fondy, Karker, Calcagnino, Emlich, 1974). Presumably this effect is mediated by the ability of this compound to deplete central monoaminergic stores (Miller, Cox, Snodgrass, Maickel, 1969) especially in the hypothalamus (Shore, Pletscher, Tomich, Carlsson, Kuntzman, Brodie, 1957; Cooper, Cranston, Honour, 1967). The peripheral effects of reserpine which include vasodilation (Nickerson, 1970) and decreased mobilization of calorogenic substrates (Mueller and McDonald, 1968) have also been well described. In view of these reports it was of interest to know the feasibility of using reserpine as a hypothermic agent for an extended period of time. Experiments were therefore conducted to answer this question. METHODS General Procedures The experimental animal used in these studies was the Swiss Webster mouse, male and female, obtained from Spartan Research Animals, Inc., Haslett, Michigan. The animals were marked for individual identification and unless otherwise noted housed six to a cage and kept at ambient temperature (22°C). Food and water were available §d_lib, To minimize circadian rhythms all experiments were conducted under conditions of 24 hour light. The experimental compounds were administered either intraperitoneally (i.p.) or sub- cutaneously (s.c.) in .852 saline, sterile double distilled water, or other suitable diluents. The dose schedule was dependent on the prop- erties of the individual compounds being tested. Dose levels chosen were those that in previous trials were shown to produce maximum effects on body temperature with minimum side effects such as sedation, torpor, and extrapyramidal involvement. Core body temperature was determined prior to injection and thereafter monitored at regular intervals with a Yellow Springs Instruments telethermometer and a Fisher Scientific small animal probe. The probe was inserted rectally at a distance of 2.5 cm and allowed to equilibrate before temperature was recorded. Each cohort of animals served as its own control. Data were analyzed by analysis of variance (completely random design), and the Student-Newman—Keuls test for unequal sample size (Sokal and 10 11 Rohlf, 1969). The level of significance was chosen as P<.05. Phenothiazines Promazine. The experimental population consisted of six mature females. Every animal received promazine intraperitoneally at 25 mg/kg in .5 m1 volumes carried in .852 saline. Injections were made at regular 24 hour intervals. Core body temperatures were determined at 30 minute intervals for three hours following the injections. The experiment ran for seven days. Triflupromazine. The experimental population consisted of six mature females and six 5-week old females. Each animal received triflupromazine intraperitoneally at 5 mg/kg in .5 m1 volumes carried in .852 saline. Injections were made at regular 24 hour intervals. Core body temperatures were determined at 30 minute intervals for three hours following injections. The experiment ran for six days. Chlorpromazine and L-Dopa. The experimental population consisted of 18 mature females divided into three cages of six animals. The first group received L-dopa subcutaneously in .2 ml volumes as a slurry in .852 saline at 266 mg/kg three times a day at regular eight hour intervals. The second cage received L-dopa via the same route and dosage schedule but also was treated at regular 24 hour intervals with intraperitoneal injections of chlorpromazine in .852 saline, .5 m1 volumes at 5 mg/kg. The remaining group received chlorpromazine alone via the same dosage schedule as above. In all cases the admin- istration of chlorpromazine coincided with the administration of L-dopa. Core body temperature was determined in all groups after chlorpromazine administration at 30 minute intervals for 180 minutes. The experiment ran three days. To investigate sex dependent response 12 differences to chlorpromazine, L—dopa, and the combination, the entire experiment was repeated using male animals. In this case, due to an amplified response, core body temperature was followed at 30 minute intervals for 240 minutes after chlorpromazine. This experiment ran 10 days. Determination of cross tolerance between triflupromazine, promazine and chlorpromazine. Triflupromazine, promazine and chlorpromazine were sequentially administered in effective initial doses (5 mg/kg, 25 mg/kg, and 5 mg/kg respectively) at 24 hour intervals. The experimental population consisted of six mature females. All compounds were carried in .85% saline and injected intraperitoneally in .5 ml volumes. Core body temperatures were recorded at 30 minute intervals for two hours following the injections. The experiment ran three days. Procaine The experimental population consisted of six males and six females. The animals were caged individually to avoid any masking of a hypothermic response because of huddling behavior. Half the males received intraperitoneal injections of procaine HCl in .5 m1 volumes at 8 mg/kg. The other half received twice this dose via the same route. The same dose schedule was followed for the female population. The procaine HCl was carried in .85 saline. Core body temperature was determined at 30 minute intervals for two hours after the injection. Parachlorophenylalanine The experimental population consisted of six mature males and 13 six mature females. Every animal received parachlorophenylalanine methyl ester intraperitoneally in .15 ml volumes carried in .851 saline, 425 mg/kg at regular 24 hour intervals. The methyl ester was used due to the insolubility of the parent compound in any suitable diluent. Core body temperature was monitored at 30 minute intervals for three hours after injections. The experiment ran six days. Reserpine The experimental population consisted of three groups of three mature females, individually housed to avoid huddling, and kept in a constant temperature cabinet at 28.00C and 45-50% humidity. (Earlier trials had shown that ambient temperatures lower than 28.00C caused reserpinized animals to experience deep hypothermic reactions which were apparently lethal.) The three experimental groups received reserpine at 0.5 mg/kg, 1.0 mg/kg, and 2 mg/kg, respectively. The reserpine was obtained in a presolubilized form as a commercial preparation (Serpasil, Ciba), diluted to the correct dosage with sterile double distilled water and injected intraperitoneally in .2 m1 volumes. The injections were made at intervals of no less than two days and no greater than six days. Core body temperature was determined at regular intervals. To alleviate the anorexia sometimes associated with the administration of this compound, a slurry of powdered mouse pellets and water was made available §§_lib, The experiment ran for 32 days. RESULTS Phenothiazines Promazine. Promazine evoked a significant hypothermia on each of the seven days it was administered to mature female mice (Table 1). In every case hypothermia appeared within 30 minutes after injection and was still evident at 180 minutes. Normothermia was always restored within 24 hours. Except on days 2 and 5, the return to normothermia began within 180 minutes after injection. Tolerance appeared after three days with respect to the hypothermic response exhibited at 90 minutes following promazine, and after two days with respect to the hypothermic response exhibited at 180 minutes. Table 4 shows the further development of tolerance. Triflupromazine. The hypothermic response induced by triflu- promazine was investigated in both mature and 5 week old female mice. In mature mice triflupromazine evoked a significant hypothermia on each of the six consecutive days it was administered (Table 2). On days 2-6 hypothermia appeared at 30 minutes following injection; on day l hypothermia did not appear until 60 minutes after injection. In all cases, however, hypothermia was still evident at 180 minutes and no return towards normothermia was begun over this interval. Normothermia was, nevertheless, always restored within 24 hours after injection. Tolerance appeared after one day with respect to the hypothermic response exhibited at both 90 and 180 minutes following 14 0 39.17:.11 39.001.13 39.331.11 39.08:.15 39.251.11 39.251.17 39.50t0.00 30 36.25:.28 36.082.08 36.331.11 36.501.26 36.75:.28 36.831.21 37.001.26 215 60 35.33:.11 gs.oa:.1s‘ 35.42:.3 36.25:.44 36.16:.28‘ 36.921.33 36.33:.31b 90 34.67:.25 35.42:.64 35.58:.65 36.33:.48 36.33:.40 37.081.30 36.91:.30 120 34.33:.21b 33753:??? 36.67:.38 36.171.42b 36.50:.48 37.83:.30 37.501.31 150 35.171.49 35.831.46 37.001.45 37.251.42 37.171.38 38.331.17 37.581.35 180 35.751.50 36.581.54 37.50:.45 37.831.21 37.501.34 38.421.15 38.16:.17 a Table 1. Mean core body temperature (cc) 18.3. of mature female nice (n-6) injected 0 39.5010.00 39.331.11 39.”:0.” 39.41i.08 39.581.08 39.5010.00 30 (37.921.40) 38.171.17 38.081.15 37.671.17 38.001.22 37.751.25 60 36.331.48 37.421.33 36.92:.24 37.081.55 37.001.47 37.001.26‘ * Table 2. Han core body temperature 0 38.75:.11 38.581.15 38.58:.15 39.33:.11c 39.08:.20 39.5010.00c 30 (36.921.51) 37.001.18 37.17:.24 37.00:.26 37.171.17 37.42:.15 i.p. with triflupromazine, 60 (35.421.72) 35.66:.44 36.50:.25b 36.83:.2sb 36.67:.17a 37.50:.13 90 35.00:.74 37.17:.28 36.42:.43‘ 36.92:.49 37.00:.53 37.00:.32 9O (34.922.37) 35.25:.46a 36.50:.44 36.83:.31 37.251.30 37.50:.37 120 34.00:.82 37.67:.38 36.67:.48 36.16:.54a 36.67:.65a 37.833.20 150 34.081.88 37.081.40a 36.67:.56 36.831.71 36.75:.74 37.331.33 i.p. with promazine, 25 mg/kg, at 24 hr intervals for 7 consecutive days. 180 33.7Stl.23 37.251.44 36.83:.65 36.92:.71 36.92:.72 37.25:.31 (OC)tS.E. of mature female mice (n-6) injected 5 mc/kg, at 24 hr intervals for 6 consecutive days. 120 150 180 (35.67tl.95) (35.0011.37) (35.4211.29) 35.50:.51 35.831.51 36.581.64 36.671.35 37.41:.24 37.751.21 37.672.21 37.58:.08 37.73:.17 37.831.43 37.501.18 37.67:.21 37.17:.30a 38.08:.30 38.08:.30 * Table 3. Mean core body temperature (0C) iS.E. of 5 week old female mice (n-6) injected *The dotted line indicates injection. i.p. with triflupromazine, 5 ms/ks, at 24 hr intervals for 6 consecutive days. Parentheses enclose mean core body temperatures not different from the intratrial preinjection value (P>;05). The deepest hypothermia reached after each injection (underlined) was compared to the intratrial mean core body temperature at 180 min. began within the daily experimental interval. between trials determined whether normothermia was restor This provided a method of determining whether a return towards normothermia The comparison of preinjection temperatures not different from intratrial value at 180 min (P>.05). value at 180 min (P<.05). Ed prior to each injection. Indicates different from intratrial cIndicates different from preinjection value on day l (P<.05). Indicates l6 1 2 3 4 5 6 7 34671.25‘ 35.421.44 35.581.45 36.331.48 36.33:.40 37.08:.30 36.91:.30 1 2 3 4 5 6 7 35.57t.50b 36.581.54 37.501.45 37.831.21 37.501.34 38.421.15 38.161.17 a Table 4. Development of tolerance after i.p. administration of promazine (25 mg/kg) at 24 hour intervals for 7 consecutive days in mature female mice. 1 2 3 4 S 6 35.001.74a 37.171.28 36.421.43 36.921.49 37.00:.53 37.00:.32 1 2 3 4 5 6 33.7511.23b 37.251.44 36.833.65 36.921.49 36.92:.72 37.25:.31 a Table 5. Development of tolerance after i.p. administration of triflupromazine (5 mg/kg) at 24 hour intervals for 6 consecutive days in mature female mice. 2 3 a 5 6 35.25:.46‘ 36.502.44 36.83:.30 37.25:.30 37.50:.37 2 3 4 S 6 36.58:.64b 37.751.21 37.75:.17 37.671.21 38.08:.30 a Table 6. Development of tolerance after i.p. administration of triflupromazine (5 mg/ks) at 24 hour intervals for 6 consecutive days in 5 week old female mice. Hypothermia did not develop at any time after injection on day l. *Days underlined by the same line were not different with respect to the hypothermic response exhibited at 90 minutes (above) or 180 minutes after injection (below). P>.05, n-6. ‘lndicates core body temperature at 90 minutes after injection. Indicates core body temperature at 180 minutes after injection. l7 triflupromazine. Further tolerance did not develop over the course of the remaining trials (Table 5). Administration of triflupromazine to 5 week old mice for six consecutive days evoked a significant hypothermia on days 2-6. The hypothermia appeared at 30 minutes after injection and was still evident at 180 minutes. Hypothermia did not develop at any time after injection on day 1 (Table 3). While a return to normothermia was not evidenced within 180 minutes following the administration of triflu- promazine on days 2, 5, and 6, in no case was hypothermia still apparent 24 hours after injection. Tolerance appeared after four days with respect to the hypothermic response exhibited at 90 minutes following triflupromazine, but further tolerance did not occur. Tolerance never developed with respect to the hypothermic response at 180 minutes following triflupromazine over the course of this experiment (Table 6). Chlorpromazine and L—dgpa. The hypothermic response evoked by chlropromazine, L-dopa, and the combination was investigated in both mature male and female mice. In female mice chlorpromazine induced significant hypothermia on each of the three consecutive days it was administered. Hypothermia appeared 60 minutes after injection on day 1, and 30 minutes after injection on days 2 and 3 (Figure 1). In all cases hypothermia was still evident at 180 minutes and no significant return to normothermia was initiated over this interval. Normothermia was, however, always restored within 24 hours. Tolerance developed after one day with respect to the hypothermic response at 90 and 180 minutes following chlorpromazine. Core body temperature at 90 and 180 minutes on day 2 was significantly different than core body 18 CPZ (5 Ins/k9) ago-K "" CPZ (5 ills/k9) \ , \ + Mom (266 mg/kg) zoo ' \\ 1__ .L a \ / S 37.0 \ \ / o \ ,, 7 \ \\ ,, ’ /T V/ >‘ 00 \\ \ \\ // 350 \ . . \\\‘ I ’ ari/ .. \ .. 2 I .JL 34.0 X \ i= \ \ «P 330 1\ \ .1 \ \ \] \ 1P ,. 32.0 ]\ z: \ \ o \ .. _ 1 ._ .. J I V V Y T r 1 Min. 0 30 so 90 120 150 ISO 39 ,J'. L-dopa (266 mg/kg) '1‘ °Uas I T 37 , , , , , . Min. 0 so so so 120 150 180 Ist'l 2 3 l 2 3 l 2 3 l 2 3 l 2 3 l 2 3 l 2 3 Figure l. Time vs core body tenperature of mature female mice injected with chlorpromazine, chlorpromazine plus L-dopa (above) or L-dopa (below). Injections were made at 24 hour intervals for three successive days. Chlorpromazine was injected i.p.; L-dopa was injected s.c. Each point (above) or bar (below) represents the mean core body temperature :S.E. of 6 mice. 19 temperature at these times on days 1 and 3. The administration of L-dopa did not significantly affect the core body temperature of female mice, nor did L-dopa alleviate tolerance with respect to the hypothermic response evoked by chlor- promazine in these animals. The core body temperature of mice receiving both chlorpromazine and L-dopa was only significantly less than animals receiving chlorpromazine alone at 30 minutes after injection on day 1. Indeed, animals receiving both Lodopa and chlorpromazine exhibited significantly higher mean core body temper- atures than animals receiving chlorpromazine alone at time 0 and 120 minutes on day 3. Figure 2 shows the effects of chlorpromazine, L—dopa and the combination on the core body temperature of male mice. The effects of chlorpromazine in female mice are also plotted on this figure for comparison. In male mice chlorpromazine induced a significant hypothermia on each of the ten consecutive days it was administered except on day 3. In all cases where hypothermia occurred it appeared 30 minutes after injection and was still evident at 180 minutes. The deepest hypothermia reached was never significantly lower than * the hypothermic response at 180 minutes suggesting that there was no trend towards normothermia over this interval. No trend towards normothermia ever developed over the 240 minute daily experimental interval on days 1, 2, 5, 6, and 7. However, normothermia was always restored within 24 hours. A trend towards normothermia did begin after 180 minutes on days 4, 8, and 10. In contrast to parameters previously defined for female mice, in male mice tolerance took six days to develop with respect to the hypothermic 20 A33... 3 Eu so ooHI m .32.. o .3 .m.m« 332363 >63 9.8 can... 05 35330.. «58 5am 6.... 830?: was Sou-.. 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Comparison of hypothermia evoked in male and female mice after chlorpromazine administration for three successive days showed that significantly deeper hypothermia appeared in males at 30 and 60 minutes after injection on day l, at all times after injection on day 2 and at 30, 60, and 90 minutes after injection on day 3. Preinjection tempteratures were never different. In male mice the continual administration of L-dopa at 8 hour intervals for 10 successive days significantly affected core body temperature from day 3 through day 10. On each of these days core body temperature at 30 minutes after injection was significantly depressed. On days 3, 4, 5, 6, 7, 8, and 10 this hypothermia was transient, normothermia was restored at 60 minutes. On day 9 the hypothermia existed for 90 minutes. Hyperthermia was exhibited at 120 and 150 minutes after injection on day 4, and 90 and 120 minutes after injection on day 6. Preinjection temperatures on days 2, 3, and 5 were also significantly elevated. In spite of these fluctuations a comparison of the L-dopa evoked temperature depression at 30 minutes after injection between trials suggested that there was no development of tolerance at this time over the 10 day experimental period (P<.05). Treatment with L-dopa, however, did not alleviate tolerance with respect to the hypothermia evoked by chlorpromazine in male mice. The mean core body temperature of animals receiving both chlorpromazine and L-dopa was only significantly less than animals receiving CPZ alone at 30 minutes after injection on day 3, 0 minutes after injection on day 7 and 30 and 90 minutes after injection on day 9. Indeed animals receiving both L-dopa and chlorpromazine exhibited a significantly 22 .m.~|a .no.Am uaauonuonmn «an ou uuonuou no“: uauuoumuv uoa onus mafia mama «so hp vocwauovus mama .Anx\wa n.v unannouou .nouuoonuw nouns mason o as vouapqnuo uncommon mo nowuuuuufinwavu .m.« o>aououu=u sound cows mamas“ casual aw huw>wuwuaoo unannouona no unannoao>on .o manna n~.now.on om.won.~n oo.H«on.nn m~.un~.¢n n~.nn~.mn “00 n u o n e "mun .mla .no.Am .uusuuuonamu Avon ouou uoouuo unaccoua van ammo on aH .aoauoonaw moamacwao «can vouuov any .vououavca uao>oa snow on ounceoun sun: .9.“ vouoofiaa Aaoaopv mafia mama can Am>onov mamamu masons mo .m.mw Auov ouauauuaaou neon ouoo and: .n uHAMH sq.nmm.mn sa.»nw.mn as.“ho.mn oo.wmm.mm " mm.uom.mm ms\ma ea 5H.nne.mn sa.nno.nn am.uoo.mm e¢.«nn.wn “oo.o«oo.on nx\wa n o~H as as on m o Assay mafia «v.«wo.on 5H.«na.an nd.«sa.on oo.on0m.mn “oo.o«on.mn wxxna on nn.u~o.mn mm.»ho.mn «a.»oo.sn “H.ann.on m ”H.unn.om ux\uu a ONH co on an n o Acaav made .olc .no.Am .AsoHonv nowuoufiaw nouns nuances one no Ao>on¢v ouu:¢«a_oo um vouaaqaxo uncommon udauoauomhn on» ou uuoauou nuva uaouomuuv was who: mafia dado «nu an voawauovd: shun .uuwa maul aways! nu chum o>wuaoouaoo OH new oHd>uounu "no: «N no Anx\wa m v onauqaououoano mo douuouuawcaavo .9.“ nouns moauuoaou mo uaoaaoao>oo .u wanna nn.nna.on Ne.«ha.on o~.a«nn.en Hm.«ne.nn ~H.Hnna.en on.a«uo.¢n «o.HuNa.Hn Na.aoc.an o h o m a n N H nn.«nu.nn ~n.«~q.nn nH.H«~H.nn es.«sa.en nH.Hmno.nn o~.~«um.nn nc.nsa.an oo.HOn.Hn n s o n e n N H 23 higher mean core body temperature than animals receiving chlorpromazine alone at 180 and 210 minutes after injection on day 5; 150, 180, and 240 minutes after injection on day 7 and at 150 and 210 minutes after injection on day 8. Determination of cross tolerance between triflupromazine, promazine and chlorpromazine. The existence of cross tolerance with respect to the hypothermic response evoked by triflupromazine, promazine and chlorpromazine was investigated to determine if the sequential administration of these compounds would provide a more efficacious method of inducing extended hypothermia than the successive admin— istration of either compound alone. Figure 3 shows the hypothermic response evoked by the sequential administration of triflupromazine, promazine and chlorpromazine at 24 hour intervals to mature female mice. Significant hypothermia developed after each compound was injected. The hypothermic response produced by triflupromazine was not significantly different than that produced in previous trials (Table 2) under the same conditions. Similarly the hypothermia evoked by chlorpromazine after pretreatment with triflupromazine and promazine did not significantly differ from the temperature depression produced in previous trials (Figure 1) involving the administration of three doses of chlorpromazine 24 hours apart. In contrast the hypothermic response produced by promazine 24 hours after triflu- promazine (Figure 4) was significantly greater than that produced in previous trials involving the administration of two doses of promazine 24 hours apart. 21% .mo—s o co .m.mu otzpogoaemu noon u.oo cums on» mucommLQo. ..3 58 d; 8...... 2038...... .2 85350333..» .0 85850.... .8 5.58.2: .3... .3: 2:9. en 85:59:. 5; “.382: 3.5. 22.8... 9.39.. .6 839.353 >68 9.8 m> as: J. 853. GNP OG 00 On 0 5:2 3...... ms 8:28... .3: m... ¢~ 3.2m... m3 05350... D Amxxme m. ocv~oeo.q:Pa—.» .3: m... cm 3.35 mm. 2.326... . an on On —n an nn in an on kn on .32.. m mo .8 58 a; 9.03 23.582: :< 053598.25 2:. 3.525.... 65.350.33.75 5:: 333538.». 80.8.2: 32.. 29.6.. 9.32. .6 9.39.353 .33 9.3 m> 85:. m- m- n~_ .m.m« 0.39.8953 .83 9.3 :8... 05 35.9.9.8. .m_a>.o»:. eso; «N on m~_>oO O Our 0.. ~ '5‘. n a... o .... I... I a... . In}... I'; v.0...- o a o . ... .‘c u n I a o n n n u - ... .:.:.::::.2:::EE?".'.‘.:::.'5.9:".'. . '5‘ ’ u:: . . o ' . ‘ . u - II'II'. o‘ '3’. . . ... a a - u .' .‘u . .‘I‘o Ga 00 F—VU/fZ/l/llllfl/Zl/ll I I-[7///////7//[///////// -:=:¢:-:- Amim... m. 9.328.335 3.3... m2 05350.... I 3.3... 3 u:.~2.o.&=Cr¢ B -:-:-:%b: $"‘-.,.,...35-.-.- . . l—Y/l/l/l/ll/llljl/ZZI/ll I [III IIIIIA .m oasmwu on on On 8 an on an on kn on On 25 0306090le50” 0306090l20150l00 TIME TIME Day I 2 33 _ 33 0 so so 90 120 I50 loo 0 so so 90 120 150 no TIME ‘ TIME Day3 4 39°C _ 39°C 38 38 }’;K\ xx‘J - 'L/ ‘L / 37 ‘47 - 36 - 35 _ 34 _ 34 33 _ as o 30 so 90 I20 150 no 0 so so 90 120 l50 100 TIME TIME Dav5 6 Figure 5. Time vs core body tenperature of mature male and female mice Injected with parachlorophenylalanine methyl ester (425 mg/kg) at 24 hour intervals for 6 consecutive days. All Injections were i.p. Each point represents the mean core body temerature .+.S.E. of 6 mice. 26 Procaine Procaine did not in any way affect core body temperature of male or female mice at either dose level administered (Table 8). Parachlorophenylalanine Figure 5 shows the effects of the sequential administration of PCPA for six consecutive days on the core body temperature of both male and female mice. PCPA evoked a hypothermic response in female mice on days 1 and 4, and in male mice on days 1 and 2, which appeared at 30 minutes after injection and was still evident at 180 minutes. In females the deepest hypothermia reached on these days was not significantly different from the hypothermic response at 180 minutes suggesting that there was no trend towards normothermia over this interval. In males this method of analysis revealed that there was a trend towards normothermia during this period. On days 3 and 6 in females and days 3 and 5 in males PCPA evoked a hypothermia which was only significant at 30 minutes after injection. And on days 2 and 5 in females and 4 and 6 in males no hypothermia occurred at all after PCPA. In females preinjection core body temperatures on days 2, 3, S, and 6 were significantly depressed with respect to these values on days 1 and 4. In males preinjection core body temperatures did not differ significantly over the course of this experiment. Reserpine Figure 6 shows the effects of the sequential administration of reserpine on the core body temperature of mature female mice. A total of eight injections were made over a 32 day period. Table 10 27 .0055 m to N we 8.:uucoa50u xuon some 8 m commcam. u=_oa scum .a.. 8.83 mcompuuncp —_< .uouuuwvcv mu . wmmwncmmo. “wwzuumuuoncw ou_5 apnea; menace mo 8.:uucogem» Avon mcou m> 85.5 o 8.3m.. .I.I oxonnd 58.. . I 9.on— co..8§ . .....I 9:95 .rom_m~_o~_ mm mm cm an mm II c 9.8 ‘ 'l" ‘o\ .-.-.k ¢~_n~_- _~_o~_m_._no._..._m__n__v._n. NM mm \\ o x... VM on mm . \ \. ...-I I. .I.I.|.0'.\ . ‘. (I ‘.‘.I.‘ . “J'.‘.f.“l."‘.‘.' II ,I‘.‘.Il. ‘ .lu.-. .. . l I 060» ~__:_o._m_o_..._no_n_.._n_~__..>chlorpromazine>promazine. Davis and Brody (1965) have found that the ability of phenothiazines to inhibit Na+K+ ATPase in the brain and therefore to stabilize neuronal membranes to Na+ and K+ flux also follows this same potency ratio. If phenothiazines limit Na+ flux in the posterior hypothalamus it is possible that their hypothermic effects may be, in part, due to their ability to effectively increase the Ca++/Na+ ratio in this area. The duration and intensity of action of many drugs are determined by the speed at which they are metabolized in the body by enzymes in the liver microsomes (Conney, 1967). Since chlorpromazine is well known to stimulate liver growth and liver microsomal enzyme levels (Silverstini, Catanese, Del Basso, 1966) and phenothiazine metabolites are largely inactive (Johri and Biachetti, 1966) it is probable that this mechanism accounted for the adaptive responses after phenothiazines seen in these studies. The induction of the liver microsomal enzyme fraction by foreign compounds is usually more marked inthe adult female rodent than in the adult male. Westfall (Westfall, Boules, Shields, 1964) has shown that male mice are affected by pentobarbitol for a longer time than females, and the duration of action can be decreased by treating the males with stilbesterol and increased by treating the females with testosterone. Likewise chronic treatment of male mice with testosterone or methyltestosterone prolongs the action of hexobarbitol and coincidentally decreases the activity of the corresponding enzyme system (Norvick, Stohler, Swagzkis, 1966). Although both male and female mice, in the studies presented here, 32 exhibited a tolerance to repeated administration of chlorpromazine, the degree of induced hypothermia and resistance to tolerance was strikingly greater in the males than in the females, presumably as a result of the greater efficacy of chlorpromazine to induce hepatic microsomal enzymes in females. Similarly repeated injections of triflupromazine produced statistically less tolerance in 5 week old female mice than in adult females. These results are consistent with the findings of Jondorf and his co-workers (Jondorf, Maickel, Brodie, 1959) who have shown that liver microsomes are poorly developed in immature mice. It is conceivable that administration of triflupromazine may also involve the induction of an enzyme system necessary for the production of active phenothiazine metabolites. This would explain the failure of this compound to evoke a hypo- thermic response in immature mice when first administered. And it would also explain the ability of prior administration of triflu- promazine to dramatically potentiate the hypothermic effect of promazine. If such a quality exists in triflupromazine it could possibly be advantageously exploited in future studies. An alternate but not mutually exclusive explanation of the tolerance produced by repeated administration of phenothiazines concerns the finding by Moore (1971) that the increased turnover of central catecholamines produced by these compounds is correlated with a decreased receptor availability. It is plausible, therefore, that tolerance results as the catecholamine receptors become increasingly less vulnerable. This mechanism would explain why in these studies L-dopa did not reliably potentiate the hypothermic effects of chlorpromazine in male or female mice but when 33 administered alone was capable of producing hypothermy at least in males. Despite its reported ability to produce rapidly appearing centrally mediated hypotension in experimental animals (Peterson, 19553), procaine, under the conditions of these investigations, did not produce any significant hypothermy in male or female mice. PCPA, however, which has been reported by numerous investigators to potentiate the effects of other hypothermic agents (Bruinvels and Kemper, 1971; Grabowska et a1, 1973) but to lack the ability to produce hypothermia when administered alone to rodents (Bruinvels and Kember, 1971; Grabowska, 1973; Maj and Pawlowska, 1973) did produce statistically significant hypothermia under the conditions of the experiments reported here when administered to mice in comparable doses. Even though destruction of central serotonergic neurons and blockage of central 5-HT receptors has been shown to produce hypo- thermia (Yehuda and Wurtman, 1972) it is doubtful that PCPA produced its hypothermic effects solely by depletion of hypothalamic 5-HT. Miller et a1 (1969) has shown that central depletion of S-HT in rats after PCPA is significant for about eight days, yet the results presented here indicate that animals treated daily with this compound exhibited only intermittent hypothermy, although a slight hypothermia may have been prolonged for 24 hours or more in female but not male mice. It is possible, therefore, that a direct action on peripheral vasculature as has been suggested by MCDonald and Mueller (1969), played a role in the hypothermia generated by this drug. Of all the compounds tested in these studies, only two, reserpine and L-dopa, exhibited the ability to produce consistent hypothermia 34 with repeated administration. Reserpine has been shown to lower the concentration of monoamines in the hypothalamic area of experimental animals for approximately 30 hours (Brodie, Finger, Orlong, Quinn, Sulser, 1960; Shellenberger and Elder, 1967), a time course which correlates well with the temporal characteristics of the hypothermic responses seen in mice injected with comparable doses of reserpine under the conditions of the experiments presented here. It is likely that the effects of this compound are mediated by the central release of catecholamines since prior depletion of these transmitters blocks or reverses reserpine induced hypothermia (Shellenberger, et a1, 1967). In this respect reserpine acts in much the same way to depress temperature as does chlorpromazine. Reserpine, however, unlike chlorpromazine, does not exhibit an adaptive response. Indeed, mice treated with .5 mg/kg of reserpine exhibited an increasing sensitivity to this compound with repeated trials, eventually reaching the same temperature depression profile as was seen in mice treated with higher doses. The finding that mice injected with elevated doses of reserpine did not show an increasing response to repeated injections but did not show tolerance could possibly suggest that the central catecholamine receptors in reserpinized mice, unlike those in chlorpromazine treated mice, reach a constant level of availability. Although it is generally acknowledged that reserpine exerts a "hit and run" effect (Brodie, Olin, Kuntzman, Shore, 1957) it is certainly possible that the profound hypothermia produced by this compound acts to inhibit the induction of specific enzyme systems and to slow its elimination from the body (Sjoqvist and Hammer, 1968; Cagen, Janoff, Bus, Gibson, 1976). 35 Like reserpine, L-dopa also produced a nonadapting hypothermic response probably mediated through a central mechanism. Butcher and his co-workers (Butcher, Engel, Fuxe, 1972) have shown that systematic ad— ministration of L-dopa in rats at doses comparable to those used in the studies reported here, reduced central S-HT levels by 30%, increased dopamine levels by 300%, but had no effect on norepinephrine levels. In addition, histochemical techniques revealed the presence of catecholamines within serotonergic terminals after L-dopa. Brittain and Handley (1967) have shown that intraventricular administration of dopamine in mice evokes hypothermia. Further, when L-dopa was administered together with a peripheral decarboxylase inhibitor on a single trial basis in rats, significant hypothermia was induced but did not occur when L-dopa was administered alone (Maj and Pawlowski, 1973). Other workers have demonstrated that systemic administration of apomorphine produces hypothermia in rats and mice which can be blocked by various dopamine antagonists (Barnett, Goldstein, Taber, 1972; Grabowska et a1, 1973). In the investigations presented here, L-dopa evoked hypothermia appeared in male mice after two days of prior administration and did not exhibit a diminishing magnitude with repeated injections over the remaining trials. It is likely that in these mice two days of L-dopa loading were required before central concentrations of catecholamines were effectively increased. Similarly, female mice which were treated with L-dopa as in males failed to show a hypothermic response within three days and may have required a longer period for central catecholamine concentrations to saturate monoaminergic neurons. Iverson (1970) has demonstrated that the ability of central 36 monoaminergic neurons to concentrate monoamines is large, and only when the concentration mechanism is saturated are monoamines released. In an intriguing experiment Cantzias and his co-workers (Cantzias, Miller, Nicholson, Maston, Tang, 1974) showed that rats fed large amounts of L-dopa in their diet (4% w/w) exhibited a 73% increase in life span and a concomitant prolongation of vitality when compared to nontreated controls. Since experiments reported here indicate that L-dopa depresses core body temperature and may eVen exhibit increased potency with long term administration, it seems highly likely that these animals were hypothermic. Thermodynamically a small decrease in body temperature could account for this increase in life span. In summary, L-dopa and reserpine both induced nonadapting hypothermy in mice under the conditions of these investigations, probably by evoking the release of catecholamines in the anterior hypothalamus. When L-dopa was administered systemically to rats, together with RO4-4602, to prevent peripheral decarboxylation, nilamide (a MAO inhibitor), and reserpine, histochemical techniques revealed a bright fluorescence indicative of catecholamines both within and around the terminals of central dopaminergic and serotonergic neurons particularly in the anterior hypothalamus (Butcher et al, 1972). Neurochemically this correlated with a 700% increase in whole brain dopamine and a 86% decrease in whole brain 5-HT. In light of the body of information presented here, it is highly probable that this L-dopa/RO4-4602/ni1amide/reserpine "cocktail" represents an extremely effective nonadapting hypothermic agent. APPENDICES APPENDIX A Pathways of Monoamine Biosynthesis COOH TYROSINE HOG- CHz-CH-NHZ 1 Tyrosine hydroxyiase H0 COOH DOPA H0 CH -CH-NH 2 2 Aromatic L-amino acid 1 decarboxylase H0 DOPAMINE HOQCHz-CHZ-NHZ l Dopamine B-hydroxylase H0 NOREPINEPHRINE HO CH-CH 0H -NH 2 2 Figure A1. Biosynthesis of Catecholamines TRYPTOPHAN m CHZ-C'H - COOH N NH H 2 l tryptophan-S-hydroxylase 5-HYDROXYTRYPTOPHAN Hm CHz-C'H - COOH N NH2 H aromatic L-mino acid decarboxylase 5-HYDROXYTRYPTAMINE H0[::]:-jr CHz-CHz-NH2 N H Figure A2. Biosynthesis of 5-hydroxytryptamine 37 APPENDIX B Chemical Structures of the Compounds Investigated ”Z. I Figure Bl. Chlorpromazine S O N H I Figure BZ. Promazine ”Z. . 3 CH -CH -CH2-N(CH 2 2 3)2 Figure B3. Triflupromazine H0 NH 0 I 2 u HO.CH2CH- c -0H Figure B4. L-dopa 38 39 Figure B5. 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